IES84582Y1 - A method for preparing microbeads for use in an assay for determining the presence of antibodies to human immunodeficiency virus in a sample - Google Patents

Description

A method for preparing microbeads for use in an assay for determining the presence of antibodies to human immunodeficiency virus in a sample Field of the invention The present invention relates to a method for preparing microbeads for use in an assay for determining the presence of antibodies to human immunodeficiency virus (HIV) in a sample. The invention also relates to an assay for determining the presence of antibodies to HIV in a sample, and the invention also relates to a suspension of the microbeads. gackqround 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 sample and 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 microbead particles are coated with antigen under denaturing conditions (guanidine HCI) since the antigen is insoluble. The antigen used in the Riggin test underwent 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 microbeads for use in an assay for determining the presence of antibodies to HIV in a sample, and the invention is also directed towards a suspension of the microbeads for use in an assay for determining the presence of antibodies to HIV in a sample.

Summary of the Invention According to the invention there is provided a method for preparing microbeads for use in an assay for determining the presence of antibodies to human immunodeficiency virus (HIV) in a sample, the method comprising: acylating a HIV antigen, coating microbeads with the acylated HIV antigen, and washing the acylated HIV antigen coated microbeads in an acidic buffer solution for deacylating the HIV antigen coated on the microbeads, wherein prior to being coated with the acylated HIV antigen, the microbeads are precoated with a protein.

In one embodiment of the invention the HIV antigen is acylated in a citraconic anhydride solution.

Advantageously, the acidic buffer solution in which the acylated HlV antigen coated microbeads are washed is a citric acid buffer solution. Ideally. the acidic buffer solution in which the acylated HIV antigen coated microbeads are washed is of pH in the range of 4.7 to 4.9.

In one embodiment of the invention the washed acylated HIV antigen coated microbeads are incubated at a deacylating temperature for a deacylating period to complete deacylation of the HIV antigen.

Preferably, the deacylating incubating temperature lies in the range of 35°C to 39°C.

Advantageously, the deacylating incubating period lies in the range of 18 hours to 24 hours.

In one embodiment of the invention the acylated HIV antigen coated microbeads are washed at least twice prior to being incubated. Preferably, the acylated HIV antigen coated microbeads are washed four times prior to being incubated.

In another 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.

In a further embodiment of the invention the microbeads are precoated with the protein by immersing the microbeads in a solution having an effective amount of the protein.

Preferably, the microbeads coated with the acylated HIV antigen are incubated at a sensitising temperature for a sensitising period prior to deacylating the HIV antigen for sensitising the HIV antigen. Preferably, the sensitising temperature is room temperature in the range of 18°C to 25°C. Advantageously, the sensitising period lies in the range of 18 hours to 24 hours.

In one embodiment of the invention the deacylated HIV antigen coated microbeads are washed in a buffer solution after completion of deacylation thereof.

Advantageously, the deacylated HIV antigen coated microbeads are washed after deacylation thereof in a Tris buffer.

Ideally, the washed deacylated HIV coated microbeads are suspended in a Tris polyethylene glycol solution with a pH in the range of 8 to 9. Preferably, the concentration of the microbead suspension in the Tris polyethylene glycol solution is adjusted to lie in the range of 0.95% to 1.05%. Advantageously, bovine serum albumin is added to the Tris polyethylene glycol/microbead suspension to bring the pH of the suspension to lie in the range of 8.5 to 8.6. Ideally, the bovine serum albumin is added to the Tris polyethylene glycol/microbead suspension in an amount of approximately 1% by volume of the Tris polyethylene glycol/microbead suspension. in one embodiment of the invention the Tris polyethylene glycol/microbead suspension of pH in the range of 8.5 to 8.6 is allowed to equilibrate at an equilibrating temperature for an equilibrating period. Preferably, the equilibrating temperature lies in the range of 2°C to 8°C. Advantageously, the equilibrating period lies in the range of 6 days to 8 days.

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

Advantageously, the microbeads are selected from one of the following materials: ‘ A polystyrene, and silica.

Preferably, the HIV antigen is a HIV specific antigen.

Advantageously, the HIV antigen is a recombinant HIV antigen.

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

In another embodiment of the invention the HIV antigen is derived from a recombinant HIV-1 envelope protein. Alternatively, the HIV antigen is derived from a recombinant HIV-2 envelope protein.

In a further embodiment of the invention the HIV antigen is selected from any one or more of the following antigens: CBRe3 (recombinant HIV-1) antigen, and K1 (HIV-2) antigen.

The invention also provides HIV antigen coated microbeads for use in an assay for determining the presence of antibodies to HIV in a sample, the microbeads being prepared by the method according to the invention.

The invention further provides a suspension of HIV antigen coated microbeads according to the invention for use in an assay for detennining the presence of antibodies to HIV in a sample.

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 suspension of HIV coated microbeads 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 microbeads according to the invention for use in an assay for determining the presence of antibodies to HIV in a sample has many advantages. In particular, the microbeads when used in an assay provide an assay which is particularly sensitive to HIV antibodies, and which provides relatively rapid results.

A particularly important advantage of the invention is achieved by deacylating the HIV antigen after the HIV antigen has been coated onto the microbeads. It has been found that by deacylating the HIV antigen after the antigen has been coated onto the microbeads, the antigen is restored to its native state, thus significantly enhancing the sensitivity of the HIV antigens to HIV antibodies. Acylation of the HIV antigens prior to coating the HIV antigens onto the microbeads solubilises the proteins of the HIV envelope without the need to use strong denaturing agents, such as urea or guanidine hydrochloride, and thereby. the acylation of the HIV antigen permits a conventional column chromatographic purification step by ion exchange chromatography. Accordingly, there is less risk of loss of functional or antigenic antigen due to irreversible denaturing of the protein.

Another particularly important advantage of the invention is achieved by precoating the microbeads with protein, and in particular, by precoating the microbeads with bovine serum albumin prior to coating with the HIV antigen. It has been found that by precoating the microbeads with protein prior to coating with the acylated HIV antigen, hydroxylation of the microbeads is not required, thus permitting the microbeads to be coated in their unmodified state. Furthermore, it has been found that bonding of the acylated HIV antigen to the microbeads is significantly enhanced by precoating the unmodified microbeads with protein over and above the bonding which can be achieved by coating the HIV antigen directly onto hydroxylated microbeads. It is believed that the method according to the invention may also provide the advantage that the antigens are oriented on the microbeads at an optimal orientation.

A further advantage of the invention is achieved by subjecting the acylated HIV antigen coated microbeads to incubation at the sensitising temperature for the sensitising period prior to deacylation of the HIV antigen. It has been found that by incubating the acylated HIV antigen coated microbeads prior to deacylation significantly increases the sensitivity of the HIV antigen after the HIV antigen has been deacylated by allowing for optimum binding and coating of the antigen to the microbeads.

A further advantage of the invention is achieved by suspending the deacylated HIV antigen coated microbeads in a low percentage bovine serum albumin Tris polyethylene glycol buffer solution comprising 1% bovine serum albumin suspension.

In particular, it has been found that by suspending the deacylated HIV antigen coated microbeads in the suspension buffer, with elevated Tris concentration of approximately 500mM containing 1% bovine serum albumin is highly effective at maintaining relatively high sensitivity/specificity and relatively long shelf life of the microbeads, which can be up to twenty months.

The invention and its many advantages will be readily apparent to those skilled in the art from the following description of embodiments thereof, which are given by way of the following non-limiting example.

Detailed Description of a Preferred Embodiment A suspension of HIV antigen coated microbeads according to the invention in a buffer solution for use in an assay also according to the invention for determining the presence of antibodies to HIV in a sample is prepared by a method according to the invention as follows. A HIV antigen corresponding to the antibodies to be detected is selected. In this case, the selected antigen is a recombinant HIV-1 antigen, which is commonly referred to as CBRe3 and a 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 HCI and subsequently alkylkated (iodoacetate). This material is then dialysed against mM borate pH 9.0. After dialysis, the antigen is recovered as a precipitate. It is then centrifuged and the resulting pellet is dissolved in BM urea, and is then acylated by adding a specified amount of citraconic anhydride is added incrementally while the pH is maintained between 8.8 to 9.2 by addition of NaOH. After 30 minutes has elapsed from the last addition of citraconic anhydride, the material is dialysed against 50 mM Borate pH 9.0 to remove urea. The acylated antigen is now soluble and can be further purified by ion exchange chromatography to remove contaminants in the absence of denaturants (urea/guanidine) which would otherwise be required to keep the protein in solution if it were used in the unmodified form.

Similarly, the soluble antigen can also be coated onto microbead beads under mild conditions which would not be the case if unmodified antigen were used.

Under alkaline conditions, the dicarboxylic acid anhydride, citraconic anhydride react 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 and the original free amine is restored. 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 unmodified 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. Prior to coating the microspheres with the acylated HIV antigen, the microspheres are precoated with a protein, which in this embodiment of the invention is bovine serum albumin. The coating of the microspheres is carried out as follows.

The microbead microspheres are initially diluted to 5% in 50 mM Sodium Carbonate buffer, pH 9.65 and mixed for 15 minutes. The beads are then suspended in an equal volume of 50 mM Tris/0.5% bovine serum albumin buffer pH 8.63 and mixed for 18 hours to 24 hours at a temperature of 21 °C to 25°C. After this time the beads are then centrifuged and the supernatant is aspirated. The beads are then resuspended in 50 mM Sodium Carbonate and after 15 minutes, centrifuged again.

After removal of the supernatant the pellet is resuspended in a Sodium Carbonate buffer. Three centrifuge washes are carried out in total and the final pellet is resuspended in 50 mM Carbonate buffer to 5%. The microbead suspension is then sonicated for 4 to 6 minutes and further mixed for 15 to 25 minutes.

On completion of the precoating of the microspheres with the bovine serum albumin, the microspheres are coated with the acylated HIV antigen. Coating of the precoated microspheres with the acylated HIV antigen is carried out as follows.

A pre—determined amount of the soluble HIV-1 antigen is added dropwise to the microbead suspension at a rate of 6 to 9 ml/minute. A pre—determined amount of the soluble HIV-2 antigen is then added dropwise to the suspension at the same rate as above. The antigen coated microbeads are then mixed and incubated for 18 to hours at a temperature of 21 °C to 25°C. After this time the incubated acylated HIV antigen coated microbeads are deactylated by centrifuging and the pellet resuspended in 50mM citric acid buffer, pH 4.8. A total of four centrifuge washes are carried out in the citric acid buffer. At each re-suspension the antigen microbead preparation is mixed for 10 to 20 minutes. After the last wash the preparation is adjusted to 5% with 50 mM citric acid and mixed for 18 to 24 hours at 35°C to 39°C.

After this time the antigen microbead suspension is washed four times in 50 mM Tris buffer pH 8.63 with alternate centrifugation steps and resuspension steps. After the last step the antigen coated microbead is resuspended to 2% in 50 mM Tris buffer, mixed and sonicated. In the next stage the microbead suspension is diluted to 1% with an equal volume of 50 mM Tris/PEG buffer, pH 8.63. In the last stage the antigen microbead suspension is diluted to 0.375% using 0.8M Tris/0.5% bovine serum albumin pH 8.5 with constant stirring. This preparation is now placed at a temperature of 2°C to 8°C for a minimum of 18 hours. The material is then sonicated for 15 minutes and mixed for one hour before it undergoes in—process testing. At this stage the microbead suspension is equilibrated for a minimum of one week at a temperature of 2°C to 8°C. The antigen microbead suspension is now ready for use diagnostis of the presence of anti HIV antibodies in a sample.

An example of a method for carrying out the assay according to the invention is as follows.

The microbead reagent is suitable for use with human serum, plasma or whole blood samples using a proprietary slide. The slide consists of a well area for mixing the microbead reagent and the sample. At one end of the mixing well, there is a capillary flow channel which leads to a viewing window. In order to carry out a test for the presence of HIV antibodies the microbead reagent (120 pl) is initially dispensed onto the slide at the edge of the mixing well furthest from the capillary channel. Using a calibrated pipette the sample (10 pl) is dispensed directly into the microbead solution on the slide. Using the pipette, the sample and microbead are mixed by pumping the mixture in and out of the tip three times followed by stirring five times. The pipette tip is then used to move the mixture of sample and sensitised microbead to the opening of the channel to allow capillary flow to commence. The microbead mixture must flow through the entire capillary channel and into the viewing window during a period of 3 minutes to 7 minutes before interpreting the result. The window is then observed for aggregation. Samples demonstrating any microbead aggregation should be considered initially reactive. Samples showing no aggregation should be interpreted as non-reactive.

Claims (5)

Claims

1. A method for preparing microbeads for use in an assay for determining the presence of antibodies to human immunodeficiency virus (HIV) in a sample, the method comprising the steps of: acylating a HIV antigen, coating microbeads with the acylated HIV antigen, and washing the acylated HIV antigen coated microbeads in an acidic buffer solution for deacylating the HIV antigen coated on the microbeads, wherein prior to being coated with the acylated HIV antigen, the microbeads are precoated with a protein.

2. A method as claimed in Claim 1 in which the protein with which the microbeads are precoated is selected from one or more of the following proteins: bovine serum albumin, casein, gelatin, human serum albumin, and ovalbumin.

3. A method for preparing microbeads for use in an assay for determining the presence of antibodies to HIV in a sample, the microbeads being substantially as described herein with reference to the accompanying examples.

4. A suspension of HIV antigen coated microbeads for use in an assay for 15 determining the presence of antibodies to HIV in a sample, the microbeads being prepared by the method as claimed in any preceding claim.

5. 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 suspension of HIV coated microbeads as claimed in Claim 4, and evaluating whether agglutination of the microbeads occurs resulting from the presence of antibodies in the sample. F.F. GORMAN & CO.