GB2436003A

GB2436003A - A method for preparing a microbead suspension for use in an assay for determining the presence of antibodies to human immunodeficiency virus

Info

Publication number: GB2436003A
Application number: GB0624280A
Authority: GB
Inventors: Ronan Patrick O'caoimh; James Walsh; Brendan Kevin Farrell; Rory Peter Nealon
Original assignee: Trinity Research Ltd
Current assignee: Trinity Research Ltd
Priority date: 2005-12-06
Filing date: 2006-12-05
Publication date: 2007-09-12
Anticipated expiration: 2026-12-05
Also published as: GB0624280D0; IE20060890A1; IES20050816A2; GB2436003B

Abstract

A method for preparing a microbead suspension for use in an assay for detecting antibodies to HIV comprising; acylating an HIV antigen in a citraconic anhydride solution for solubilising thereof, precoating polystyrene microbeads with a protein (e.g. BSA, casein, gelatin), coating the precoated microbeads with the acylated HIV antigen and deacylating the HIV antigen coated onto the microbeads in an acidic buffer. After washing, the deacylated HIV antigen coated microbeads are suspended in a Tris/polyethylene glycol buffer and further diluted to 0.375% using 800 mM Tris/1 % bovine serum albumin of pH of 8.5 with constant stirring. The microbead suspension is maintained at a temperature in the range of 2 {C to 8{C for a minimum of one week for equilibration thereof.

Description

"A method for preparing a microbead suspension for use in an assay for

determining the presence of antibodies to human immunodeficiency virus in a sample and an assay using the microbead suspension"

Field of the Invention

The present invention relates to a method for preparing a microbead suspension for use in an assay for determining the presence of antibodies to human inimunodeficiency virus (H IV) in a sample. The invention also relates to a microbead suspension prepared by the method, and to an assay for determining the presence of antibodies to HIV in a sample.

Background to the Invention

Assays for determining the presence of antibodies to HIV in a sample, for example, a sample of blood, blood plasma, serum, urine, saliva, teardrops, cerebrospinal fluid, tissue, faeces, sperm or vaginal fluids, are known. Such an assay is disclosed in U.S. Patent Specification No. 4,921,787 of Riggin, et al, assigned to Cambridge Bioscience Corporation. In the assay of Riggin, latex microbeads are hydroxylated prior to coating with a recombinant HIV antigen in order to facilitate bonding of the HIV antigen to the microbeads. The HIV antigen coated microbeads of Riggin are suspended in a phosphate buffered saline solution having a pH of approximately 7.6 and containing approximately 1% bovine serum albumin. The sample to be tested is diluted in a bovine serum albumin buffer solution and is mixed with the HIV antigen coated microbead suspension. After mixing for approximately 3 to 5 minutes, the mixture of the sample and the HIV coated microbead suspension of Riggin is inspected for agglutination of the microbeads which would result from the presence of antibodies in the sample. Thus, agglutination is an indication of a positive reaction for antibodies to HIV, while a negative reaction is indicated by no agglutination.

While the assay of Riggin does detect the presence of antibodies to HIV in a sample, it suffers from some disadvantages. In the Riggin method microbeads are coated with the HIV antigen under denaturing conditions using, for example, guanidine hydrochloride to solubilise the antigen since the antigen is insoluble. The antigen used in the Riggin test is subject to purification by gel filtration chromatography, also under denaturing conditions. Thus, there is a risk of loss of functional or antigenic antigen due to permanent or irreversible denaturing of the protein. There is therefore a need for an assay which is more sensitive to a sample containing HIV antibodies than those known heretofore.

The present invention is directed towards providing such an assay, and the invention is also directed towards a method for preparing microbead suspension for use in an assay for determining the presence of antibodies to HIV in a sample. I0

Summary of the Invention

According to the invention there is provided a method for preparing a microbead suspension for use in an assay for determining the presence of antibodies to HIV in a sample, the method comprising: acylating an HIV antigen for solubilising thereof, precoating microbeads with a protein, coating the precoated microbeads with the acylated HIV antigen, and deacylating the HIV antigen coated onto the microbeads in an acidic buffer solution.

In one embodiment of the invention the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen.

Preferably, the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen for a time period in the range of eighteen hours to twenty-four hours.

Advantageously, the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen at a temperature in the range of 35 C to 39 C.

Ideally, the acidic buffer solution in which the acylated HIV antigen coated microbeads is suspended during deacylation of the HIV antigen is maintained at a pH in the range of 4.7 to 4.9.

In one embodiment of the invention the acylated HIV antigen coated microbeads are washed at least once prior to being incubated during deacylation thereof.

Preferably, the acylated HIV antigen coated microbeads are washed four times prior to being incubated during deacylation thereof.

In another embodiment of the invention the acylated HIV antigen coated microbeads are washed in an acidic buffer solution prior to being incubated during deacylation thereof.

In another embodiment of the invention the acidic buffer solution in which the acylated HIV antigen coated microbeads is suspended during deacylation of the NIV antigen is a citric acid buffer solution.

In one embodiment of the invention the acylated HIV antigen coated microbead suspension is incubated prior to being subjected to deacylation for subsequently increasing the sensitivity of the microbead suspension. Preferably, the acylated HIV antigen coated microbead suspension is incubated for a time period in the range of eighteen hours to twenty hours prior to being subjected to deacylation.

Advantageously, the acylated HIV antigen coated microbead suspension is incubated at a temperature in the range of 21 C to 25 C prior to being subjected to deacylation.

In one embodiment of the invention the deacylated HIV antigen coated microbeads are suspended in a Tris buffer solution of pH in the range of 8 to 9. Preferably, the concentration of the deacylated HIV coated microbeads suspended in the Tris buffer solution is adjusted to lie in the range of 0.25% to 1.05%. Advantageously, the p1-I of the deacylated HIV antigen coated microbead suspension is adjusted to lie in the range 8.5 to 8.6.

Preferably, bovine serum albumin is added to the deacylated HIV antigen coated microbead suspension in the Iris buffer solution for stabilising the suspension.

Advantageously, the bovine serum albumin is added to the deacylated t-IIV antigen S coated microbead suspension in an amount in the range of 0.25% to 1.75% bovine serum albumin.

In another embodiment of the Tris buffer solution of the deacylated HIV antigen coated microbead suspension comprises a polyethylene glycol solution.

Preferably, the Iris buffer solution of the deacylated HIV antigen coated microbead suspension is of high molarity. Advantageously, the Tris buffer solution of the deacylatecJ HIV antigen coated microbead suspension is a 800 mM Iris buffer solution.

In one embodiment of the invention the deacylated HIV antigen coated microbeads are washed in a Tris buffer solution after deacylation thereof and prior to suspension in the buffer solution.

Preferably, the deacylated HIV antigen coated microbeads constitute approximately 0.3% to 0.4% of the suspension.

Advantageously, the deacylated HIV antigen coated microbead suspension is maintained at a temperature in the range of 2 C to 8 C for a time period of not less than eighteen hours for equilibration thereof.

Ideally, the deacylated HIV antigen coated microbead suspension is maintained at a temperature in the range of 2 C to 8 C for a time period in the range of six days to eight days for equiliberating thereof.

In one embodiment of the invention the protein with which the microbeads are precoated is selected from one or more of the following proteins: bovine serum albumin, casein, gelatine, human serum albumin, and ovalbumin.

Preferably, the protein with which the microbeads are precoated is bovine serum albumin. Advantageously, the microbeads are precoated with the protein by immersing the microbeads in a solution having an effective amount of the protein.

In one embodiment of the invention the HIV antigen is acylated by citraconylation in a citraconic anhydride solution. Preferably, the citraconylation of the HIV antigen is carried out by adding citraconic anhydride to a solution containing the HIV antigen.

In one embodiment of the invention the HIV antigen is a HIV specific antigen.

In another embodiment of the invention the HIV antigen is a recombinant HIV antigen.

In a further embodiment of the invention the HIV antigen is derived from a recombinant HIV envelope protein.

In a still further embodiment of the invention the HIV antigen is derived from a recombinant HIV-1 envelope protein.

In a further embodiment of the invention the HIV antigen is derived from a recombinant HIV-2 envelope protein.

Preferably, the HIV antigen is selected from any one or more of the following HIV antigens: CBRe3 (recombinant HIV-1) antigen, and Ki (HIV-2) antigen.

In one embodiment of the invention the microbeads are of size in the range of 0.476 microns to 0.575 microns.

Preferably, the microbeads are selected from one or more of the following materials: polystyrene, and silica.

The invention also provides an HIV antigen coated microbead suspension prepared io by the method according to the invention.

Additionally the invention provides an assay for determining the presence of antibodies to HIV in a sample, the assay comprising contacting a sample suspected of containing antibodies to HIV with the microbead suspension according to the invention, and evaluating whether agglutination of the microbeads occurs resulting from the presence of antibodies in the sample.

Advantages of the Invention The advantages of the invention are many. The method for preparing the microbead suspension according to the invention for use in an assay for determining the presence of antibodies to HJV in a sample has many advantages, in particular, the microbead suspension provides an assay which is particularly sensitive to HIV antibodies, and which provides relatively rapid results.

Acylating the HIV antigen solubilises the protein without the need to use a strong denaturing agent such as urea or guanidine hydrochloride, and this, thus, allows a conventional column chromatographic purification step, in other words, ion exchange chromatography. Once solubilised by acylation, the HIV antigen can be readily coated onto the protein precoated microbeads. Accordingly, there is less risk of loss of functional antigenic antigen due to irreversible denaturing of the protein.

A particularly important advantage is achieved by precoating the microbeads with a protein, and in particular, by precoating the microbeads with bovine serum albumin prior to coating with the HIV antigen. Precoating the microbeads with a protein, and in particular, bovine serum albumin avoids the need for hydroxylation of the microbeads, thus permitting the microbeads to be coated with the HIV antigen in their unmodified state. Furthermore, the precoating of the microbeads with bovine serum albumin enhances the bonding of the acylated HIV antigen onto the microbeads. It is also believed that the precoating of the microbeads with bovine serum albumin may orient the HIV antigen on the microbead in an optimal orientation.

Deacylation of the acylated HIV antigen after coating thereof onto the microbeads produces a particularly important advantage, in that the HIV antigen is restored to its native state, thus significantly enhancing the sensitivity of the HIV antigen to HIV antibodies. A further advantage is achieved by subjecting the acylated HIV antigen coated microbeads to incubation prior to deacylation of the HIV antigen, in that it has been found that the incubation prior to deacylation significantly enhances the sensitivity of the microbead suspension in an assay.

It has been found that by suspending the deacylated HIV antigen coated microbeads in a low percentage bovine serum albumin Tris polyethylene glycol buffer solution whereby the bovine serum albumin constitutes 1 % or less of the suspension stabilises the suspension, and the bovine serum albumin in the buffer solution also acts as a blocker for blocking the free sites on the microbeads, thereby further enhancing the sensitivity and specificity of the microbead suspension and also enhancing the shelf life of the microbead suspension. Indeed, by suspending the deacylated HIV antigen coated microbeads in an elevated Iris concentration of approximately 800 mM containing 1% or less bovine serum albumin produces a microbead suspension with a shelf life of up to twenty months.

The invention and its many advantages will be readily apparent from the following description of a preferred embodiment thereof which given by way of the following non-limiting example.

Detailed description of a preferred embodiment

A suspension of HIV antigen coated microbeads in a buffer solution according to the invention is prepared for use in an assay, also according to the invention for determining the presence of antibodies to HIV in a sample. The HIV antigen coated microbead suspension is prepared by a method according to the invention as follows. An HIV antigen corresponding to the HIV virus, the antibodies of which are to be detected is selected. In this case the selected antigen is a recombinant envelope protein of HIV-1, which is commonly referred to as CBRe3, and a recombinant envelope protein of HIV-2 antigen. The recombinant HIV envelope antigens are typically expressed as insoluble inclusion bodies in genetically engineered E.coli.

The recombinant HIV-1 antigen is reduced with 2-mercaptethanol in guanidine hydrochloride, and subsequently alkylated with an alkylating agent, namely, lodoacetate. This material is then dialysed using 50mM borate of pH 9.0. after dialysis the antigen is recovered as a precipitate by centrifuging. The resulting antigen pellet is dissolved in 8M urea, and is then solubilised by acylation. The acylation of the antigen is carried out by incrementally adding citraconic anhydride to the urea solution to a total amount of 0.01 82ml citraconic anhydride pair mg of protein. The pH of the solution is maintained between 8.8 to 9.2 by the addition of sodium hydroxide during the addition of the citraconic anhydride to the protein urea solution. The protein urea solution is citraconylated for approximately 30 minutes, and is then dialysed using 50 mM borate with a pH of approximately 9.0 to remove the urea. At this stage the acylated antigen is soluble, and can thus, be further purified by ion exchange chromatography to remove contaminants in the absence of denaturants, such as urea/guanidine, which would otherwise be required in order to keep the protein in solution, if it were used in the unmodified form. Additionally, by acylating the antigen, the antigen can readily be coated onto the microbeads under mild conditions, which would not be possible if unmodified antigen were used.

Under alkaline conditions, the dicarboxylic acid anhydnde citraconic anhydride reacts with amine groups of proteins to form amide linkages with an extending terminal carboxylate. If desired, the modified protein can be subjected to acidic conditions, which causes the bonds to hydrolyze and to release citraconic acid, thereby restoring the original free amine. Acylation of proteins with citraconic anhydride, results in the lowering of the isoelectric point of proteins as multiple amine groups are converted to carboxylate groups.

The HIV-2 antigen is treated similarly as the HIV-1 antigen, and is similarly acylated.

In this embodiment of the invention the microbeads are latex microspheres of polystyrene material and are of diameter in the range of 0.476 microns to 0.575 microns, and predominantly of diameter of the order of 0.525 microns. The microbeads are unmodified. Prior to coating the microbeads with the acylated HIV antigen, the microbeads are precoated with a protein, which in this embodiment of the invention is bovine serum albumin. Initially, the microbeads are diluted to 5% in a 50 mM sodium carbonate buffer of pH 9.65 and mixed for fifteen minutes. The microbeads are then suspended in an equal volume of 50 mM Tris/0.5% bovine serum albumin buffer of pH 8.63 and mixed for eighteen hours to twenty-four hours at a temperature in the range of 21 C to 25 C. The solution is then centrifuged, and then the supernatant is aspirated. The microbeads are then resuspended in 50 mM sodium carbonate, and after fifteen minutes are again centrifuged. After removal of the supernatant, the microbeads are resuspended in a sodium carbonate buffer, and are subjected to two further centrifuged washes in sodium carbonate. The precoated microbeads are then resuspended in 50 mM carbonate buffer to 5% and the microbead suspension is sonicated for four to eight minutes and mixed for a further fifteen to twenty-five minutes.

The precoated microbeads are next coated with the acylated HIV antigens. The acylated HIV-1 and HIV-2 antigens are diluted separately in 50 mM sodium borate, pH 9, and the dilutions of the HIV-1 and HIV-2 antigens are determined using a BCA assay (bicinchoninic acid assay) using bovine serum albumin as a standard. In this case the HIV-1 and HIV-2 antigens are diluted to a concentration of 0.55 mg antigen per millilitre in 50 mM sodium borate, pH 9, and the solutions of diluted antigens are added drop wise to the precoated microbead suspension, namely the suspension of the precoated microbeads in the carbonate buffer. The actual amount of each of the two antigens required is calculated based on the volume of the precoated microbead suspension, and it has been found that an optimal coating concentration is 0.0225 pg per cm2 of HIV-1 recombinant antigen, and 0.0405 pg per cm2 of HIV-2 recombinant antigen. The diluted solution of HIV-1 and HIV-2 antigens are added to the precoated microbead suspension at the rate of 6 mI/per minute to 9 mI/per minute.

The precoated microbead suspension with the HIV-1 antigen and HIV-2 antigen added thereto is mixed and incubated at a temperature in the range of 21 C and 25 C for between eighteen and twenty-four hours in order to sensitise the subsequently produced HIV antigen coated microbead suspension. On completion of the incubation at the temperature in the range of 21 C to 25 C the microbeads are adequately coated with the acylated HIV antigens.

The acylated HIV antigen coated microbead suspension is centrifuged and the acylated HIV antigen coated microbeads are resuspended in a 50mM citric acid buffer of pH of the order of 4.8 for deacylating the acylated HIV antigens coated onto the precoated microbeads, for washing thereof, and are again centrifuged. In total, the HIV antigen coated microbeads are subjected to a total of four such centrifuge washes in a citric acid buffer solution. On each resuspension of the HIV antigen coated microbeads the suspension is mixed for approximately ten to twenty minutes.

After the final one of the four centrifuge washes, the HIV antigen coated microbeads are again suspended in a 50mM citric acid buffer solution and the HIV antigen coated microbeads in the suspension is adjusted to approximately 5%. The suspension is incubated at a temperature in the range of 35 C to 39 C for a time period of approximately eighteen to twenty-four hours in order to fully deacylate the HIV antigens coated onto the precoated microbeads so that the HIV antigens are restored to their original native states. The deacylated HIV antigen coated microbeads suspension is washed four times in 50mM Tris buffer of pH of approximately 8.63 with alternate centrifugation steps and re-suspension steps.

After the final of the four washing and centrifugation steps, the deacylated HIV antigen coated microbeads are resuspended to 2% in 50mM Tris buffer, which is mixed and sonicated. The deacylated HIV antigen coated microbead suspension is then diluted to 1% with an equal volume of 50mM Tris/polyethylene glycol buffer of pH of the order of 8.63. The deacylated HIV antigen coated microbead suspension is then diluted to 0.375% using 800mM Tris/1 % bovine serum albumin of pH in the o order of 8.5 which is added to the suspension with constant stirring. The deacylated HIV antigen coated microbead suspension is then maintained at a temperature in the range of 2 C to 8 C for a minimum of eighteen hours and is then sonicated for fifteen minutes and mixed for one hour before undergoing in-process testing. After testing, the antigen coated microbead suspension is maintained at a temperature in the range of 2 C to 8 C for a minimum of one week for equilibration of the suspension, and the suspension is then ready for use in an assay for determining the presence of antibodies to the specific HIV virus.

An assay for determining if a sample containing antibodies to the specific HIV virus, which typically would be a sample of blood plasma or whole blood is carried out by mixing the sample with the HIV antigen coated microbead suspension on a proprietary slide. The proprietary slide is one which comprises a well area within which the sample may be mixed with the HIV anitgen coated microbead suspension, and a capillary flow channel extends from the mixing well to a viewing window. The assay is carried out as follows. 1 20p1 of the HIV antigen coated microbead suspension if initiafly placed on the slide in an area of the mixing welt remote from the capillary channel. Using a calibrated pipette lOpI of the sample is dispensed directly into the HIV antigen coated microbead suspension in the mixing well of the slide. The sample and the suspension are mixed by pumping the mixture into and out of the tip of the pipette three times followed by stirring five times. The pipette tip is then used to urge the mixture of sample and HIV coated microbead suspension to the opening of the capillary channel in order to allow capillary flow to commence.

The mixture of the sample and the microbead suspension is allowed to flow through the entire capillary channel and into the viewing window during a period of three minutes to seven minutes before interpreting the results. The viewing window is then observed for identifying a agglutination of the microbeads. Samples which demonstrate any microbead agglutination are considered initially reactive. Samples showing no agglutination of the microbeads are interpreted as being non-reactive.

Claims

Claims 1. A method for prepanng a microbead suspension for use in an

assay for determining the presence of antibodies to human immunodeficiency virus (HIV) in a sample, the method comprising: acylating an HIV antigen for solubilising thereof, precoating microbeads with a protein, coating the precoated microbeads with the acylated HIV antigen, and deacylating the HIV antigen coated onto the microbeads in an acidic buffer solution.

2. A method as claimed in Claim 1 in which the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen.

3. A method as claimed in Claim 2 in which the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen for a time period in the range of eighteen hours to twenty-four hours.

4. A method as claimed in Claim 2 or 3 in which the suspension of acylated HIV antigen coated microbeads in the acidic buffer solution is incubated during deacylation of the HIV antigen at a temperature in the range of 35 C to 39 C.

5. A method as claimed in any of Claims 2 to 4 in which the acidic buffer solution in which the acylated HIV antigen coated microbeads is suspended during deacylation of the HIV antigen is maintained at a pH in the range of 4.7 to 4.9.

6. A method as claimed in any of Claims 2 to 5 in which the acylated HIV antigen coated microbeads are washed at least once prior to being incubated during deacylation thereof.

7. A method as claimed in Claim 6 in which the acylated HIV antigen coated microbeads are washed four times prior to being incubated during deacylation thereof.

8. A method as claimed in Claim 6 or 7 in which the acylated HIV antigen coated microbeads are washed in an acidic buffer solution prior to being incubated dunng deacylation thereof.

9. A method as claimed in any of Claims 2 to 8 in which the acidic buffer solution in which the acylated HIV antigen coated microbeads is suspended during deacylation of the HIV antigen is a citric acid buffer solution.

10. A method as claimed in any preceding claim in which the acylated HIV antigen coated microbead suspension is incubated prior to being subjected to deacylation for subsequently increasing the sensitivity of the microbead suspension.

11. A method as claimed in Claim 10 in which the acylated HIV antigen coated microbead suspension is incubated for a time period in the range of eighteen hours to twenty hours prior to being subjected to deacylation.

12. A method as claimed in Claim 10 or 11 in which the acylated HIV antigen coated microbead suspension is incubated at a temperature in the range of 21 C to 25 C prior to being subjected to deacylation.

13. A method as claimed in any preceding claim in which the deacylated HIV antigen coated microbeads are suspended in a Ins buffer solution of pH in the range of 8 to9.

14. A method as claimed in Claim 13 in which the concentration of the deacylated HIV coated microbeads suspended in the Ins buffer solution is adjusted to lie in the range of 0.25% to 1.05%.

15. A method as claimed in Claim 13 or 14 in which the pH of the deacylated HIV antigen coated microbead suspension is adjusted to lie in the range 8.5 to 8.6.

16. A method as claimed in any of Claims 13 to 15 in which bovine serum albumin is added to the deacylated HIV antigen coated microbead suspension in the Iris buffer solution for stabilising the suspension.

17. A method as claimed in Claim 16 in which the bovine serum albumin is added to the deacylated HIV antigen coated microbead suspension in an amount in the range of 0.25% to 1.75% bovine serum albumin.

18. A method as claimed in any of Claims 13 to 17 in which the Tris buffer solution of the deacylated HIV antigen coated microbead suspension comprises a polyethylene glycol solution.

19. A method as claimed in any of Claims 13 to 18 in which the Tris buffer solution of the deacylated HIV antigen coated microbead suspension is of high molarity.

20. A method as claimed in Claim 19 in which the Tris buffer solution of the deacylated HIV antigen coated microbead suspension is a 800 mM Tris buffer solution.

21. A method as claimed in any of Claims 13 to 20 in which the deacylated HIV antigen coated microbeads are washed in a Iris buffer solution after deacylation thereof and prior to suspension in the buffer solution.

22. A method as claimed in any of Claims 131021 in which the deacylated HIV antigen coated microbeads constitute approximately 0.3% to 0.4% of the suspension.

23. A method as claimed in any of Claims 13 to 22 in which the deacylated HIV antigen coated microbead suspension is maintained at a temperature in the range of 2 C to 8 C for a time period of not less than eighteen hours for equilibration thereof.

24, A method as claimed in Claim 23 in which the deacylated HIV antigen coated microbead suspension is maintained at a temperature in the range of 2 C to 8 C for a time period in the range of six days to eight days for equilibrating thereof.

25. A method as claimed in any preceding claim in which the protein with which the microbeads are precoated is selected from one or more of the following proteins: bovine serum albumin, casein, gelatine, human serum albumin, and ovalbumin.

26. A method as claimed in Claim 24 in which the protein with which the microbeads are precoated is bovine serum albumin.

27. A method as claimed in any preceding claim in which the microbeads are precoated with the protein by immersing the microbeads in a solution having an effective amount of the protein.

28. A method as claimed in any preceding claim in which the HIV antigen is acylated by citraconylation in a citraconic anhydride solution.

29. A method as claimed in Claim 28 in which the citraconylation of the HIV antigen is carried out by adding citraconic anhydride to a solution containing the HIV antigen.

30. A method as claimed in any preceding claim in which the HIV antigen is a l-IIV specific antigen.

31. A method as claimed in any preceding claim in which the HIV antigen is a recombinant HIV antigen.

32. A method as claimed in any preceding claim in which the HIV antigen is derived from a recombinant HIV envelope protein.

33. A method as claimed in any preceding claim in which the HIV antigen is derived from a recombinant HIV-1 envelope protein.

34. A method as claimed in any preceding claim in which the HIV antigen is derived from a recombinant HIV-2 envelope protein.

35. A method as claimed in any preceding claim in which the HIV antigen is selected from any one or more of the following HIV antigens: CBRe3 (recombinant HIV-1) antigen, and Ki (HIV-2) antigen.

36. A method as claimed in any preceding claim in which the microbeads are of size in the range of 0.476 microns to 0.575 microns.

37. A method as claimed in any preceding claim in which the microbeads are selected from one or more of the following materials: polystyrene, and silica.

38. A method for preparing a microbead suspension for use in an assay for determining the presence of antibodies to HIV in a sample, the method being described herein with reference to the example.

39. An HIV antigen coated microbead suspension prepared by the method as claimed in any preceding claim.

40. An HIV antigen coated microbead suspension substantially as described herein with reference to the example.

41. An assay for determining the presence of antibodies to HIV in a sample, the assay comprising contacting a sample suspected of containing antibodies to HIV with the microbead suspension as claimed in Claim 39 or 40, and evaluating whether agglutination of the microbeads occurs resulting from the presence of antibodies in the sample.

42. An assay for determining the presence of antibodies to HIV in a sample, the assay being substantially described with reference to the example.