WO1992022816A1

WO1992022816A1 - Solid phase immunoassay

Info

Publication number: WO1992022816A1
Application number: PCT/AU1992/000271
Authority: WO
Inventors: Isabel Margaret Roberts
Original assignee: Csl Limited
Priority date: 1991-06-11
Filing date: 1992-06-10
Publication date: 1992-12-23

Abstract

The inventors have found that it is possible to achieve binding of highly hydrophobic immunoglobulins to a solid phase without loss of sensitivity caused by binding of the F(Ab)2 region, by using as the binding agent a substance which stabilizes hydrophobic interactions in solution while inhibiting hydrophilic interactions. While not wishing to be bound by any proposed mechanism for the observed beneficial effects, it is believed that the use of such binding agents results in a stabilisation of the hydrophobic region of the immunoglobulin. The method of the invention is applicable to any immunoassay in which it is essential that the F(Ab)2 region be highly hydrophobic, i.e. any immunoassay system in which the antigen is highly hydrophobic. The method of the invention is applicable to immunoassay supports for both qualitative and quantitative assays, and is suitable for the preparation of microtitre plates intended for use in immunoassays which require the plates to be centrifuged. The invention also provides a method of screening binding agents for suitability for use in the invention, assay kits, and immunoassays.

Description

SOLID PHASE IMMUNOASSAY

This invention relates to methods of immunoassay, especially solid phase immunoassay, and in particular to a method of detection of Rh antigens.

Background and Prior Art

For the past few decades the exquisite specificity of antibodies has been widely exploited for the detection of a very large variety of substances, ranging from pathogens such as bacteria and viruses to cell surface components such as blood group antigens and receptors for hormones and neurotransmitters, to metabolites and pharmacological agents. Solid phase immunoassays using antibodies labelled with radioactive or fluorescent markers have enabled convenient, economical assay kits to be prepared for the screening of large numbers of samples for a very wide variety of antigens. Our earlier application, International Patent Application No. PCT/AU89/00530 (WO 90/07114) describes a rapid, simple and convenient solid phase assay which can be used for detection of several markers. This assay is particularly useful for detection of blood group antigens of the A-B-O system or of the Rh system.

Hitherto, oι: particular aspeαt of solid phase immunoassays has restricted their universal applicability. In a situation where the antigen to be detected relies for its i munogenicity on highly hydrophobic regions of the target molecule, ie. highly hydrophobic epitopes, the antibody which must be used to detect the antigen must also possess an antigen combining site which is hydrophobic. Thus the F(Ab)₂ region of the antibody molecule must be highly hydrophobic. A solid phase for a solid phase immunoassay is normally a plastic surface, and thus is itself hydrophobic; this results in preferential binding of the antibody to the solid phase via its hydrophobic region, and conseςpαently the antigen binding site of the detector antibody is obscured. n order to avoid this preferential loss of activity, hitherto it has been customary to subject the solid phase to a prior coating step with an anti- binding antibody, normally from a species different from that from which the binding antibody is obtained, directed against the Fc region of the capture antibody. The binding antibody (e.g. anti-human antibody) must frequently be preabsorbed to prevent it from inadvertently capturing the detector antibody (e.g. (enzyme-labelled) mouse monoclonal antibody) through cross-reactive epitopes. This absorption step, apart from being time-consuming, can also significantly reduce the potency of the anti-human antibody, and has so far limited mass production of microtitre plates for use in solid phase antibody kits.

The conventional wisdom in the art has been that the second, binding antibody is an absolute requirement to attain acceptable sensitivity of a solid phase immunoassay for detection of a highly hydrophobic antigen. For example, see P.L. Mollison, C.P. Engelfriet and M. Contreras (1987), "Blood Transfusion in Clinical Medicine", Blackwell Scientific Publications, 8th ed. p. 90; S.D. Rolih (1986), New Frontiers in Serologic Testing, Ch.5 in Wallas, C.H. and McCarthy, L.J., eds "New Frontiers in

Blood Banking" Arlington, VA, American Association of Blood Banks, p.139.

Previously, the only way in which to overcome this problem has been to bind the antigen, rather than the antibody, to the solid phase using reagents such as 8M urea, 60% methanol, or 6M guanidine. This expedient suffers from the disadvantages that the method is cumbersome, requiring extensive washing and dialysis of the plates in order to remove the binding agent, the agents used are toxic, and, in the case of urea and guanidine, the agent is expensive. We have now unexpectedly found that it is possible to achieve binding of highly hydrophobic immunoglobulins to a solid phase without loss of sensitivity caused by binding of the F(Ab)_a region, by using as the binding agent a substance which stabilizes hydrophobic groups in solution and thus promotes interaction of less hydrophobic regions with the solid phase while inhibiting hydrophilic interactions in solution. While not wishing to be bound by any proposed mechanism for the observed beneficial effects, it is believed that the use of such binding agents results in a stabilisation of the hydrophobic region of the immunoglobulin.

The method of the invention is applicable to any immunoassay in which it is essential that the F(Ab)₂ region be highly hydrophobic, ie. any immunoassay system in which the antigen is highly hydrophobic.

Summary of the Invention

According to a first aspect of the present invention, there is provided a method of binding of immunoglobulin to a solid phase immunoassay support comprising the step of incubating the immunoglobulin with the immunoassay support in the presence of an agent which stabilizes hydrophobic groups in solution and inhibits hydrophilic interactions in solution.

The antibody may be an immunoglob lin of any type, but is preferably IgM. It may be of polyclonal or monoclonal origin, but is preferably monoclonal.

The agent to be used may be any agent which is successful in promoting hydrophobic interactions and inhibiting hydrophilic interactions, and is most suitably a non-polar solvent. However, certain ionic agents have been found also to be unexpectedly successful. The suitability of the agent may readily be tested, as described below. Suitable agents include 1 to 3 M sodium chloride, preferably 3M sodium chloride, and 10 to 80%, preferably 60%, 2-methyl-butan-2-ol (2-methyl-2-butanol) ; and 20 to 60%, preferably 60%, isopropanol. Combinations of agents may also be used.

Preferred agents are also preferably non-toxic, miscible with water, and non-flammable. It may also be advantageous in certain circumstances if the binding agent is volatile.

Preferably the immunoassay support is a plastic surface, such as a tube, plate, microtitre plate, bead , slide or dipstick. The method of the invention is applicable to immunoassay supports for both qualitative and quantitative assays, and is suitable for the preparation of microtitre plates intended for use in immunoassays which require the plates to be centrifuged. A widely used plastic is polystyrene. Preferably the support is a microtitre plate or dipstick. Any immunoassay detection system known in the art, such as radioactive labelling, fluorescent labelling, or enzyme labelling, may be used. Enzyme labels, such as horseradish peroxidase, are particularly convenient.

It has also unexpectedly been found that the more non-polar the solvent, the stronger the binding of immunoglobulin to the surface.

In a second aspect, the invention provides a method of screening binding agents to determine their binding of immunoglobulin, comprising the steps of incubating the putative binding agent with a plastic surface in the presence of an immunoglobulin with a hydrophobic Fab region, and subjecting the treated plastic surface to an immunoassay capable of detecting binding by the immunoglobulin.

The person skilled in the art will recognise that it is necessary to test putative binding compounds for their ability to promote binding of the target immunoglobulin, and that the immunoglobulin is subjected to the conditions of the actual assay, so that the sensitivity of the bound immunoglobulin in the assay and the correct orientation of the immunoglobulin is assessed. The screening is conveniently performed by using a standard 96 well microtitre plate as the plastic surface, since this enables the simultaneous testing of up to 12 binding agents at 8 concentrations per agent.

It is best to use a higher concentration than normal of the capture (binding) antibody. A suitable binding agent still needs to be selected in order for the bound antibody to be oriented correctly, since using conventional buffers (e.g. 0.05M bicarbonate buffer, pH 9.5) and higher concentrations of capture antibody does not allow the bound antibody to capture the relevant antigen. It will be clearly understood that the invention also includes within its scope assay kits prepared using the method of the invention, and immunoassays carried out using immunoglobulin bound by the method of the invention.

Although the invention is specifically described with reference to anti-D immunoglobulin (ie. anti-Rh immunoglobulin) , it will also be clearly understood that the invention is applicable to antibody directed against any highly hydrophobic antigen, and in particular antigens such as zein, gluten, and other food antigens.

Detailed Description of the Invention

The invention will now be described in detail by way of reference only to the following non-limiting examples.

Example 1 - Meaning of binding agents.

Putative binding agents were tested for their ability to promote the binding of hydrophobic antibody to a plastic surface, using the following protocol:

1. A sample of purified anti-D immunoglobulin was mixed with the sample of binding agent to be tested; 2. The mixture was incubated overnight at 4°C in the presence of a plastic surface, for example in a polystyrene tube or in a well of a polystyrene microtitre plate; 3. The mixture was poured off, and a solution of an inert protein, for example bovine serum albumin or casein, dissolved in the same solvent as that used in Step 1, was added and incubated for one hour at room temperature; 4. This solution in turn was poured off, and the tube or well was washed 2 to 10 times. A preferred washing agent is binding solution containing 0.05% Tween 20.

5. A red blood cell reagent, such as whole blood, and anti-red blood cell conjugate were added, and incubated as in the simultaneous immunoassay described in International Patent Application No. PCT/AU89/00530 (WO 90/07114). For detection of Rh antigen, whole blood diluted 1/20, or an equivalent dilution of packed red cells, may suitably be used.

6. The tubes or plates were washed and detector substrate added for a standard detection step.

Suitable concentrations of inert protein may readily be ascertained using routine methods.

Example 2 Detection of Rh antigen

The method outlined for the preparation of the solid phase was followed, using a 96-well polystyrene microtitre plate. The inert protein used was casein, diluted in corresponding binding solution. The wells were washed 5 times with the binding solution containing 0.05% Tween 20. In all cases the prepared plates were tested by adding 50μl of an appropriate dilution of horseradish peroxidase-labelled anti-red blood cell antibody conjugate and 50μl of whole blood diluted 1/10. The blood used in these experiments was type O, with either the R_tR₂ phenotype (D positive - this generated the signal, "S") or the rr phenotype (D negative - this generated the background noise, "N"). After incubation with this mixture of red blood cells and conjugate for 5 mins, the solid phase was washed 5 times with phosphate buffered saline. Substrate was added, and the reaction with enzyme was stopped after 2-5 mins. The optical density of each well was read by standard means.

The results in Table 1 indicate that in addition to choosing the appropriate binding agent, a higher concentration of the bound antibody is desirable or essential for good results. Specificity is expressed as signal:noise ratio (S:N) .

Example 3 Comparison of Different Binding Agents

A number of different binding agents was compared, using the same method as in Example 2. The results, summarised in Table 2, show that the more hydrophobic the binding agent is, the more likely it is that the antibody will retain its ability to capture the relevant antigen. The best results were obtained with 1.0 to 3.0M NaCl and with 60% 2-methyl butan-2-ol. Very poor results were obtained with urea ana guanidine, which!have previously been used to bind antigen to solid supports.

Example 4 Detection of the Antigen with Antibody- Captured Anti-D

For comparison, an assay was prepared using a standard prior art-type system, in which anti-D antibody was bound to plates by means of anti-human antibody.

Different anti-human immunoglobulin preparations were used, and the buffer was 0.05M NaHC0₃, pH 9.5. Table 3 shows that the results of capturing the human anti-D with an anti-human Ig are relatively poor, due largely to high backgrounds generated by binding of the conjugate (horseradish peroxidase-labelled mouse antibody) to the anti-human antibody on the solid phase. This activity could be removed by absorption, but the potency of the anti-human antibody was significantly reduced and therefore there was no improvement in the S:N ratio. Additionally, the cross-reactivity was not removed, and hence the S:N ratio was not improved, by addition of irrelevant mouse monoclonal antibody. Thus the best sensitivity obtained was far inferior to the best results obtained in Examples 2 and 3.

TABLE 1 Effect of concentration of IgM anti-D on detection of Rh

TABLE 2 Comparison of different binding agents

TABLE 3 Detection of Rh with antibody captured anti-D

* Although the background (N) was much lower, the signal (S) was reduced also, indicating that a large portion of anti-Hu activity had been removed, as well as anti-mouse.

It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

Claims

1. A method of binding of immunoglobulin to a solid phase immunoassay support comprising the step of incubating the immunoglobulin with the immunoassay support in the presence of an agent which stabilizes hydrophobic groups in solution and inhibits hydrophilic interactions in solution.

2. A method according to Claim 1 wherein the agent is a non-polar solvent.

3. A method according to Claim 2 wherein the solvent is 2-methyl-butan-2-ol.

4. A method according to Claim 2 wherein the solvent is isopropanol.

5. A method according to Claim 1 wherein the agent is sodium chloride.

6. A method according to any one of Claims 1 to 5 wherein the immunoglobulin is of monoclonal origin.

7. A method according to any one of Claims 1 to 6 wherein the immunoglobulin is of IgM type.

8. A method of screening binding agents to determine their binding of immunoglobulin, comprising the steps of incubating a putative binding agent with a plastic surface in the presence of an immunoglobulin with a hydrophobic Fab region, and subjecting the treated plastic surface to an immunoassay capable of detecting binding by the immunoglobulin.

9. An immunoassay kit in which immunoglobulin is coupled to a solid phase support by the method of any one of Claims 1 to 7.

10. An immunoassay wherein immunoglobulin is coupled to a solid phase support by the method of any one of Claims 1 to 7.

11. An immunoassay kit or an immunoassay according to Claim 9 or Claim 10 wherein the immunoglobulin is directed against an antigen selected from the group consisting of D antigen (Rh antigen), zein, and gluten.

12. An immunoassay kit or an immunoassay according to Claim 11 wherein the detection marker is an enzyme.