ANTIB OD Y-FUNC TIONALIZED CARBON NANOTUBE-BASED FIELD EFFECT TRANSISTORS AND THEIR USE FOR F MUNO AS SAY OF PACLITAXEL

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of US Application No. 62/344,431, filed

June 2, 2016, expressly incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 58650_Seq_Listing_Final_2017-06-01.txt. The text file is 103 KB; was created on June 1, 2017, and is being submitted via EFS-Web with the filing of the specification.

BACKGROUND OF THE INVENTION

Biosensors are analytical devices that incorporate a biological recognition element in contact with a transduction element and provide rapid conversion of biological events to detectable signals. Among biosensing architectures, devices based on field effect transistors (FETs) have attracted great attention because they are a type of biosensor that can directly translate interactions between target molecules (e.g., biological molecules) and the transistor surface into readable electrical signals without the requirement of a label. In a standard field effect transistor, current flows along a conducting path (the channel) that is connected to two electrodes, (the source and the drain). The channel conductance between the source and the drain is switched on and off by a third (gate) electrode that is capacitively coupled through a thin dielectric layer. Field effect transistors detect target chemicals and measure chemical concentrations for a wide range of commercial applications.

A problem with field effect transistors is their limit of sensitivity. Increasing sensitivity has been explored by incorporating carbon nanotubes, such as single-walled carbon nanotubes (SWNTs), into field effect transistors.

In the field of molecular nanoelectronics, carbon nanotubes, which comprise hollow cylinders of graphite, angstroms in diameter are attractive materials due to their electrical characteristics. Nanotubes have been implemented in electronic devices such as diodes and transistors. Nanotubes are unique for their size, shape, and physical properties. Structurally a carbon nanotube resembles a hexagonal lattice of carbon rolled into a cylinder.

In the context of devices, carbon nanotubes exhibit at least two important characteristics: a nanotube can be either metallic or semiconductor. Metallic nanotubes can carry large current densities with constant resistivity. Semiconducting nanotubes can be electrically switched on and off as field effect transistors.

Despite the advances in the use of carbon nanotubes in field effect transistor architectures, a need exists for improved devices having increased sensitivity for the detection of analytes in physiological samples. The present invention seeks to fulfill this need and provides further related advantages.

SUMMARY OF THE INVENTION

The present invention provides field effect transistor sensors based on a carbon nanotube functionalized with an antibody and methods for using the field effect transistor sensors in a label-free immunoassay.

In one aspect, the invention provides an antibody-functionalized carbon nanotube field effect transistor sensor.

In one embodiment, the sensor comprises:

(a) a first electrode (source electrode);

(b) a second electrode (drain electrode);

(c) a conduction channel operably coupled to the first and second electrodes; and

(d) a paclitaxel antibody or a functional fragment thereof coupled to the conduction channel (gate).

In one embodiment, the conduction channel is a carbon nanotube. Representative carbon nanotubes include a single-walled carbon nanotube.

In another embodiment, the conduction channel is a film comprising a plurality of carbon nanotubes.

In certain embodiments, the sensor is a single-molecule sensor.

In certain embodiments, the paclitaxel antibody or a functional fragment thereof is non-covalently coupled to the conduction channel. In certain embodiments, the paclitaxel antibody or a functional fragment thereof is coupled to the conduction channel by a bifunctional linker. Suitable bifunctional linkers include bifunctional linkers having a first functional group for non-covalently coupling the linker to the conduction channel and a second functional group for covalently coupling the antibody or a functional fragment thereof to the linker. Representative paclitaxel antibodies include 3C6, 8A10, or a functional fragment thereof. In certain embodiments, the paclitaxel antibody or a functional fragment thereof is a single-cysteine variant.

In another aspect, the invention provides a method for detecting paclitaxel in a physiological sample.

In one embodiment, the sensor comprises contacting a sample containing paclitaxel with an antibody-functionalized carbon nanotube field effect transistor sensor of the invention.

In certain embodiments, the method effectively determines paclitaxel concentration from about 20 pM to about 100 nM. In certain embodiments, the paclitaxel concentration is from about 1 to about 10 nM.

In certain embodiments, the physiological sample comprises blood or a blood product (e.g., plasma, serum).

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

FIGURE 1A is a side view of a representative embodiment of a sensor in accordance with an aspect of the disclosure.

FIGURE IB is a top view of the sensor illustrated in FIGURE 1 A.

FIGURE 2 is a top view of another representative embodiment of a sensor in accordance with an aspect of the disclosure.

FIGURES 3A and 3B illustrate two examples of 3C6 antibody (a representative paclitaxel antibody useful in the field effect transistors and methods of the invention) attachment (arrows) to single-walled carbon nanotube (SWNT) field effect transistors (FETs). Each device has a single SWNT labeled with multiple 3C6 antibodies. The left and right sides of each image show the edges of a passivating polymer layer of PMMA. Electrical source and drain connections to each SWNT exist underneath the PMMA, approximately 0.5 μπι beyond the limits of each image.

FIGURES 4A-4D compares electrical signals from a representative single- molecule antibody device, using four paclitaxel concentrations: 0 nM (4A), 0.2 nM (4B), 2 nM (4C), and 200 nM (4D). Current levels of about 35 and about 55 nA correspond to the unbound and bound configurations, respectively. Fluctuations between these two levels correspond to single binding and unbinding events.

FIGURE 5 is a high magnification representation of representative binding- unbinding dynamics by 3C6 antibody at a paclitaxel concentration of 70 nM.

FIGURES 6A and 6B compare mean duration of bound and unbound states (4A) and averaged turnover rate for C36 antibody versus paclitaxel concentration (4B).

FIGURE 7 shows electrical signals for a representative ensemble sensing by SWNT FETs with multiple antibodies. Each shade shows multiple overlaid I(VQ) characteristics measured in a small droplet of phosphate buffered saline (PBS) or PBS with paclitaxel. Data are acquired at V _§D = 50 mV by ramping the electrochemical liquid potential with platinum wire counter and reference electrodes. Inset shows same data on semi -logarithmic axes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides field effect transistor sensors based on a carbon nanotube functionalized with an antibody and methods for using the field effect transistor sensors in a label-free immunoassay.

Antibody-Functionalized Carbon Nanotube-Based Field Effect Transistor

In one aspect, the invention provides a paclitaxel antibody-functionalized carbon nanotube (CNT)-based field effect transistor (FET) that is effective for the detection and quantitation of paclitaxel in a physiological sample. The device takes advantage of the discovery that the binding of paclitaxel present in physiological samples to an antibody that binds paclitaxel can be effectively measured at pharmacologically relevant concentrations by a paclitaxel antibody-functionalized carbon nanotube-based field effect transistor.

In the practice of the invention, carbon nanotube-based devices are modified to incorporate a paclitaxel antibody to provide carbon nanotube-based field effect transistor sensors for assaying paclitaxel. Thus, in another aspect, the invention provides label-free immunoassay methods for paclitaxel detection using a CNT-based FET sensor.

Representative carbon nanotube-based field effect transistors useful for modification in accordance with the invention include those described in U.S. Patent Nos. 6,891,227, 7,897,960, 8, 138,491, and 9, 164,053, each expressly incorporated herein by reference in its entirety. Representative paclitaxel antibody-functionalized carbon nanotube (CNT)-based field effect transistors of the invention that are useful in the methods of the invention are illustrate schematically in FIGURES 1 A, IB, and 2.

Turning to FIGURES 1A and IB, an exemplary antibody-functionalized carbon nanotube field effect transistor sensor is illustrated. As shown, the antibody- functionalized carbon nanotube field effect transistor sensor 20 includes a first electrode 22, a second electrode 24, and a conduction channel 26 connected to and in conductive communication with the first electrode 22 and the second electrode 24. The sensor 20 further includes a paclitaxel antibody or functional fragment thereof 28 coupled to the conduction channel 26. As shown, the paclitaxel antibody or functional fragment thereof 28 is coupled to the conduction channel 26, here a single single-walled carbon nanotube, through a linker 36. As described elsewhere herein, the linker 36 can be a covalent linker or a non-covalent linker.

In an embodiment, the first electrode 22 and the second electrode 24 are coated with and covered by an insulator 30. Such an insulator electrically insulates the first electrode 22 and second electrode 24 from an electrolyte solution, such as a sample, in contact with the conduction channel 26, thereby preventing an electrical short in the antibody-functionalized carbon nanotube field effect transistor sensor 20. In an embodiment, the insulator 30 is an insulating polymer, such as poly(methylmethacrylate). In an embodiment, the insulator 30 is a metal oxide, such as aluminum oxide.

As shown, the insulator 30 covers the first electrode 22 and the second electrode 24, leaving exposed at least a portion of the conduction channel 26. In an embodiment, the exposed portion of the conduction channel is between about 1 nm and about 50 nm wide. The exposed portion of the conduction channel 26 includes the portion of the conduction channel 26 coupled to the paclitaxel antibody or functional fragments thereof 28. Because the paclitaxel antibody or functional fragment thereof 28 is coupled to the exposed portion of the conduction channel 26, the paclitaxel antibody 28 is free to bind with paclitaxel in a sample contacting the antibody-functionalized carbon nanotube field effect transistor sensor 20.

As described elsewhere herein, the sensor 20 is configured to generate a signal indicative of paclitaxel binding to the sensor. In an embodiment, the sensor 20 is configured to generate a signal indicative of a single paclitaxel molecule binding to the sensor 20. As described elsewhere herein, in operation a potential is applied to an electrode chosen from the first electrode 22 and the second electrode 24. In an embodiment, the voltage is between about 0 mV and about 100 mV. This drives DC current between about 10 nA and about 100 nA. When a paclitaxel molecule couples to the paclitaxel antibody or functional fragment thereof 28 a discrete jump in current is generated, which can be measured and correlated to a paclitaxel molecule binding event.

In an embodiment, the first electrode 22, the second electrode 24, and the conduction channel 26 are carried by a substrate. In an embodiment, the substrate includes a silicon oxide layer 32 layered on top of a silicon layer 34.

In an embodiment, the carbon nanotube-based sensor field effect transistor sensor 20 includes a conduction channel including a plurality of conduction elements coupled to paclitaxel antibodies or functional fragments thereof. Such a configuration allows many paclitaxel binding antibodies and functional fragments thereof to be exposed to a sample. In certain embodiments, the sensor including a plurality of conduction elements coupled to paclitaxel antibodies or functional fragments thereof is configured to produce quasi-ensemble signals indicative of multiple paclitaxel binding events.

Turning to FIGURE 2 a carbon nanotube-based sensor field effect transistor sensor 20 is illustrated, which includes a first electrode 22 and a second electrode 24 and a conduction channel comprising a plurality of single-walled carbon nanotubes 26A, 26B, and 26C in conductive communication with the first electrode 22 and the second electrode 24. As shown, each of the of the single-walled carbon nanotubes 26A, 26B, and 26C are coupled to a paclitaxel antibody or functional fragment thereof 28A, 28B, and 28C. In an embodiment, not all of the single-walled carbon nanotubes are coupled to a paclitaxel antibody or functional fragment thereof (not shown). In an embodiment, one or more of the single walled carbon nanotubes are coupled to two or more paclitaxel antibodies or functional fragments thereof (not shown). As shown, the paclitaxel antibodies or functional fragments thereof 28A, 28B, 28C are coupled to the single- walled carbon nanotubes 26 A, 26B, and 26C through linkers 36 A, 36B, and 36C, respectively.

In an embodiment, the plurality of conduction channel including a plurality of conduction elements forms a dilute film carried by a substrate. As shown in FIGURE 2, the substrate can include a silicon oxide layer 32 layered on top of a silicon layer 34 carrying the first electrode 22, the second electrode 24, and dilute film of conduction elements.

As above, in an embodiment the first electrode 22 and second electrode 24 are covered by an insulator, leaving exposed at least a portion of the plurality of conduction elements 26A, 26B, and 26C, thereby facilitating label-less paclitaxel immunoassay.

The following is a description of representative CNT -based FET sensors useful for modification in accordance with the invention.

In one embodiment, the carbon nanotube-based field effect transistor comprises a carbon nanotube deposited on a substrate, a source and a drain formed at a first end and a second end of the carbon nanotube, respectively, and a gate formed substantially over a portion of the carbon nanotube, separated from the carbon nanotube by a dielectric film. The substrate can include a thermal oxide deposited over a silicon substrate. The gate can be further separated from the carbon nanotube by an oxide layer. A portion of the gate is separated from the source and the drain by a nitride spacer. The device further comprises a passivation dielectric layer over the device.

In another embodiment, the carbon nanotube-based filed effect transistor comprises a vertical carbon nanotube wrapped in a dielectric material, a source and a drain formed on a first side and a second side of the carbon nanotube, respectively, a bilayer nitride complex through which a band strap of each of the source and the drain is formed connecting the carbon nanotube wrapped in the dielectric material to the source and the drain, and a gate formed substantially over a portion of the carbon nanotube. The device can include a metal catalyst at a base of the carbon nanotube.

In a further embodiment, the carbon nanotube-based filed effect transistor is a single molecule sensing device that includes a first electrode, a second electrode and a carbon nanotubes, such as a single-walled carbon nanotube (SWNT), connected to the first and second electrodes.

In the practice of the invention, the representative CNT -based FET sensors described above can be modified to include a paclitaxel antibody (i.e., an antibody that binds paclitaxel) or functional fragment thereof.

Suitable paclitaxel -binding antibodies and functional fragments and derivatives thereof include those known in the art. In certain embodiments, the paclitaxel antibody is a monoclonal antibody. Representative paclitaxel antibodies useful in the invention are commercially available as 3C6 and 8A10. Other representative paclitaxel antibodies and functional fragment thereof include affinity reagents, such as described in US 2017/0102401, expressly incorporated herein by reference in its entirety.

As used herein, the term "antibody" encompasses whole antibodies and functional antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, camelid, and primate, including human) or synthetically or recombinantly produced, that specifically binds to a target of interest (i.e., paclitaxel) or epitopes thereof. Exemplary antibody types include polyclonal, monoclonal, and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact molecule or fragment thereof, such as an antigen binding fragment. As described herein, monoclonal antibodies are advantageous because they provide for increased specificity in binding of paclitaxel. However, "clonal" compositions comprising only one antibody fragment or derivative are also possible.

As used herein, the term "antibody fragments" can refer to antigen-binding (i.e., paclitaxel-binding) fragments. The term "antigen" or "paclitaxel binding fragments" refers to the antigen binding or variable region from or related to a full-length antibody. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, nanobodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the Vfj and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VJJ and VL domains, which enables the scFv to form the desired structure for antigen binding.

The term "chimeric antibody" refers to a recombinant protein that contains the variable domains and/or complementarity-determining regions derived from one species' (e.g., rodent) antibody, while the remainder of the antibody molecule is derived from another species' (e.g., a human) antibody. Chimeric antibodies are typically utilized in therapeutic applications, but can also be directed to detection technologies as disclosed herein. As used herein, a "humanized antibody" is a chimeric antibody that comprises a minimal sequence that conforms to specific complementarity-determining regions derived from non-human immunoglobulin that is transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining regions are of non-human origin.

As used herein, the term "derivative" indicates that the antibody or antibody fragment has been produced from a reference antibody. For example, sometimes it is desirable to modify or enhance binding characteristics of a reference antibody (or fragment thereof) by mutation. The resulting antibody products with altered properties are then referred to as a "derivative" of the reference antibody. For example, an antibody derivative can be an antibody that contains mutations resulting from affinity maturation processes that were applied to the reference antibody (or the nucleic acids encoding the reference antibody). Such mutations can result in antibodies with altered (e.g., improved) binding affinity, selectivity, and the like.

Accordingly, the devices and methods of the invention can include affinity reagents, such as antibody-based compositions including antibody variants, antibody fragments, and antibody derivatives that bind to paclitaxel. In certain embodiments, the paclitaxel antibody is a monoclonal antibody or a paclitaxel -binding fragment or derivative thereof. In one embodiment, the affinity reagent comprises six complementary determining regions, namely three on the light chain framework (i.e., also referred to as CDRL1, CDRL2, and CDRL3) and three on the heavy chain framework (i.e., also referred to as CDRH1, CDRH2, and CDRH3). Non-limiting, representative paclitaxel antibodies useful in the invention are commercially available, such as 3C6 and 8A10. The 3C6 and 8A10, as well as fragments and derivatives thereof (including mutant variants with modified binding characteristics) are described in more detail in US 2017/0102401, incorporated by reference in its entirety.

As described in more detail in US 2017/0102401, the representative 8A10 and 3C6 monoclonal antibodies were subjected to various modifications, including mutations subjected to the encoding DNA, to alter binding properties. The monoclonal antibodies, 8A10 and 3C6, and their derivatives were demonstrated to bind to paclitaxel and to be useful for the detection of paclitaxel in a biological sample. Libraries were constructed that incorporated single amino acid sequence variations for each position in each CDR of the reference 8A10 and 3C6 anti-paclitaxel antibodies. The libraries were screened for variant Fab domains exhibiting binding to paclitaxel antigen. Specific mutations of "positive" variants were then combined in a combinatorial library and screened again to confirm binding. These results indicate that affinity reagents that are useful, for example, as compositions for the binding, isolation, and/or detection of paclitaxel, can be generated from known paclitaxel-binding antibodies, such as 8A10 and 3C6.

In accordance with the foregoing, in one aspect the present disclosure incorporates antibody, antibody fragments, and/or antibody derivative affinity reagents that bind to paclitaxel to facilitate detection of paclitaxel using the disclosed methods, devices, and system. In any of the affinity reagent embodiments encompassed by this disclosure, the affinity reagent binds to paclitaxel, as can be determined by any technique known in the art.

A non-limiting description of exemplary 8A10 and 3C6 antibodies, fragments, and derivatives that are useful for the present disclosure is provided below for illustration purposes only.

8A10 and 8A10-derived affinity reagents

In one embodiment, the affinity reagent comprises one, two, three, four, five, or all six of the complementary determining regions contained in the 8A10 mAb. Specifically, the affinity reagent can comprise a CDRL1 with the amino acid sequence set forth in SEQ ID NO: 11, a CDRL2 with the amino acid sequence set forth in SEQ ID NO:31, a CDRL3 with the amino acid sequence set forth in SEQ ID NO:45, a CDRH1 with the amino acid sequence set forth in SEQ ID NO: 58, a CDRH2 with the amino acid sequence set forth in SEQ ID NO:68, and/or a CDRH3 with the amino acid sequence set forth in SEQ ID NO: 99, and/or any combination thereof. In some embodiments, the affinity reagent comprises the amino acid sequence of the 8A10 antibody variable light chain (VLC) and/or the 8A10 variable heavy chain (VHC) domains. The 8A10 amino acid sequence of the 8A10 VLC domain can comprise the sequence set forth in SEQ ID NO:8 and can be encoded by the nucleic acid set forth in SEQ ID NO:7. The amino acid sequence of the 8A10 antibody VHC can be the sequence set forth in SEQ ID NO: 10 and can be encoded by the nucleic acid set forth in SEQ ID NO:9. The sequence of the entire 8A10 mAb is known and discernable by persons of ordinary skill in the art.

In a further embodiment, the affinity reagent comprises at least one amino acid difference relative to the amino acid sequence of 8A10 mAb, as described in more detail below. In one embodiment, the affinity reagent comprises at least one amino acid difference in the framework (i.e., non-CDR) sequence of the variable region of the 8A10 heavy chain or light chain. In another embodiment, the affinity reagent comprises one, two, three, four, five or all six of the CDRs corresponding to the CDRs of the 8A10 mAb, but also comprises at least one mutation, e.g., an amino acid difference, in any one or more of the six CDRs relative to 8A10 mAb, in any combination. Specifically, the affinity reagent can comprise at least one amino acid difference in at least one of the CDRs relative to: a CDRLl with the amino acid sequence set forth in SEQ ID NO: 11, a CDRL2 with the amino acid sequence set forth in SEQ ID NO:31, a CDRL3 with the amino acid sequence set forth in SEQ ID NO:45, a CDRHl with the amino acid sequence set forth in SEQ ID NO:58, a CDRH2 with the amino acid sequence set forth in SEQ ID NO:68, and/or a CDRH3 with the amino acid sequence set forth in SEQ ID NO:99, and/or any combination thereof.

In one embodiment, the affinity reagent specifically comprises:

a light chain complementary determining region CDR1 with the amino acid sequence KPXQXVXSXVX, as set forth in SEQ ID NO: 1,

wherein X at position 3 is S or V,

wherein X at position 5 is N, T, D, M, R, or K,

wherein X at position 7 is G or F,

wherein X at position 9 is A, P, or R,

wherein X at position 11 is T, N, or A;

a light chain complementary determining region CDR2 with the amino acid sequence XXXXRYX, as set forth in SEQ ID NO:2,

wherein X at position 1 is S or Y

wherein X at position 2 is A, H, or T,

wherein X at position 3 is S or T,

wherein X at position 4 is N or R,

wherein X at position 7 is T, M, or R;

a light chain complementary determining region CDR3 with the amino acid sequence QQYXSXPYX, as set forth in SEQ ID NO:3,

wherein X at position 4 is S or P,

wherein X at position 6 is Y, K, R, or V,

wherein X at position 9 is T or R;

a heavy chain complementary determining region CDR1 with the amino acid sequence GXXFXDXXXX, as set forth in SEQ ID NO:4, wherein X at position 2 is Y or S,

wherein X at position 3 is T or R,

wherein X at position 5 is T, S, or H,

wherein X at position 7 is S or Y,

wherein X at position 8 is T or R,

wherein X at position 9 is M or T,

wherein X at position 10 is N or K;

a heavy chain complementary determining region CDR2 with the amino acid sequence XIXPXXXXXXXNQXFXX, as set forth in SEQ ID NO:5,

wherein X at position 1 is E or K

wherein X at position 3 is D, F, W, or A

wherein X at position 5 is N, T, M, S, K, W, or R

wherein X at position 6 is N, S, D, or R

wherein X at position 7 is G or L

wherein X at position 8 is G, W, or R

wherein X at position 9 is T or A

wherein X at position 10 is N, R, or A

wherein X at position 11 is Y or T

wherein X at position 14 is K or N

wherein X at position 16 is K or S

wherein X at position 17 is G or L; and/or

a heavy chain complementary determining region CDR3 with the amino acid sequence ARXXWG, as set forth in SEQ ID NO: 6,

wherein X at position 3 is G, R, or P

wherein X at position 4 is V, P or S;

wherein the monoclonal antibody, antibody fragment, or antibody derivative binds to paclitaxel.

In some embodiments, the light chain complementary determining region CDR1 has the amino acid sequence KPXQXVXSXVX, as set forth in SEQ ID NO: l, wherein X at position 3 is S or V, wherein X at position 5 is N, R, or K, wherein X at position 7 is G, wherein X at position 9 is A, P, or R, and wherein X at position 11 is T, N, or A.

In some embodiments, the light chain complementary determining region CDR2 has the amino acid sequence XXXXRYX, as set forth in SEQ ID NO:2, wherein X at position 1 is S, wherein X at position 2 is A or T, wherein X at position 3 is S or T, wherein X at position 4 is N or R, and wherein X at position 7 is T or R.

In some embodiments, the light chain complementary determining region CDR3 has the amino acid sequence QQYXSXPYX, as set forth in SEQ ID NO:3, wherein X at position 4 is S, wherein X at position 6 is Y, K, R, or V, and wherein X at position 9 is T.

In some embodiments, the heavy chain complementary determining region CDR1 has the amino acid sequence GXXFXDXXXX, as set forth in SEQ ID NO: 4, wherein X at position 2 is Y, wherein X at position 3 is T or R, wherein X at position 5 is T or H, wherein X at position 7 is S, wherein X at position 8 is T or R, wherein X at position 9 is M, and wherein X at position 10 is N.

In some embodiments, the heavy chain complementary determining region CDR2 has the amino acid sequence XIXPXXXXXXXNQXFXX, as set forth in SEQ ID NO:5, wherein X at position 1 is E, wherein X at position 3 is D, F, W, or A, wherein X at position 5 is N, S, K, W, or R, wherein X at position 6 is N or R, wherein X at position 7 is G, wherein X at position 8 is G, W, or R, wherein X at position 9 is T, wherein X at position 10 is N, wherein X at position 11 is Y, wherein X at position 14 is K, wherein X at position 16 is K, and wherein X at position 17 is G.

In some embodiments, the heavy chain complementary determining region CDR3 has the amino acid sequence ARXXWG, as set forth in SEQ ID NO: 6, wherein X at position 3 is G, and wherein X at position 4 is V or S.

In some embodiments, the affinity reagent comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations within the 6 CDR domains relative to the CDR domains of the 8A10 mAb, as described herein.

As indicated above, in some embodiments, the affinity reagent can be the 8A10 monoclonal antibody, an 8A10 antibody fragment, or antibody derivative that is distinct from the 8A10 antibody. In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that is distinct from an 8A10 antibody fragment (e.g., contains one or more mutations). In this embodiment, the affinity reagent has an amino acid sequence that is distinct from any single contiguous subsequence of the 8 A10 mAb.

In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that comprises at least one amino acid difference in a CDR amino acid sequence from a corresponding CDR amino acid sequence of the 8A10 antibody as set forth in SEQ ID NOS: l 1, 31, 45, 58, 68, and 99.

In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that comprises an amino acid sequence in a CDR selected from the following SEQ ID NOS: 12-30, 32-44, 46-57, 59-67, 69-98, and 100-103.

In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that comprises one or more of the following amino acid substitutions N5R, N5K, A9R, TUN, and Ti l A with respect to SEQ ID NO: 11, A2T, S3T, N4R, and T7R with respect to SEQ ID NO: 31, Y6R, Y6K, and Y6V with respect to SEQ ID NO:45, T3R, T5H, and T8R with respect to SEQ ID NO:58, D3F, D3W, D3A, N6R, G8R and G8W with respect to SEQ ID NO: 68. In a further embodiment, the monoclonal antibody, antibody fragment, or antibody derivative otherwise comprises the same CDR sequences of the 8A10 antibody as set forth in SEQ ID NOS: 11, 31, 45, 58, 68, and 99.

Further embodiments of individual CDRs will now be described, which reflect desirable derivatives of the 8A10 CDRs that can be integrated into the disclosed methods, devices, and systems for detecting paclitaxel. It will be apparent that any particular embodiment of one specific CDR can be combined within the affinity reagent with any other particular embodiment of another specific CDR described herein, unless stated otherwise.

In some embodiments, the affinity reagent comprises a light chain CDR1 with an amino acid sequence selected from SEQ ID NOS: 104-110.

In some embodiments, the affinity reagent comprises a light chain CDR2 with an amino acid sequence selected from SEQ ID NOS: 111-117.

In some embodiments, the affinity reagent comprises a heavy chain CDR1 with an amino acid sequence selected from SEQ ID NOS: 118-124.

In some embodiments, the affinity reagent comprises a heavy chain CDR2 with an amino acid sequence selected from SEQ ID NOS: 125-132.

In some embodiments, the affinity reagent comprises a VLC and/or VHC variant of the 8A10 mAb. Representative amino acid sequences of 8A10 VLC region variants are set forth in SEQ ID NOS: 190-197. Representative amino acid sequences of 8A10 VHC region variants are set forth in SEQ ID NOS: 198-205. As generally indicated above, embodiments of the disclosed anti-paclitaxel affinity reagents can be derived from the 8A10 antibody. The affinity reagents can comprise one or more mutations, e.g., amino acid substitution deletion, addition, and/or substitution, relative to a CDR of the 8A10 mAb (as set forth in SEQ ID NOS: 1 1, 31, 45, 58, 68, and 99), the framework (non-CDR) regions of the 8A10 variable light or heavy chain sequences, or other domains of the 8A10 mAb. The 8A10 variable light or heavy chain sequences are set forth herein as SEQ ID NOS:8 and 10, respectively. In some embodiments, the affinity reagent has a combined CDR sequence (considering all six CDR sequences) that is at least about 60, 65, 70, 75, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the combined CDR sequence of the 8A10 antibody. In other embodiments, the affinity reagent has a variable light or heavy chain with an amino acid sequence that is at least about 60, 65, 70, 75, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% to the sequence of the variable light or heavy chain sequences of the 8A10 antibody. It will be apparent to persons of ordinary skill that the anti-paclitaxel affinity reagents can still be distinct from the 8A10 reference monoclonal antibody, and when one domain has perfect identify with the corresponding domain of the 8A10 reference sequence, a difference is incorporated in a distinct domain.

As used herein, the term "percent identity" or "percent identical," when used in connection with a polypeptide, is defined as the percentage of amino acid residues in a polypeptide sequence that are identical with the amino acid sequence of a specified reference polypeptide (such as the amino acid sequence of SEQ ID NO: 8), after aligning the sequences to achieve the maximum percent identity. Amino acid sequence identity can be determined according to any algorithm or technique known in the art.

As used herein, an "amino acid" refers to any of the 20 naturally occurring amino acids found in proteins, D-stereoisomers of the naturally occurring amino acids (e.g., D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these types of amino acids is not mutually exclusive. a-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain. " The side chains of naturally occurring amino acids are well-known in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in histidine).

The following abbreviations are used for the 20 naturally occurring amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Val; V).

Often, desirable amino acid substitutions relative to any portion of the reference 8A10 sequence (or 3C6 sequence, as described below) include a substitution with a similar amino acid as defined by a similar characteristic exhibited by the reference and substituted residues. Thus, in some embodiments, the variant affinity reagent comprises a conservative amino acid substitution as compared to the reference 8A10 sequence (or 3C6 sequence). Any substitution mutation is conservative in that it minimally disrupts the biochemical properties of the protein. Non-limiting examples of mutations that are introduced to substitute conservative amino acid residues include: positively-charged residues (e.g., H, K, and R) substituted with positively-charged residues; negatively-charged residues (e.g., D and E) substituted with negatively-charged residues; neutral polar residues (e.g., C, G, N, Q, S, T, and Y) substituted with neutral polar residues; and neutral non-polar residues (e.g., A, F, I, L, M, P, V, and W) substituted with neutral non-polar residues. Nonconservative substitutions can be made as well (e.g., proline for glycine).

Amino acids, and, more specifically, their side chains, can be characterized by their chemical characteristic(s). For example, amino acid side chains can be positively charged, negatively charged, or neutral. The pH of a solution affects the charged nature of certain side chains, as is known by those of skill in the art. Non-limiting examples of side chains that can be positively charged include histidine, arginine, and lysine. Non-limiting examples of side chains that can be negatively charged include aspartic acid and glutamic acid. Non-limiting examples of side chains that can be characterized as neutral include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, methionine, proline, and tryptophan. Sterics of side chains can also be used to characterize an amino acid. Tables of atom diameters can assist one in determining whether one side chain is larger than another. Computer models may also help with this determination.

Amino acids can also be characterized by the polarity of their side chains. Polar side chains, which are typically more hydrophilic than non-polar side chains, include, for example, those of serine, threonine, tyrosine, cysteine, asparagine, and glutamine. Non-polar side chains, which are typically more hydrophobic than polar side chains, include, for example, those of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. One can determine polarity of a side chain using conventional techniques known in the art involving atom electronegativity determinations and three-dimensional structural assessments of side chains. One can also compare hydrophobicities/hydrophilicities of side chains using conventional techniques known in the art, such as comparing the octanol/water partition coefficient of each amino acid.

Alternatively, one may consider the hydropathic index of amino acids. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and/or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and/or arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and/or score and/or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices may be within ±2; within ±1, or within ±0.5.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. As detailed in U.S. Patent 4,554, 101, incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, it is contemplated that the substitution of amino acids whose hydrophilicity values may be within ±2, within ±1, or those within ±0.5.

3C6 and 3C6-derived affinity reagents

In one embodiment, the affinity reagent comprises one, two, three, four, five, or all six of the complementary determining regions contained in the 3C6 mAb. Specifically, the affinity reagent can comprise a CDRLl with the amino acid sequence set forth in SEQ ID NO: 143, a CDRL2 with the amino acid sequence set forth in SEQ ID NO: 155, a CDRL3 with the amino acid sequence set forth in SEQ ID NO: 160, a CDRH1 with the amino acid sequence set forth in SEQ ID NO: 166, a CDRH2 with the amino acid sequence set forth in SEQ ID NO: 172, and/or a CDRH3 with the amino acid sequence set forth in SEQ ID NO: 184, and/or any combination thereof. In some embodiments, the affinity reagent comprises the amino acid sequence of the 3C6 antibody variable light chain (VLC) and/or the 3C6 variable heavy chain (VHC) domains. The 3C6 amino acid sequence of the 3C6 VLC domain can comprise the sequence set forth in SEQ ID NO: 140 and can be encoded by the nucleic acid set forth in SEQ ID NO: 139. The amino acid sequence of the 3C6 antibody VHC can be the sequence set forth in SEQ ID NO: 142 and can be encoded by the nucleic acid set forth in SEQ ID NO: 141. The sequence of the entire 3C6 mAb is known and discernable by persons of ordinary skill in the art.

In a further embodiment, the affinity reagent comprises at least one amino acid difference relative to the amino acid sequence of 3C6 mAb. In one embodiment, the affinity reagent comprises at least one amino acid difference in the framework (i.e., non-CDR) sequence of the variable region of the 3C6 heavy chain or light chain.

In another embodiment, the affinity reagent comprises one, two, three, four, five or all six CDRs corresponding to the CDRs of the 3C6 mAb, but also comprises at least one mutation, e.g., an amino acid difference, in any one or more of the six CDRs relative to 3C6 mAb, in any combination. Specifically, the affinity reagent can comprise at least one amino acid difference in at least one of the CDRs relative to: a CDRLl with the amino acid sequence set forth in SEQ ID NO: 143, a CDRL2 with the amino acid sequence set forth in SEQ ID NO: 155, a CDRL3 with the amino acid sequence set forth in SEQ ID NO: 160, a CDRH1 with the amino acid sequence set forth in SEQ ID NO: 166, a CDRH2 with the amino acid sequence set forth in SEQ ID NO: 172, and/or a CDRH3 with the amino acid sequence set forth in SEQ ID NO: 184, and/or any combination thereof.

In one embodiment, the affinity reagent specifically comprises:

a light chain complementary determining region CDR1 with the amino acid sequence XSXQXLXHXXGNXYXH, as set forth in SEQ ID NO : 133 ,

wherein X at position 1 is R or H

wherein X at position 3 is R, G, or N

wherein X at position 5 is S, M, or G

wherein X at position 7 is V or L

wherein X at position 9 is S or I

wherein X at position 10 is N or V

wherein X at position 13 is T or S

wherein X at position 15 is L or W;

a light chain complementary determining region CDR2 with the amino acid sequence XVSXXXS, as set forth in SEQ ID NO: 134,

wherein X at position 1 is K or N

wherein X at position 4 is N or R

wherein X at position 5 is R or L

wherein X at position 6 is F or R;

a light chain complementary determining region CDR3 with the amino acid sequence SXSTHXXPX, as set forth in SEQ ID NO: 135,

wherein X at position 2 is Q or P

wherein X at position 6 is V or G

wherein X at position 7 is P or S

wherein X at position 9 is T or R;

a heavy chain complementary determining region CDR1 with the amino acid sequence XDSITXGYXX, as set forth in SEQ ID NO: 136,

wherein X at position 1 is G or P

wherein X at position 6 is S or I

wherein X at position 9 is W or F

wherein X at position 10 is N, R, or K;

a heavy chain complementary determining region CDR2 with the amino acid sequence XISYXGXXYXXPXLKX, as set forth in SEQ ID NO: 137, wherein X at position 1 is Y or F

wherein X at position 5 is S, R, or T

wherein X at position 7 is S or D

wherein X at position 8 is T or I

wherein X at position 10 is Y or F

wherein X at position 11 is N or K

wherein X at position 13 is S or F

wherein X at position 16 is S or N; and/or

a heavy chain complementary determining region CDR3 with the amino acid sequence XXXXY, as set forth in SEQ ID NO : 138,

wherein X at position 1 is G, A, or E

wherein X at position 2 is D or W

wherein X at position 3 is G or T

wherein X at position 4 is A, D, G, or Q;

wherein the monoclonal antibody, antibody fragment, or antibody derivative binds to paclitaxel.

As indicated above, in some embodiments, the affinity reagent can be a 3C6 monoclonal antibody, a 3C6 antibody fragment, or antibody derivative that is distinct from the 3C6 antibody. In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that is distinct from a 3C6 antibody fragment (e.g., contains one or more mutations). In this embodiment, the affinity reagent has an amino acid sequence that is distinct from any single contiguous subsequence of the 3C6 mAb.

In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that comprises at least one amino acid difference in a CDR amino acid sequence from a corresponding CDR amino acid sequence of the 3C6 antibody as set forth in SEQ ID NOS: 143, 155, 160, 166, 172, and 184.

In some embodiments, the affinity reagent is a monoclonal antibody, antibody fragment, or antibody derivative that comprises an amino acid sequence in a CDR selected from the following SEQ ID NOS: 144-154, 156-159, 161-165, 167-171, 173-183, 185-189, and 246-248.

In some embodiments, the affinity reagent comprises a VLC and/or VHC variant of the 3C6 mAb. Representative amino acid sequences of 3C6 VLC region variants are set forth in SEQ ID NOS:206-225. Representative amino acid sequences of 3C6 VHC region variants are set forth in SEQ ID NOS:226-245.

As generally indicated above, embodiments of the disclosed affinity reagents can be derived from the 3C6 antibody, and has at least some amino acid sequence difference from the 3C6 antibody. The affinity reagents can comprise one or more mutations, e.g., amino acid substitution deletion, addition, and/or substitution, relative to a CDR of the 3C6 mAb (as set forth in SEQ ID NOS: 143, 155, 160, 166, 172, and 184), the framework (non-CDR) regions of the 3C6 variable light or heavy chain sequences, or other domains of the 3C6 mAb. The 3C6 variable light and heavy chain sequences are set forth herein as SEQ ID NOS: 140 and 142, respectively. In some embodiments, the affinity reagent has a combined CDR sequence (considering all six CDR sequences) that is at least about 60, 65, 70, 75, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the combined CDR sequence of the 3C6 antibody. In other embodiments, the affinity reagent has a variable light or heavy chain with an amino acid sequence that is at least about 60, 65, 70, 75, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% to the sequence of the variable light or heavy chain sequences of the 3C6 antibody.

In one embodiment, the affinity reagent derived from the 3C6 antibody is a monoclonal antibody, antibody fragment, or antibody derivative that comprises one or more of the following amino acid substitutions S5G with respect to SEQ ID NO: 143 or SEQ ID NO: 144 and A4Q and A4G with respect to SEQ ID NO: 184.

INDEX OF SEQUENCES

The following is an index of the sequences set forth in the Sequence Listing submitted herewith. The sequences are referred to by their respective SEQ ID NOS as listed in the Sequence Listing

1. Consensus amino acid sequence for 8A10 CDRLl

2. Consensus amino acid sequence for 8A10 CDRL2

3. Consensus amino acid sequence for 8A10 CDRL3

4. Consensus amino acid sequence for 8A10 CDRHl

5. Consensus amino acid sequence for 8A10 CDRH2

6. Consensus amino acid sequence for 8A10 CDRH3

7. Nucleic acid encoding 8A10 VLC region

8 Amino acid sequence of 8A10 VLC region 9. Nucleic acid encoding 8A10 VHC region

10. Amino acid sequence of 8A10 VHC region

11. Amino acid sequence of 8A10 CDRLl

12. Amino acid sequence of 8A10 CDRLl variant 13. Amino acid sequence of 8A10 CDRLl variant

14. Amino acid sequence of 8A10 CDRLl variant

15. Amino acid sequence of 8A10 CDRLl variant

16. Amino acid sequence of 8A10 CDRLl variant

17. Amino acid sequence of 8A10 CDRLl variant 18. Amino acid sequence of 8A10 CDRLl variant

19. Amino acid sequence of 8A10 CDRLl variant

20. Amino acid sequence of 8A10 CDRLl variant

21. Amino acid sequence of 8A10 CDRLl variant

22. Amino acid sequence of 8A10 CDRLl variant 23. Amino acid sequence of 8A10 CDRLl variant

24. Amino acid sequence of 8A10 CDRLl variant

25. Amino acid sequence of 8A10 CDRLl variant

26. Amino acid sequence of 8A10 CDRLl variant

27. Amino acid sequence of 8A10 CDRLl variant 28. Amino acid sequence of 8A10 CDRLl variant

29. Amino acid sequence of 8A10 CDRLl variant

30. Amino acid sequence of 8A10 CDRLl variant

31. Amino acid sequence of 8A10 CDRL2

32. Amino acid sequence of 8A10 CDRL2 variant 33. Amino acid sequence of 8A10 CDRL2 variant

34. Amino acid sequence of 8A10 CDRL2 variant

35. Amino acid sequence of 8A10 CDRL2 variant

36. Amino acid sequence of 8A10 CDRL2 variant

37. Amino acid sequence of 8A10 CDRL2 variant 38. Amino acid sequence of 8A10 CDRL2 variant

39. Amino acid sequence of 8A10 CDRL2 variant

40. Amino acid sequence of 8A10 CDRL2 variant

41. Amino acid sequence of 8A10 CDRL2 variant 42. Amino acid sequence of 8A10 CDRL2 variant

43. Amino acid sequence of 8A10 CDRL2 variant

44. Amino acid sequence of 8A10 CDRL2 variant

45. Amino acid sequence of 8A10 CDRL3

46. Amino acid sequence of 8A10 CDRL3 variant

47. Amino acid sequence of 8A10 CDRL3 variant

48. Amino acid sequence of 8A10 CDRL3 variant

49. Amino acid sequence of 8A10 CDRL3 variant

50. Amino acid sequence of 8A10 CDRL3 variant

51. Amino acid sequence of 8A10 CDRL3 variant

52. Amino acid sequence of 8A10 CDRL3 variant

53. Amino acid sequence of 8A10 CDRL3 variant

54. Amino acid sequence of 8A10 CDRL3 variant

55. Amino acid sequence of 8A10 CDRL3 variant

56. Amino acid sequence of 8A10 CDRL3 variant

57. Amino acid sequence of 8A10 CDRL3 variant

58. Amino acid sequence of 8A10 CDRH1

59. Amino acid sequence of 8A10 CDRH1 variant

60. Amino acid sequence of 8A10 CDRH1 variant

61. Amino acid sequence of 8A10 CDRH1 variant

62. Amino acid sequence of 8A10 CDRH1 variant

63. Amino acid sequence of 8A10 CDRH1 variant

64. Amino acid sequence of 8A10 CDRH1 variant

65. Amino acid sequence of 8A10 CDRH1 variant

66. Amino acid sequence of 8A10 CDRH1 variant

67. Amino acid sequence of 8A10 CDRH1 variant

68. Amino acid sequence of 8A10 CDRH2

69. Amino acid sequence of 8A10 CDRH2 segment A

70. Amino acid sequence of 8A10 CDRH2 segment A variant

71. Amino acid sequence of 8A10 CDRH2 segment A variant

72. Amino acid sequence of 8A10 CDRH2 segment A variant

73. Amino acid sequence of 8A10 CDRH2 segment A variant

74. Amino acid sequence of 8A10 CDRH2 segment A variant 75. Amino acid sequence of 8A10 CDRH2 segment A variant

76. Amino acid sequence of 8A10 CDRH2 segment A variant

77. Amino acid sequence of 8A10 CDRH2 segment A variant

78. Amino acid sequence of 8A10 CDRH2 segment A variant

79. Amino acid sequence of 8A10 CDRH2 segment A variant

80. Amino acid sequence of 8A10 CDRH2 segment A variant

81. Amino acid sequence of 8A10 CDRH2 segment A variant

82. Amino acid sequence of 8A10 CDRH2 segment A variant

83. Amino acid sequence of 8A10 CDRH2 segment A variant

84. Amino acid sequence of 8A10 CDRH2 segment A variant

85. Amino acid sequence of 8A10 CDRH2 segment A variant

86. Amino acid sequence of 8A10 CDRH2 segment A variant

87. Amino acid sequence of 8A10 CDRH2 segment A variant

88. Amino acid sequence of 8A10 CDRH2 segment A variant

89. Amino acid sequence of 8A10 CDRH2 segment A variant

90. Amino acid sequence of 8A10 CDRH2 segment A variant

91. Amino acid sequence of 8A10 CDRH2 segment B

92. Amino acid sequence of 8A10 CDRH2 segment B variant

93. Amino acid sequence of 8A10 CDRH2 segment B variant

94. Amino acid sequence of 8A10 CDRH2 segment B variant

95. Amino acid sequence of 8A10 CDRH2 segment B variant

96. Amino acid sequence of 8A10 CDRH2 segment B variant

97. Amino acid sequence of 8A10 CDRH2 segment B variant

98. Amino acid sequence of 8A10 CDRH2 segment B variant

99. Amino acid sequence of 8A10 CDRH3

100. Amino acid sequence of 8A10 CDRH3 variant

101. Amino acid sequence of 8A10 CDRH3 variant

102. Amino acid sequence of 8A10 CDRH3 variant

103. Amino acid sequence of 8A10 CDRH3 variant

104. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP2

105. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP3

106. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP4

107. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP6 108. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP7

109. Amino acid sequence of CDRLl of 8A10 combinatorial variant CP8

110. Amino acid sequence of CDRLl 8A10 of combinatorial variant CP9

111. Amino acid sequence of CDRL2 of 8A10 combinatorial variant CP2 112. Amino acid sequence of CDRL2 8A10 of combinatorial variant CP3

113. Amino acid sequence of CDRL2 of 8A10 combinatorial variant CP4

114. Amino acid sequence of CDRL2 of 8A10 combinatorial variant CP6

115. Amino acid sequence of CDRL2 of 8A10 combinatorial variant CP7

116. Amino acid sequence of CDRL2 of 8A10 combinatorial variant CP8 117. Amino acid sequence of CDRL2 of 8 A10 combinatorial variant CP9

118. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP2

119. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP3

120. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP4

121. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP6 122. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP7

123. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP8

124. Amino acid sequence of CDRHl of 8A10 combinatorial variant CP9

125. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP2

126. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP3 127. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP4

128. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP5

129. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP6

130. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP7

131. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP8 132. Amino acid sequence of CDRH2 of 8A10 combinatorial variant CP9

133. Consensus amino acid sequence for 3C6 CDRLl

134. Consensus amino acid sequence for 3C6 CDRL2

135. Consensus amino acid sequence for 3C6 CDRL3

136. Consensus amino acid sequence for 3C6 CDRHl

137. Consensus amino acid sequence for 3 C6 CDRH2

138. Consensus amino acid sequence for 3C6 CDRH3

139. Nucleic acid encoding 3C6 VLC region

140. Amino acid sequence of 3C6 VLC region 141. Nucleic acid encoding 3C6 VHC region

142. Amino acid sequence of 3C6 VHC region

143. Amino acid sequence of 3C6 CDRLl

144. Amino acid sequence of 3C6 CDRLl segment A

145. Amino acid sequence of 3C6 CDRLl segment A variant

146. Amino acid sequence of 3C6 CDRLl segment A variant

147. Amino acid sequence of 3C6 CDRLl segment A variant

148. Amino acid sequence of 3C6 CDRLl segment A variant

149. Amino acid sequence of 3C6 CDRLl segment A variant

150. Amino acid sequence of 3C6 CDRLl segment B

151. Amino acid sequence of 3C6 CDRLl segment B variant

152. Amino acid sequence of 3C6 CDRLl segment B variant

153. Amino acid sequence of 3C6 CDRLl segment B variant

154. Amino acid sequence of 3C6 CDRLl segment B variant

155. Amino acid sequence of 3C6 CDRL2

156. Amino acid sequence of 3C6 CDRL2 variant

157. Amino acid sequence of 3C6 CDRL2 variant

158. Amino acid sequence of 3C6 CDRL2 variant

159. Amino acid sequence of 3C6 CDRL2 variant

160. Amino acid sequence of 3C6 CDRL3

161. Amino acid sequence of 3C6 CDRL3 variant

162. Amino acid sequence of 3C6 CDRL3 variant

163. Amino acid sequence of 3C6 CDRL3 variant

164. Amino acid sequence of 3C6 CDRL3 variant

165. Amino acid sequence of 3C6 CDRL3 variant

166. Amino acid sequence of 3C6 CDRH1

167. Amino acid sequence of 3C6 CDRH1 variant

168. Amino acid sequence of 3C6 CDRH1 variant

169. Amino acid sequence of 3C6 CDRH1 variant

170. Amino acid sequence of 3C6 CDRH1 variant

171. Amino acid sequence of 3C6 CDRH1 variant

172. Amino acid sequence of 3C6 CDRH2

173. Amino acid sequence of 3C6 CDRH2 segment A 174. Amino acid sequence of 3C6 CDRH2 segment A variant

175. Amino acid sequence of 3C6 CDRH2 segment A variant

176. Amino acid sequence of 3C6 CDRH2 segment A variant

177. Amino acid sequence of 3C6 CDRH2 segment A variant

178. Amino acid sequence of 3C6 CDRH2 segment A variant

179. Amino acid sequence of 3C6 CDRH2 segment B

180. Amino acid sequence of 3C6 CDRH2 segment B variant

181. Amino acid sequence of 3C6 CDRH2 segment B variant

182. Amino acid sequence of 3C6 CDRH2 segment B variant

183. Amino acid sequence of 3C6 CDRH2 segment B variant

184. Amino acid sequence of 3C6 CDRH3

185. Amino acid sequence of 3C6 CDRH3 variant

186. Amino acid sequence of 3C6 CDRH3 variant

187. Amino acid sequence of 3C6 CDRH3 variant

188. Amino acid sequence of 3C6 CDRH3 variant

189. Amino acid sequence of 3C6 CDRH3 variant

190. Amino acid sequence of 8A10 VLC region variant

191. Amino acid sequence of 8A10 VLC region variant

192. Amino acid sequence of 8A10 VLC region variant

193. Amino acid sequence of 8A10 VLC region variant

194. Amino acid sequence of 8A10 VLC region variant

195. Amino acid sequence of 8A10 VLC region variant

196. Amino acid sequence of 8A10 VLC region variant

197. Amino acid sequence of 8A10 VLC region variant

198. Amino acid sequence of 8A10 VHC region variant

199. Amino acid sequence of 8A10 VHC region variant

200. Amino acid sequence of 8A10 VHC region variant

201. Amino acid sequence of 8A10 VHC region variant

202. Amino acid sequence of 8A10 VHC region variant

203. Amino acid sequence of 8A10 VHC region variant

204. Amino acid sequence of 8A10 VHC region variant

205. Amino acid sequence of 8A10 VHC region variant

206. Amino acid sequence of 3C6 VLC region variant 207. Amino acid sequence of 3C6 VLC region variant

208. Amino acid sequence of 3C6 VLC region variant

209. Amino acid sequence of 3C6 VLC region variant

210. Amino acid sequence of 3C6 VLC region variant

211. Amino acid sequence of 3C6 VLC region variant

212. Amino acid sequence of 3C6 VLC region variant

213. Amino acid sequence of 3C6 VLC region variant

214. Amino acid sequence of 3C6 VLC region variant

215. Amino acid sequence of 3C6 VLC region variant

216. Amino acid sequence of 3C6 VLC region variant

217. Amino acid sequence of 3C6 VLC region variant

218. Amino acid sequence of 3C6 VLC region variant

219. Amino acid sequence of 3C6 VLC region variant

220. Amino acid sequence of 3C6 VLC region variant

221. Amino acid sequence of 3C6 VLC region variant

222. Amino acid sequence of 3C6 VLC region variant

223. Amino acid sequence of 3C6 VLC region variant

224. Amino acid sequence of 3C6 VLC region variant

225. Amino acid sequence of 3C6 VLC region variant

226. Amino acid sequence of 3C6 VHC region variant

227. Amino acid sequence of 3C6 VHC region variant

228. Amino acid sequence of 3C6 VHC region variant

229. Amino acid sequence of 3C6 VHC region variant

230. Amino acid sequence of 3C6 VHC region variant

231. Amino acid sequence of 3C6 VHC region variant

232. Amino acid sequence of 3C6 VHC region variant

233. Amino acid sequence of 3C6 VHC region variant

234. Amino acid sequence of 3C6 VHC region variant

235. Amino acid sequence of 3C6 VHC region variant

236. Amino acid sequence of 3C6 VHC region variant

237. Amino acid sequence of 3C6 VHC region variant

238. Amino acid sequence of 3C6 VHC region variant

239. Amino acid sequence of 3C6 VHC region variant 240. Amino acid sequence of 3C6 VHC region variant

241. Amino acid sequence of 3C6 VHC region variant

242. Amino acid sequence of 3C6 VHC region variant

243. Amino acid sequence of 3C6 VHC region variant

244. Amino acid sequence of 3C6 VHC region variant

245. Amino acid sequence of 3C6 VHC region variant

246. Amino acid sequence of 3C6 CDRL1 segment A variant

247. Amino acid sequence of 3C6 CDRH3 variant

248. Amino acid sequence of 3C6 CDRH3 variant

To optimize functionalization of the CNT -based FET, in certain embodiments, the paclitaxel antibody is a single-cysteine variant (i.e., the paclitaxel antibody includes a single cysteine and coupling of the antibody to the carbon nanotube, optionally through a bifunctional linker, occurs through the thiol group of the single cysteine).

In the practice of the invention, the antibody is coupled to carbon nanotube component (i.e., conductor) of the FET. The antibody can be coupled to the CNT -based FET sensor by a variety of methods, including those known in the art. The preferred method of coupling the antibody to the carbon nanotube component preserves the binding affinity of the antibody to paclitaxel and enables the measurement of the binding of paclitaxel to the coupled antibody by the sensor. In certain embodiments, the antibody is covalently coupled to the carbon nanotube component of the FET, either directly or through the use of a linker. In other embodiments, the antibody is non-covalently coupled to the carbon nanotube component of the FET, either directly or through the use of a linker. In non-covalent embodiments that use a linker, the linker includes at least one functional group that effectively couples the antibody to the carbon nanotube component by non-covalent association (e.g., electrostatic, hydrophobic/hydrophobic association) and at least a second functional group that effectively couples the antibody to the linker. The coupling of the antibody to the linker can be achieved either covalently or non-covalently. In certain embodiments, the antibody is coupled to the carbon nanotube component with a linker having a first functional group that couples the linker (and ultimately antibody) to the carbon nanotube by non-covalent association and a second functional group that covalently couples the linker to the antibody. Suitable functional groups for the linker are known in the art. For embodiments that include a linker having a first functional group that provides for non-covalent coupling to the carbon nanotube and a second functional group that provides covalent coupling to the antibody, representative first functional groups include hydrocarbon groups, such as a pyrene, a benzene, a cyclohexane, or a 2,3-dichloro-5,6-dicyano-l,4- benzoquinone group, and representative second functional groups include groups that react with the amino acid residues of the antibody (e.g., amino and thiol reactive groups), such as a maleimide or other activated ester group.

In one embodiment, the CNT -based FET sensor of the invention is a single molecule (e.g., paclitaxel) sensing device that includes a first electrode, and a second electrode. A carbon nanotube (e.g., single-walled carbon nanotube (SWNT) is connected to the first electrode and the second electrode, respectively. The device includes at least one linker molecule having first and second functional groups, the at least one linker molecule having the first functional group non-covalently coupled to a sidewall of the carbon nanotube. A single sensitizing molecule (e.g., paclitaxel antibody) having at least one functional group, said at least one functional group of the single sensitizing molecule being capable of reaction with the second functional group of the at least one linker molecule.

In such an embodiment, the single molecule sensing device is a field effect transistor with the coupled antibody serving as a "gate" to an electrical circuit. In this embodiment, a single sensitizing molecule serves as single molecule gate for the device. The transistor embodiment may include a two or three terminal transistor. The conduction channel of the device may also be formed from metals, metal oxides, semiconductors, or nanometer-scale conductors such as nanowires, graphene, or carbon nanotubes. In certain embodiments, the conduction channel is a single carbon nanotube (e.g., SWNT).

In the practice of the invention, the exposed portion of the carbon nanotube sidewall is non-covalently functionalized with the one or more functional groups of linker molecule(s). Processes for non-covalently functionalizing a carbon nanotube sidewall are well known in the art, and the present invention includes any suitable process for coating carbon nanotubes with a dilute coating. Achieving a dilute coating includes (a) preparing a solution containing the linker molecule, (b) soaking the prepared devices containing the exposed carbon nanotube in the prepared solution containing the linker molecules, (c) rinsing the devices to remove excess linker molecules, (d) rinsing the device to remove excess reagent, and (e) rinsing the device under flowing de-ionized water.

Generally, the dimensions of the exposed portion of the carbon nanotube is chosen such that statistically, the devices that are manufactured have only a small number (e.g., 1 to 1,000) of linker molecules associated with the carbon nanotube. The parameters that affect the number of linker molecules (and thus potential sites for the attachment of sensitizing molecules) that are associated with the carbon nanotube include the length of the carbon nanotube, the properties of the linker molecule, the incubation time, and the concentration of the linker molecules. Generally, a shorter incubation time or lower concentration of linker molecules translates into fewer association sites on the carbon nanotube.

After attachment of the linker molecules, the device is next exposed to a solution of sensitizing molecules (e.g., antibody). The properties of the sensitizing molecule, the particular attachment chemistry (e.g., maleimide-to-thiol), the properties of the solution (e.g., pH, temperature, salt and surfactant concentrations), the incubation time, and the concentration of the sensitizing molecules affect the yield with which sensitizing molecules will successfully bind to linkers. A representative procedure for coupling an antibody to a CNT-based FET device is described in Example 1.

Method for Immunoassay Using the Antibody -Functionalized Carbon Nanotube- Based Field Effect Transistor

In another aspect, the invention provides a paclitaxel antibody-funtionalized carbon nanotube field effect transistor sensor that is effective for the detection and quantitation of paclitaxel in a physiological sample. In this aspect, the invention provides a method for detecting paclitaxel in a physiological sample.

In one embodiment, the method comprises contacting a sample containing paclitaxel with an antibody-functionalized carbon nanotube field effect transistor sensor described herein.

The method is effective for determining paclitaxel in the sample at concentrations from about 20 pM to about 100 nM. In certain embodiments, the paclitaxel concentration is from about 1 to about 10 nM.

Suitable physiological samples include blood and blood products, such as plasma and serum. Representative Antibody -Functionalized Carbon Nanotube-Based Field Effect Transistor Sensors and Related Immunoassays

Nanotube-based paclitaxel sensors were built and evaluated. The 3C6 antibody was attached to individual nanotubes and to nanotube films and electrical signals were generated and compared from both cases.

In the single-molecule case, binding and unbinding of paclitaxel to the antibody generated a time-varying signal with two distinct levels. Consequently, the electrical signal represented a real-time recording of single-molecule binding and unbinding events. The number of events depended on paclitaxel concentration, with binding events being rare at low concentrations and unbinding rare at high concentrations. Using a comparator circuit calibrated to enumerate these events, paclitaxel was monitored down to a concentration of 20 pM.

Sensors comprising 5 to 500 antibody molecules achieved poorer sensitivity. The real-time sensitivity to individual binding events was lost, and the total signal magnitude generated by 100 antibody molecules was not substantially larger than the signal generated by a single antibody.

Consequently, the time resolution was lost without any compensating improvement in signal magnitude. The time-averaged signal was used to detect high paclitaxel concentrations (>10 nM), and calibrated sensing was estimated to be possible over a limited dynamic range of 10 nM down to about 500 pM.

The results demonstrate that single-molecule sensing of paclitaxel can be achieved using carbon nanotube transistors. Single-molecule resolution provided detailed information on paclitaxel binding kinetics, variability among different antibody copies, and the mechanisms at work in ensemble sensors. Proof-of-principle devices operated with dynamic ranges of 20 pM to 100 nM (single-molecule) and 1 to 10 nM (ensemble).

Attachment of Antibodies to Carbon Nanotube Transistors. Biofunctionalization protocols were adapted from work with other proteins. Pyrene-maleimide linkers were adhered to single-walled carbon nanotubes (SWNTs) on clean silicone dioxide (Si0 ₂ ) surfaces, and then cysteine-maleimide reactions conjugated antibodies to the SWNT sidewalls.

Atomic force microscopy (AFM) with a resolution of 0.4 nm was used to directly image and count the number of antibodies with specific attachments. Reasonable attachment yields were obtained by diluting antibodies to about 15 μg/mL in phosphate buffered saline (PBS) and then incubating surfaces with the diluted solutions for 30 minutes at room temperature and without shaking.

Acceptable attachment yields using the maleimide chemistry requires active cysteine groups. The aging of antibody resulted in yield decreases that can be attributed to cysteine oxidation. Reproducible yields were obtained using antibody that had been stored at 4 °C for less than 1 month; no successful attachment occurred after 3 months of storage. These values are comparable to the shelf life provided by the commercial supplier (Abcam7).

Once the attachment protocols had been tailored for the desired functionalization surface density, the steps were repeated on electrically connected SWNTs in field-effect transistor (FET) devices. These devices were fabricated using standard techniques known in the art. Each SWNT FET was imaged by AFM before biofunctionalization to confirm the absence of contaminants (e.g., from lithographic processing) and again after biofunctionalization to determine the number of newly attached molecules.

FIGURE 3 shows two SWNT FET devices labeled with multiple 3C6 antibodies.

The images show that the number of antibodies on each SWNT is much higher than the background concentration of nonspecific attachments to the silicone dioxide surface, indicating acceptable specificity via the pyrene-maleimide linker scheme.

FIGURE 3 illustrates an attachment yield of 5-10 molecules per μπι with a large variability in the apparent height of each attached molecule.

Commercial antibodies 3C6 and 8A10, which contained multiple surface cysteines, were used and the images were consistent with multiple possible attachment sites and attachment orientations.

Following success with the AFM technique, the functionalization scheme was repeated on multiply connected SWNTs and SWNT films. Surface disorder within SWNT films precluded single-molecule resolution of the individual antibodies, so the absolute number of attachments was estimated from the single-SWNT average.

Signal Generation by Antibody Binding. Devices prepared as described above were measured to determine their electronic sensitivity to paclitaxel. First, the active area of the device was submerged in a particular concentration of paclitaxel suspended in phosphate buffered saline (PBS). The lithographic electrodes were protected by a polymer layer of poly(methyl methacrylate) (PMMA), so only the SWNTs and their attached antibodies were exposed to the solution. A small potential (0-100 mV) was applied to the drain terminal, driving a DC current in the range of 10-100 nA. The FET backgate and electrolyte provided two additional terminals for controlling the current set point. The current I(t) was recorded continuously over multiple minutes while a device was exposed to different test solutions.

The first goal was to determine whether paclitaxel binding and unbinding would generate changes in I(t). The binding affinity of 3C6 is reported to be K _D = 10 nM, so devices were probed with paclitaxel concentrations in this range.

FIGURE 4 shows representative data subsets from a single-molecule device. In the absence of paclitaxel, I(t) was steady around 35 nA (FIGURE 4A), with a noise band that was typical of SWNTs in solution.

As paclitaxel was added in concentrations of 0.2 nM and 2 nM, a growing number of discrete jumps in I(t) occurred from the 35 nA baseline up to about 55 nA (FIGURES 4B and 4C). These excursions probably represented individual binding and unbinding events of paclitaxel to the antibody-FET hybrid device. Increasing the concentration of paclitaxel to 200 nM (i.e., 20 KD) pinned the current at the higher, 55 nA level (FIGURE 4D). At this concentration, very few unbinding events were observed and the current represented the bound antibody-paclitaxel complex.

FIGURE 5 is a higher-magnification signal that illustrates the clarity of two-level fluctuations at faster rates. After signal acquisition, a digital filter and comparator tuned to the midpoint between the two levels was used to convert raw data into the simpler, binary signal. The binary signal simplified automated analysis of each event, allowing the bound and unbound durations to be studied individually or as probability distributions at any concentration. In this data, for example, the binary signal clarifies 17 binding events occurring in merely 60 ms, producing an average turnover rate of 280 s ^"1 . The bound state in this data was more probable than the unbound state by a ratio of approximately 2: 1. The bound state duration followed a Poisson probability distribution with an average lifetime of approximately 2.5 ms.

Similar analysis was applied to data sets acquired at different paclitaxel concentrations. Average single-molecule kinetic values for the turnover rate, the bound state lifetime, and the bound state probability were each determined by semi-automated analysis of 10-minute data records. FIGURE 6A shows an example of the concentration dependence of bound and unbound lifetimes for one 3C6 molecule. Below K _D , the duration of binding events was roughly constant at 0.2 to 0.5 ms. The number of binding events, on the other hand, decreased proportionally to concentration and led the average unbound time to grow to long durations. Above K _D , a different mechanism turned on.

The bound duration lengthened dramatically, suggesting that paclitaxel crowding near the antibody inhibited single-molecule release events. At the highest concentrations, the unbound state had average durations below 0. 1 ms because paclitaxel in excess was readily bound by the antibody. FIGURE 6B illustrates the effects of these instantaneous kinetics on the average turnover rate. The rate exceeded 1000 s ^"1 close to K _D and fell below 10 s ^"1 when the antibody was predominantly stuck in its bound or unbound conformation.

For the purposes of paclitaxel sensing, any of these parameters could be used successfully for determining paclitaxel concentration. At very low concentrations, the duration of the unbound state was a good proxy for the diffusion-limited arrival of paclitaxel molecules. Tests extended to 20 pM concentrations, but lower concentrations could be probed with the same technique. Closer to K _D , the exponential rise in turnover rate suggests that type of analysis would be a sensitive indicator of concentration.

Ensemble Sensing by Nanotube Films. Electrical tests focused on ensembles of small numbers of antibodies.

Experiments used similar devices to FIGURE 3 except having multiple SWNTs in a very dilute film. The configuration allowed many antibodies to be exposed simultaneously to produce quasi-ensemble signals from 10 to 1000 molecules.

FIGURE 7 shows a representative response of one device to the presence or absence of paclitaxel in solution. The measurement illustrates how semiconducting SWNT FETs are sensitive to both the electrolyte potential VQ and to paclitaxel binding. The shape of the I(VQ) transistor characteristics is primarily determined by the SWNT device physics, but the absolute x-axis position is set by local (molecular) charges near the SWNT. In this example, paclitaxel added to the measurement solution shifted the I(VQ) characteristics left by 50-70 mV along the x-axis. The inset to FIGURE 5 depicts the same information on a logarithmic scale, showing how enhanced sensitivity might be obtained near the turn-off threshold of the FET. Similar shifts and modest changes are described throughout the scientific literature.

The following examples are provide for the purpose of illustrating, not limiting, the invention. EXAMPLE

Representative Antibody-Functionalized CNT -based FET Sensor In this example, a representative method for preparing an antibody-functionalized CNT -based FET sensor of the invention is described.

Carbon nanotube functionalization. In this example, pyrene maleimide is used as the linker molecule. The pyrene functional group is known to non-covalently functionalize a carbon nanotube sidewalk A solution of 1 mM N-(l-pyrenyl)maleimide in ethanol is prepared. Devices are soaked in the 1 mM N-(l-pyrenyl)maleimide in ethanol solution for 30 minute without agitation. Devices are washed with 0.1% polysorbate 20 in ethanol for 30 minutes with shaking to remove excess 1 mM N-(l-pyrenyl)maleimide. Devices are then rinsed in a solution of 50% polysorbate 20 (0.1%) in ethanol and 50% phosphate buffer (20 mM Na ₂ HP0 ₄ , pH 7) for ten minutes without shaking to remove excess reagent. Devices are finally rinsed under flowing de-ionized water for 5 minutes.

Antibody coupling to functionalized carbon nanotube. The one or more functional groups of the linker molecules of the functionalized carbon nanotube prepared as described above, are reacted with one or more functional groups of the paclitaxel antibody.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.