This invention relates to a method of attaching biological substances to solid substrates, and relates particularly, but not exclusively, to the attachment of biological materials such as antigens and/or antibodies to solid-phase substrates for use in . solid-phase immunoassays. The invention also relates to the products which are produced by using the said method.

The attachment of one of the partners in an immunological reaction to a solid support facilitates the qualitative and/or quantitative measurement of the reaction. Generally the immobilization of one of the partners allows for easy separation of the reaction products from unreacted components. In effect the immobilised partner in the reaction captures its complementary reactant from solutions and binds it to a solid-phase. Once bound to this solid-phase its presence may then be measured either quantitatively or qualitatively.

In the past, the immobilisation of one of the partners in an immunological reaction has been achieved by a number of different methods. Support materials

used have included cellulose, agarose, dextran, polyacrylamide, glass, rubber and plastics including nylon, polystyrene and polyesters etc. Direct chemical bonding to produce covalent attachment may be used. Chemical methods of attaching either an antigen or an antibody to the support material include the use of linking agents such as glutaraldehyde, cyanogen bromide, carbodiimides and aminoalkylsilanes. These techniques are well known to those skilled in the art.

Attachment of antigens or antibodies to plastic surfaces may be achieved by direct adsorption. This is a simple method of attachment however it has several disadvantages. The adsorption of the antigen or antibody to the surface is achieved by hydrophobic interaction and van der aals forces. These are relatively weak attractive forces and as a result the attached material may elute from the surface during subsequent handling of the solid support (1) .

With solid supports such as microtitre plates, the efficiency of attachment of different antigens may vary dependent on their biochemical composition.

Additionally, antigen attachment to such plates may be highly variable between different wells on the same plate (2) .

Techniques to overcome these problems have involved pre-activation of the plastic surface. Two chemical methods of preactivation are disclosed in

European patent No. 83111144 and European Patent No. 84103367. These methods involve preactivation with phenylalaninelysine copolymer and diphenylene-bis- diazonium compounds respectively.

A physical method of activating plastic microtitre plates is described in European Patent No. 82102257. This method entails the exposure of the microtitre plates to γ-rays. Gamma irradiation produces better attachment (and also sterilization) due to ionization effects leading to the presence of some free radicals/bonds (3) .

In addition to soluble materials such as antibodies and antigens, it may also be necessary to immobilise whole cells or cell extracts for use in immunological or biological reaction systems. Attachment may be facilitated using techniques known to those skilled in the art (4) . These techniques generally involve pre-treatment of the solid support with agents such as glutaraldehyde, poly-L-lysine, phenylalaninelysine copolymer or lectins derived from sources such as Glycine max, Limulus polyphemus, Lens culinaris and Phaseolus vulgaris, etc. Cells may also be attached to a solid support by first attaching to the support an antibody which is specific for the cell. Attachment of the antibody may be achieved by the techniques previously mentioned.

All of the attachment techniques mentioned above rely on the presence of reactive groups on the substrate. If not involved in the bonding process, such reactive groups need to be neutralised before the bonded antigen, antibody, cells or the like are used in immunoassays because they can allow the non-specific attachment of other material which is used in later steps of the immunoassay procedure. This non-specific attachment of material can lead to high background readings thus limiting the sensitivity of the test procedure. The biochemistry of the biological material

will also determine which particular bonding process may be used, therefore there has been no universally applicable bonding technique which can effectively bond all types of biological materials to a particular substrate.

It has now been discovered that the attachment of biological substances to solid-phase substrates can be achieved by use of the technique of ion beam bombardment.

According to the present invention, there is provided a method of attaching a biological substance to a solid-phase substrate which comprises the steps of:- (i) subjecting a solid-phase substrate to ion bombardment, and (ii) contacting the treated substrate with a biological substance to be attached thereto.

The present invention also provides a product produced by the method described above. In this aspect, the invention includes a solid-phase product comprising a solid-phase substrate, the surface of which has been treated by ion bombardment, and a biological substance attached to said treated surface.

Suitable biological materials for use in accordance with the present invention include protein materials such as antigens, antibodies, immunoglobulins such as IgG, IgM and IgE, and enzymes, as well as cellular material such as red blood cell membranes, whole cells such as bacteria and viruses, and hormones such as thyroid stimulating hormone (TSH) . Suitable solid-phase substrates include in particular, but not

exclusively, plastics materials such as polypropylene. Mylar, polystyrene, latex and Teflon; glass and silica; and cellulose and cellulose derivatives.

It has been found that the use of the ion beam bombardment technique enables specific bonding of biological substances to the solid-phase substrates with high affinity, stability and reproducibility.

The technique of ion beam bombardment is generally well known (5) , although it has not previously been used in the pretreatment of substrates for the attachment of biological substances. By way of example, U.S. Patents Nos. 3,682,729, 4,256,780 and 4,457,972 all illustrate the use of ion bombardment in the formation or deposition of films of metals and similar materials on substrates (See also Bodβ and Sundgren (6)). These prior disclosures do not, however, contain any reference whatsoever to the use of this technique in the attachment of biological substances to substrates.

The step of ion bombardment of the solid-phase substrate may, for example, be carried out by bombarding the substrate with a beam of energetic Ar ions. Whilst the exact mechanism which leads to the strong specific attachment of biological substances to such a treated substrate is not well understood at present, it is believed that ion bombardment could produce one or more of the following effects which are conducive to strong bonding at the interface:

(a) removal of surface impurities by sputtering;

(b) of etastable chemical "dangling" bonds which attach to the biological material; and

(c) modification of surface composition by preferential sputtering.

The effects of ion bombardment are quite unique as compared to the well known interaction of milder ionizing radiation (γ-rays. X-rays, high energy electrons, pile irradiation, etc.) with polymer materials (7) . Since ion bombardment produces a much higher ionization density (stopping power) and can also produce atomic recoils, it is capable of generating metastable chemical surface states/compositions not attainable by milder forms of ionizing radiation or by any conventional equilibrium chemistry. In this sense, the ion bombardment technique is believed to be quite unique in that, it can not only produce effects similar to those generated by milder ionizing radiation, but also can produce many new metastable chemical states (dangling bonds) . This feature is believed to be responsible for more universal success of ion bombardment in producing enhanced attachment of many different types of biological materials to several different types of plastics.

Since the technique of ion beam bombardment is known, the performance of this step in the method of the present invention may be accomplished by use of any standard apparatus (5) .

Following bombardment of the solid-phase substrate material, the biological substance is then brought into contact with the treated substrate. The unique bonding process of the present invention may be conducted at room temperature and does not require any extraneous chemical bonding agents. In this respect,

there is thus provided a direct bonding between the biological substance and the treated substrate material, and furthermore, the biological material is not degraded or destroyed in any way since it is not irradiated or chemically treated during the attachment process.

In one particular embodiment of the present invention, to be described in greater detail below, the method of the present invention is utilised to attach red blood cell membranes to transparent plastic solid-phase substrates chosen as test substrates, the first being polypropylene (which possesses no reactive groups) , and the second being Mylar (which has some reactive groups) . Following bombardment of the plastic substrates with an energetic Ar+ ion beam, red blood cell membranes in colloidal suspension in a suitable liquid are then injected in situ (in vacuum) onto the surface which has been treated by ion bombardment. The liquid quickly spreads to a fine coating with the red blood cell membranes being bonded so strongly to the substrate surface that even extended ultrasonic treatment in water has no effect in removal of the membranes from the surface.

Further features of the present invention will be apparent from the following Examples which illustrate, but in no way limit, the present invention. In these Examples, small strips (1cm x 1.5cm) of polypropylene. Teflon, polystyrene and Mylar were cleaned with trichlorethylene, followed by rinses in methanol and air dried. Samples were then mounted in an ion bombardment chamber (Figure 1) which was evacuated to a pressure <lxlO -5 Torr. Samples were bombarded with

60keV Ar or He ions in the dose range of 5x10 to

5x1016 ions/cm2. The rectangular strip samples were bombarded over half the area and were used for the red blood cell experiment described in Example 1. Circular discs were over the entire area and were used for experiments described in other Examples. Several bombardment parameters studied in detail were ion dose, dose rate, ion species and ambient storage environment after bombardment (till the biological substance is brought in contact) . Extensive reproducibility checks were made.

EXAMPLE 1

A. Preparation of Red Blood Cell Membranes

Red blood cell membranes were prepared from fresh anticoagulated blood by the process of calcium phosphate cosedimentation (8) . Sodium azide was incorporated at a final concentration of 0.1% to inhibit bacterial contamination during the processing of the sample for attachment to the plastic substrate surface.

B. Attachment of Membranes to Substrates

Polypropylene strips were treated with Ar ions in apparatus illustrated in Figure 1. The strips were placed in sample holder 11 within the target chamber 10 with the system maintained totally under vacuum, and bboommbbaarrddeedd with accelerated Ar ions 12 through aperture plate 13.

Following ion bombardment of the strips, the sample holder 11 was transferred through air lock valve 20 to an adjacent air lock 21 without breaking vacuum (Figure 1). In air lock 21, a controlled volume of the red blood cell membrane calcium phosphate suspension was then injected onto the plastic strips through biological

material injection port 22. The liquid did not wet the unbombarded regions of the polypropylene and quickly spread to produce a coating on the bombarded regions. The plastic strips were removed from the air lock and excess liquid removed. After allowing to air dry the strength of adhesion of the red cell membrane/calcium phosphate mixture was assessed by immersing the strip in water and subjecting it to ultrasonic cleaning for five minutes. The strips were dried in air and then examined by optical microscopy. Figures 2a and 2b show the results for bombarded and unbombarded regions on polypropylene and Mylar respectively.

The adherent membrane/calcium phosphate mixture appears darker in these micrographs. It is clearly seen that the membrane/calcium phosphate mixture is attached to bombarded regions and is removed from unbombarded virgin regions.

EXAMPLE 2

IgG was purified from human serum by

Sepharose-Staph A affinity column chromatography. An aliquot of the purified IgG was radiolabelled with 125I by the Chloramine T method. Radiolabelled IgG was added to the purified IgG as a tracer to give a ratio of labelled to unlabelled IgG of between 1:20 and 1:30.

This radiolabelled material was then used to examine the influence of IgG concentration, buffer pH and reaction time on the interaction of IgG with ion bombarded plastic surfaces.

In these experiments IgG was diluted in phosphate buffer pH 7.0 and carbonate buffer pH 9.6 over a

concentration range of lOμg/ml to lOOμg/ml. 40μl samples were applied to wells (0.6cm diameter) impressed on ion bombarded and virgin plastic strips. Following incubation at room temperature the strips were rinsed with deionised water, the wells cut from the strip and the radioactivity due to adherent IgG counted in a γ counter.

It was found that buffer pH did not significantly influence the amount of IgG bound to the ion bombarded plastic surfaces. Longer reaction times (18 to 20 hours) permitted the attachment of more IgG. The higher concentrations of IgG resulted in more IgG attaching to the ion bombarded plastics.

Table 1 indicates the influence of different forms of ion bombardment and different plastics on the uptake of IgG. This clearly shows that ion bombardment can increase the uptake of IgG to plastic surfaces and that the efficiency varies depending on the species of the ion employed and the type of plastic used.

TABLE 1 Typical results on ion beam induced enhancement (in per cent) in attachment of IgG to various plastic substrates.

Bombarded Ion Species

Plastic Substrate Helium Argon

Polypropylene 288 349

Teflon 128 42

Polystyrene 113 89

It will be appreciated that the above Examples are given only by way of illustration of the present invention, and that many modifications and variations may be made thereto without departing from the scope of the invention as broadly described herein.

REFERENCES

1. Engvall, E. , Methods in Enzymology 1980; 7_0 : 419-439.

2. Chessum, B.S. & Denmark, J.R., Inconstant ELISA Lancet, 1978; i:161.

3. du Plessis, T.A. and Schnauz, N.G. - J.Oil Col. Chem.Assoc. , 1975; 58 _^ , pp 85-89.

4. Heuser, C.H., Stocker, J.W., and Gisler, R.H. - Methods of binding cells to plastics: Application to solid phase immunoassays for cell-surface antigens. Methods in Enzymology, 1981; 7_3_, 406.

5. Dearnaley, G. , Freeman, J.H., Nelson, R.S. and Stephen, J. Ion Implantation (North Holland, Amsterdam, 1973) .

6. Bodδ, P. and Sundgren, J.-E. - Adhesion of evaporated titanium to polyethylene: Effects of ion bombardment pretreatment. J.Vac.Sci.Technol. A. Vol.2, No.4., Oct-Dec. 1984.

7. Frank, H.P., 'Polypropylene' (McDonald Tech., London, 1968) , Chapter 5.

8. Klarkowski, D.B. - A calcium phosphate co-sedimentation method for the preparation of haemoglobin free human erythrocyte stro a. Australian Journal of Medical Laboratory Science 1984, 5_ pp.124-126.