FIELD OF THE INVENTION

[0001] This invention relates generally to molecular biology and biochemistry. More specifically, this invention relates to the detection and quantification of competent baculovirus using ELISA.

BACKGROUND OF THE INVENTION

[0002] Baculovirus is a common pathogen of insects such as the Chinese silkworm. The virus is widely used in the academia and industry for expressing recombinant proteins and in the agriculture for pest control. These applications require detection and/or quantification of the virus. Current methods for doing so take at least two to seven days. The results are of limited value because they indicate the viral load as of several days ago and the virus could have already multiplied exponentially. As a result, farmers fail to detect insect diseases early enough to prevent their spread. And scientists have difficulties in developing protein expression protocols because identifying the optimal virus-to-cell ratio and monitoring virus quality from batch to batch are critical to optimizing expression conditions.

SUMMARY OF THE INVENTION

[0003] We have developed methods for determining viral titre by quantifying an anti-apoptotic protein that is produced by a virus early in infection. In some embodiments, the methods are for determining baculoviral titre by quantifying P35, an anti-apoptotic protein produced by baculovirus. In some embodiments, the methods are enzyme-linked immunosorbent assays (ELISA) that allow one to detect baculoviral infection or to measure baculoviral load within five to seven hours of the initial infection. In some embodiments, the methods allow one to detect the amount of P35, if any, in a test sample suspected of being infected by or contaminated with baculovirus. As used hereinafter, P35 denotes a protein encoded by the p35 gene. [0004] Accordingly, the invention embraces a method for determining the titre of wildtype or recombinant baculovirus in a test sample, comprising the steps of providing a plurality of insect cells; contacting said insect cells with the test sample to allow infection of the cells by baculovirus to occur; detecting the amount of P35 in said insect cells with an anti-P35 antibody; and comparing said amount with known amounts of P35 (e.g., the amounts of P35 in two or more groups of control cells wherein each group of control cells are infected with a baculovirus sample of a different known titre), thereby determining the titre of baculovirus in the test sample. The test sample can be, e.g., tissue culture, or obtained from an insect such as silkworm, a moth, or a honeybee. The insect cells used in the method can be derived from silkworm, moth, or bees. Exemplary cells are sf9 cells, sf21 cells, and BTI-TN-5B1-4 cells. [0005] The invention further embraces a method for determining the titre of baculovirus in a test sample, comprising the steps of detecting the amount of P35, if any, in the test sample with an anti-P35 antibody; and comparing said amount with known amounts of P35, thereby determining the titre of baculovirus in the test sample. The test sample can be, e.g., obtained from any organism suspected of being infected by or contaminated with baculovirus. The test sample can be, e.g., obtained from an insect such as a silkworm, a worm or a honeybee. [0006] In some embodiments, the detecting step comprises: providing a solid surface (e.g., a microtitre plate) on which a first anti-P35 antibody has been immobilized; contacting the solid surface with the lysate of the insect cells or test samples to allow binding of P35 to the first antibody; removing unbound lysate; contacting the solid surface with a second anti-P35 antibody to allow binding of the second anti-P35 antibody to the bound P35, wherein the second antibody is detectably labeled; and detecting signal from the second antibody, wherein the signal indicates the amount of P35 in the insect cells or test samples. [0007] In some embodiments, the detecting step comprises: providing a solid surface on which a first anti-P35 antibody has been immobilized; contacting the solid surface with the lysate of the insect cells or test samples to allow binding of P35 to the first antibody; removing unbound lysate; contacting the solid surface with a second anti-P35 antibody to allow binding of the second anti-P35 antibody to the bound P35; removing any unbound second anti-P35 antibody; contacting the solid surface with a third antibody which binds to the second antibody, wherein the third antibody is detectably labeled; and detecting signal from the third antibody, wherein said signal indicates the amount of P35 in the insect cells or test samples. [0008] In some embodiments, the second antibody is biotinylated, and the signal detecting step comprises contacting the solid surface with a horseradish peroxidase conjugated to avidin; removing unbound avidin-conjugated horseradish peroxidase; contacting the solid surface with a horseradish peroxide substrate to give rise to light emission or absorption signal; and determining the amount of the light emission or absorption signal with a spectrophotometer or the naked eye. [0009] In some embodiments, the second antibody is detectably labeled with a moiety selected from the group consisting of a fluorescent moiety, a luminescent moiety, a radioactive moiety and an enzyme that catalyzes a chromogenic or fluorogenic reaction. [0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety, hi case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and paragraphs, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The materials, methods, and examples are illustrative only and not intended to be limiting. [0011] Other features and advantages of the invention will be apparent from the following drawing, and detailed description.

BRIEF DESCRIPTION OF THE FIGURES [0012] Fig. 1 is a line graph showing the correlation between P35 ELISA signal and baculovirus titre in a sample.

DETAILED DESCRIPTION OF THE INVENTION [0013] Eukaryotic cells often initiate apoptosis in response to viral infection. This is a defense mechanism to prevent replication of the virus. To survive and propagate, many viruses express anti-apoptotic proteins (i.e., cell death inhibitors) early in infection to prevent apoptosis of their host cells. We have discovered that such a viral anti-apoptotic protein is a reliable indicator of infectious viral titre. This discovery was surprising because the interplay between a host cell's apoptotic defense system and a virus's anti-apoptotic system is complex, and it was heretofore unknown whether the amount of a viral anti-apoptotic protein would be proportional to the number of infectious virus particles present in a host cell. It was heretofore unknown whether a threshold amount of viral anti-apoptotic protein would quickly shut down the host's defense mechanism such that a larger number of virus particles would necessarily produce a larger amount of the anti-apoptotic protein. [0014] P35 is an anti-apoptotic protein produced by baculovirus. See, e.g., Hisahara et al., The EMBO Journal 19:341-8 (2000); Clem, Science 254:1388-90 (1991). The P35 protein is expressed upon activation by the viral transcriptional activator IEl and appears in the host cytosol during the early stages of infection, usually within the first six hours, before any morphological signs of infection or the budded progeny virus can be observed (Gershburg et al., J Virol 71 :7593-7599 (1997)). P35 was originally identified as a protein required to prevent apoptosis of infected cells, and later was found to be a strong inhibitor of caspases, the proteases that disassembles cells during apoptosis (Xu et al., Nature 410:494-497 (2001)). It has also been reported that P35 is associated with the virus. The association of P35 with baculovirus was demonstrated by Hershberger et al., J Viol 68:3467-3477 (1994) when purification and immunoblot analysis of baculovirus demonstrated that, unlike the major virion structural proteins such as gp64 and vp39, P35 represented a minor component of the virus. Hershberger et al. further suggested that additional studies will need to be performed to determine P35's precise location within the enveloped baculovirus. [0015] We have discovered that P35 can be used as an indicator for detecting and quantifying the titre of competent (i.e., infectious) baculovirus. Specifically, P35 amounts correlate with the titre of infectious viral particles rather than the total viral particles. This is important because not all viral particles are infectious. For example, only 1-10% of total human immunodeficiency virus (HIV) particles are infectious (Vogt, V. M., "Retroviral Virions and Genomes", Retroviruses. Eds. Coffin, J. M., Hughes, S. H. & Varmus, H. E., Cold Spring Harbor Lab. Press, Plainview, NY, p. 27-70 (1997); Rusert et al., Virology 326:113-129 (2004)). Factors that may contribute to the generation of non-infectious viral particles include, without limitation, the improper assembly of the virus particle, failure of cellular mechanisms, the effect of different culturing conditions, presence of light, either fluorescent or UV radiation and detergents (Jarvis et al., Biotechniques 16:508-513 (1994); Martin et al., J Econ Entomol. 95:261-268 (2002)). [0016] The fact that the P35 protein is expressed early also allows the detection of viral infection before the first daughter virus is produced. The amount of P35 in the host, as quantified by, e.g., ELISA, SDS PAGE/Western blot analysis, can be converted to infectious virus titre or infectivity titre (also known as viral infectivity). [0017] Other anti-apoptotic viral proteins that can be used as markers for viral titres include, without limitation, the crmA/crmB/C5L protein in cowpoxvirus, ElB 19k in adenovirus, IAPs in baculovirus, v-FLIPs in gamma-herpesvirus or molluscipox virus, and NS2 in hepatitis C virus. [0018] We have developed a P35-specifϊc ELISA for detecting and quantifying baculoviral titre in as little as a few hours. This is a drastic improvement over the conventional end-point dilution and plaque- forming assays, which take as long as a week to estimate viral load. The new assay can be used, e.g., to determine wildtype and recombinant baculovirus titre, to detect baculovirus infection of silkworm, and to test baculovirus-based pest control agents. The new assay can also be used, e.g., to detect P35 in test samples suspected of being infected by or contaminated with baculovirus. For example, the amount of P35 produced in infected silkworms may be sufficient to detect this protein directly in the worm homogenate, thereby diagnosing the infection without the need to expand the virus by infecting healthy silkworm or its cells. Similarly, the method can be used to detect infected cells, organisms, or any other entities of any kind that accumulate the P35 protein. The method may also be used to detect cells, organisms, or any other entities of any kind that accumulate the P35 protein that are suspected of being contaminated by baculovirus. [0019] The ELISA of this invention uses two monoclonal P35 antibodies (e.g., #32-1-176 and #26-1-196, both of which were developed using a full-length P35 polypeptide). One of the two antibodies is used to coat a microtitre plate (bottom antibody). A test sample is then added to the coated plate, where the P35 protein is bound to the plate by the immobilized antibody. A second antibody is added to the plate and binds to the captured P35 protein (top antibody). Any unbound antibody is washed off. The bound second antibody can then be detected via an attached detectable moiety, such as a fluorescent or luminescent moiety, a radioactive moiety, or an enzyme that catalyzes a chromogenic or fluorogenic reaction (e.g., horseradish peroxidase, or alkaline phosphatase). Alternatively, the second antibody can be detected by a third antibody, e.g., polyclonal sheep anti-mouse IgG antibodies in the case that the second antibody is a mouse IgG antibody. Thus, the signal from the second antibody correlates with the amount of P35 in the test sample. By comparing the signal curves of serially diluted test samples and serially diluted virus stocks with known titres, the titre of the original test sample can be calculated. EXAMPLES [0020] The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters normally encountered in the art are within the spirit and scope of the present invention.

Example 1 [0021] In an exemplary protocol, insect cells sf9 (a commercial Spodoptera frugiperda ovarian cell line) are first seeded on a 96-well microtitre cell culture plate at about 70% confluency. After the cells have attached to the plate (about 20 minutes), the cells are incubated with serially diluted test samples and baculovirus stocks with known titres for about three to five hours. Meanwhile, ELISA plates are prepared by coating the plates with monoclonal antibody #32-1-176. The sf9 cells are mixed with a lysis buffer containing 1% Triton, 0.1% SDS, 136 mM NaCl, and 50 mM Tris (pH 8). The mixture is shaken vigorously for about 5 minutes to disrupt cell membrane. The cell lysate is then added onto the coated ELISA plates. The plates are then washed to remove unbound proteins and cell debris. A second, biotinylated anti-P35 antibody #26-1-196 is added onto the ELISA plates. After unbound antibody is washed off, the amount of bound second antibody is measured by adding avidin-horseradish peroxidase conjugate (HRP) (Pierce Cat. Log. No. 31001). A chromogenic HRP substrate, e.g., 4-chloro-l- naphthol, or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma Cat. Log. No. A3219), a chemifluorescent substrate, or a chemiluminescent substrate is added to give rise to signal that can be quantified by a spectrophotometer. A standard curve is prepared from readouts of the known virus stock. The virus titre from the test sample is calculated based on the standard curve. [0022] Fig. 1 shows an exemplary a standard curve. The data indicate that at a titre of about 105 cfu/ml to about 107 cfu/ml determined by colorimetric substrates, the P35 concentration correlates linearly with the baculoviral titre. In other words, the dynamic range of the assay is at least two orders of magnitude. However, the upper limit of detection is infinite because a test sample can always be diluted to fit the dynamic range of the assay. Example 2 [0023] In another exemplary protocol, insect cells sf9 were seeded in 96-well culture plates (Falcon Cat. Log. No. 353072) in culture medium (55 mL of heat inactivated fetal bovine serum (FBS) (Sigma Cat. Log. No. F6178) per 500 mL of Grace's Insect Cell Medium (Invitrogen Cat. Log. No. 11605-094)) at about 90% confluency. For a 96-well plate, approximately 105 cells are seeded per well, and the cells are allowed to attach for at least 15 minutes. Alternatively, approximately 7x104 cells are seeded per well and incubated overnight at 270C in an incubator. It is important to bear in mind that the amount of P35 in each well will depend not only on the viral titre but also on the number of cells in each well. Therefore, it is important to have an equal number of cells in each well. After the cells have attached to the plate, the cells are incubated with serially diluted test samples and baculovirus stocks with known titres at 270C for approximately 5 hours. The infection time may be varied, however, 3 hours is the minimal time for measuring P35 amounts reliably by ELISA. [0024] Meanwhile, ELISA plates (Nunc Maxisorp 96 well plate or 8-well modules, Cat. Log. Nos. 446612 or 445101) are coated with monoclonal antibody #32-1-176 at a concentration of 0.66 ng/μL in TBS. The antibody solution is added to the wells and incubated at room temperature for at least two hours, or at 4°C overnight. It is convenient to coat the plates in advance, as they can be stored, firmly covered, with the antibody solution for at least 30 days if plates do not dry out. ELISA plates are then washed four times with TBS-T. During the last wash, the plates are incubated with TBS-T for 5 minutes before shaken off, making sure not to let the ELISA plate dry out. [0025] At the end of the infection period, the sf9 cells are lysed using 50μL of J-RIPA buffer containing 1% Triton, 0.1% SDS, 136 mM NaCl and 50 mM Tris (pH 8). The mixture is shaken vigorously (900 rev/min) on a microplate shaker for 10 minutes to lyse the cells. The sf9 cell lysate is then added onto the coated ELISA plates at 50 μL per well and incubated on a shaker for 30 minutes. The plates are then washed with TBS-T to remove any unbound proteins and cell debris. A second, biotinylated anti-P35 antibody #26-1-196 at a concentration of 1 ng/μL in TBS-T is added onto the ELISA plate and incubated on a shaker for 30 minute. After unbound antibody is washed off with TBS-T, the amount of bound second antibody is measured by adding avidin-HRP (Pierce Cat. Log. No. 21123) at a dilution of 1 : 10,000, or as the manufacturer suggests, with TBS-T and incubated on a shaker for 15 minutes. The plates are then washed with TBS-T to remove any unbound avidin-HRP and a commercially available peroxidase substrate solution, such as 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma Cat. Log. No. A3219), is added to give rise to a signal that can be quantified using a microplate reader. The time required for development will depend on how much P35 is in the sample and varies between 1 minute and 1 hour. It is important to try to measure the absorbance before it reaches OD 0.5 to insure that the signal depends linearly on the P35 concentration. This may require that the ELISA plate be quantitated at 5-10 minute intervals up to 1 hour to choose an appropriate time. [0026] A standard curve is generated from readouts of the known virus stock. The virus titre from the test sample is calculated based on the standard curve. For example, titre estimation is done by calculating the difference between the ELISA standard curve and the test sample curve. To compare viral stocks, the dilution of virus which achieves the same OD405 absorbance reading is used. For example, if a viral standard having a known titre of 107 cfu/ml has the same OD405 absorbance reading as test sample A when the standard and test sample A are at a dilution of 24 and 2 , respectively, then the viral titre of test sample A can be calculated using the following formula:

Titre of test sample A = IO7 cfu/ml X (28/24) = 1.6 X 108 cfu/ml

Example 3 [0027] The new ELISA can also be used to detect baculovirus infection of silkworm. In an exemplary protocol, homogenized silkworm larva is the infection target. The homogenized larva is mixed with an alkaline-treated suspect source for about three to five hours at room temperature. At the end of the incubation period, a lysis buffer is added to the mixture. Cell lysates are then transferred to an ELISA and detection of P35 is performed as described above. If the top antibody is detectable by a chromogenic reaction, the infection result can be read by the naked eye.

Example 4 [0028] The new ELISA can also be used to detect P35 in samples that may already be infected with baculovirus. The infected samples may be any organism, cell, or any other entity that accumulates P35 protein as a result of baculoviral infection. In an exemplary protocol, homogenized silkworm larva that is suspected of being infected by baculovirus is used as the cell lysate and is transferred to an ELISA and detection of P35 is performed as described above. If the top antibody is detectable by a chromogenic reaction, the infection result can be read by the naked eye. This method has the advantage of being able to diagnose the infection without the need to expand the virus by infecting healthy silkworms or its cells.

[0029] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. [0030] The new ELISA method is fast. It detects baculovirus within five to seven hours of initial infection, hi contrast, it takes a few days for the prior art methods to detect infection. The new ELISA method can detect both wild type and recombinant competent (infectious) baculoviruses. hi contrast, the conventional ELISA methods, which use antibodies to baculoviral surface proteins or antibodies to occlusion bodies, may fail to detect recombinant virus because recombinant virus sometimes is engineered to carry nonbaculoviral surface protein and it also typically does not form occlusion bodies in the host cells. Further, the new ELISA method detects only competent infectious virus. The conventional methods, however, cannot distinguish competent virus from incompetent virus. See, e.g., U.S. Patent 6,284,455; Parola et al., J Virol Methods 112:13-21 (2003). The new method is also highly sensitive, and can detect baculovirus at a titre as low as 104 cfu/ml using colorimetric substrates. The sensitivity is even higher when chemifluorescent or chemiluminescent substrates are used. Further, the new method does not required sterile techniques due to the short operation time. It can be used by people with no scientific training because it does not require cell foci counting, morphology observation, or cell culture facilities, unlike the traditional end-point dilution and plaque forming assays and the commercially available BacPAK™ Baculovirus Rapid Titre Kit (BD Biosciences). Also, the viral detection and quantification results can be evaluated visually, providing an advantage to farmers working in the field. Finally, due to the short operation time, the new ELISA method does not detect daughter virus, which does not emerge within 24 hours of initial infection. Thus, the new method provides an accurate reading on the original viral titre.