GB2396008A

GB2396008A - A test for detection of antibodies to HIV

Info

Publication number: GB2396008A
Application number: GB0327865A
Authority: GB
Inventors: Eoin Martin O'nuallainn; James Walsh; Ronan Patrick O'caoimh; Brendan Kevin Farrell; Arcy Marie D
Original assignee: Trinity Research Ltd
Current assignee: Trinity Research Ltd
Priority date: 2002-12-03
Filing date: 2003-12-02
Publication date: 2004-06-09
Anticipated expiration: 2023-12-02
Also published as: IE20030883A1; IES20020938A2; GB0327865D0; GB2396008B

Abstract

A test for detecting antibodies to HIV in serum, plasma or whole blood comprises mixing a modified human serum albumin with recombinant HIV antigens to form an assay. The human serum albumin is modified by reacting the albumin with citraconic anhydride at a relatively high pH which results in the formation of amide linkages with a terminal carboxylate. The assay is incorporated into devices for testing for HIV.

Description

l PA test for detection of antibodies to HIV" The present invention

relates to a test for detecting antibodies to HIV, and in particular, for detecting antibodies to HIV in serum, plasma or whole blood. The 5 invention also relates to a method for preparing the test.

Tests for antibodies to HIV and other disease-causing agents are commonly used as part of the process for diagnosing infectious diseases. Many such tests incorporate recombinant protein antigens derived from the diseasecausing agent 1o which are often expressed in E. coli. In some cases the protein antigens are expressed as insoluble inclusion bodies and denaturing agents such as urea are required for the solubilisation of such proteins. Alternatively, the protein antigens may be solubilised by chemical modification at side-chain amino groups with dicarboxylic anhydrides resulting in the formation of amide linkages with a terminal 15 carboxylate. This results in a negatively charged protein antigen which is readily soluble in aqueous buffers. Such antigens may then be incorporated in diagnostic tests. A recombinant HIV test which is sold by Trinity Biotech of Ireland under the Trade 20 Mark UNIGOLD is based on the principle of immunochromatography and incorporates recombinant HIV antigens in a sandwich type assay for the detection of antibodies to HIV in serum, plasma or whole blood. If antibodies to HIV are present in the sample, they combine with HIV antigen/colloidal gold conjugate and this complex captures the immobilized antigens in test line region of the nitrocellulose

strip. A positive result is detected as a pink/red band at the test line. In the absence of antibodies to HiV in the sample no pink/red line develops in the test region. Non-

specific binding of components present in negative patient samples can bind to antigens in a sandwich test such as that described, and have the effect of causing a 5 false positive reaction.

Accordingly, there is a need for a test for detecting antibodies to HIV in serum, plasma or whole blood which overcomes this problem, and has a relatively high sensitivity and specificity, and is stable over a reasonable time period.

The present invention is directed towards providing such a HIV test, and in particular, the invention is directed towards providing a test for detecting antibodies to HIV in serum, plasma or whole blood, and the invention is also directed towards a method for preparing the test.

According to the invention there is provided a test for the detection of antibodies to HIV comprising recombinant HIV antigens and albumin modified by reacting the albumin with citraconic anhydride.

20 In one embodiment of the invention the albumin is human serum albumin.

In another embodiment of the invention the recombinant HIV antigen comprises a modified recombinant protein.

In a further embodiment of the invention the modified protein of the recombinant HIV antigen is modified by reacting the protein with citraconic anhydride.

In one embodiment of the invention the human serum albumin is modified by s reacting the human serum albumin with dicarboxylic anhydride, and preferably, the human serum albumin is modified at amine groups with the dicarboxylic anhydride.

Preferably, the reaction is carried out at a pH sufficient for the formation of amide linkages with a terminal carboxylate, and preferably, the pH of the initial reaction buffer is 11.5 before addition of citraconic anhydride.

In another embodiment of the invention the test the reagents are impregnated on a sheet of material.

In another embodiment of the invention the sheet of material is a glass fibre sheet.

In another embodiment of the invention the recombinant HIV antigen comprises a modified recombinant protein, which has been modified with citraconic anhydride.

In one embodiment of the invention the modified human serum albumin is mixed with 20 the recombinant HIV gold conjugate in the ratio 0.5mg modified human serum albumin to 1 ml of impregnation solution.

Additionally the invention provides a method for preparing a test for detecting antibodies to HIV, the method comprising modifying albumin by reacting the albumin

with citraconic anhydride and mixing the modified albumin with a recombinant HIV antigen to form the test.

In one embodiment of the invention the albumin is human serum albumin and is s modified by reacting protein of the albumin with a dicarboxylic anhydride, and preferably, the reaction of the protein with the dicarboxylic anhydride is carried out at a pH sufficient to form amide linkages with a terminal carboxylate. In one embodiment of the invention the pH of the reaction mixture is adjusted to pH 11.5 before the addition of citraconic anhydride.

In another embodiment of the invention the reacted solution is dialysed.

In one embodiment of the invention the test is impregnated in a sheet of material, and preferably, in a sheet of fibre glass material.

The advantages of the invention are many. Comparative tests which have been carried out on tests prepared according to the invention which comprise the recombinant HIV antigens with the modified human serum albumin, which are hereinafter referred to as tests according to the invention, were compared with 20 control tests which contained the recombinant HIV antigens only. Human serum and plasma samples which were known to be HIV negative were applied to the control tests and the tests according to the invention. It was found that there was no difference in the sensitivity of the tests according to the invention and control tests, however, the specificity and the stability of the tests according to the invention were

s found to be significantly improved over the control tests. Known HIV negative human serum and plasma samples were applied to the control tests and the tests according to the invention, and in the vast majority of cases the tests according to the invention yielded negative results. However, the control tests yielded false 5 positive results in a significant number of cases. Additionally, the tests according to the invention held the results without deterioration over the time period of the test, which was twenty minutes.

The invention will be more clearly understood from the following description of a non-

10 limiting example.

Human serum albumin for the test according to the invention is modified by chemically reacting the human serum albumin with citraconic anhydride. The chemical reaction is illustrated by the following formula: Nacylation of HSA with citraconic anhydride R-NH + PH R N W

HSA O Ring Opening Citraconic with Amide Bond Anhydnde fomabon

() The following reagents were utilised during the modifying process for modifying the human serum albumin.

Quantity Description

Required Human Serum Albumin 2.4 9/250 ml Sodium Carbonate Buffer (1.0M) 250 ml Sodium Hydrogen Carbonate Buffer (1.0M) As Required 25X Phosphate Buffer 1.6 L Citraconic anhydride 12 ml/250 ml 1 N Sodium Hydroxide As Required 1 N Hydrochloric Acid As Required The following calculations are for the formulation of a 250ml batch.

The pH of 250 ml solution of 1.OM sodium carbonate buffer was adjusted to 11.50 + 0.1 using 1.0M sodium hydrogen carbonate. The 1.0M sodium hydrogen carbonate to was added in 0.2ml aliquots. Human serum albumin was then added to the solution, 2.4g/250ml, and allowed to fall into solution.

Citraconic Anhydride, 12ml/250ml, was then added, in 1 ml aliquots, while stirring the human serum albumin solution on a magnetic stir plate at a high speed. The solution 15 was stirred at room temperature for 10 mins i 1min at a high speed to disperse the citraconic anhydride droplets. On formation of a homogenous solution the stirring speed was lowered and the citraconylated-human serum albumin was stirred for 170 mins + 5 mins. The citraconylated-human serum albumin was then dialysed in 10.0 litres of phosphate buffer, pH 8.0 + 0.1, with agitation for 48 hours at 2-8 C. The

dialysis buffer was changed once after 24 hours. The collected dialysed modified human serum albumin, which is hereinafter referred to as citraconylated human serum albumin was then 0.2'um filtered in the laminar flow hood.

s The citraconylated human serum albumin was mixed with recombinant HIV gold conjugate in the ratio 0.5mg citraconylocated humeri serum albumin to 1 ml of impregnation solution, and the resulting mixture is incorporated into suitable devices for testing for HIV.

lo Evidence for effectiveness of citraconvlated human serum albumin -

preliminarv testing Initial results were obtained using selected HIV negative serum and plasma samples from members of four panels. The samples from the members of the first panel were positive and negative samples, while the samples from the members of the 15 remaining second to the fourth panels were negative samples. However, the negative samples were shown to be reactive on control recombinant HIV antigen tests which did not contain the citraconylated human serum albumin on in-house testing (i.e., false positives were observed). Similar HIV negative serum samples were applied to the test according to the invention, and compared to the control test.

20 Table 1 outlines results of the comparisons which were made at 10 minutes and 20 minutes after the HIV negative serum was applied to the control test and the test according to the invention.

x Table 1: Preliminary testing of citraconylated HSA j _ _ _, 10 MINUTE RESULTS_ _ 20 MINUTI RESULTS __

CONTROL HIV HIV DEVICES CONTROL HIV HIV DEVICES

DEVICES CONTAINING CONTAINING

| CITRA HSA DEVICES CITRA HSA

i SAMPLE'; FROM FIR iT PANEL MEMBEI _ = _. l SITIVE PANEL 1 1+,10 _1+, 10 1 1+,10 2, 10

LPOSIT E PANEL 2 F+,10 1,10 1 F+,10 _ 1,10

j POSITIVE PANEL 3 L 6, 8 6+,10 _ j 6, 9 6+, 10 NEGATIVE PANEL 4 0,10 0, 10 1 0, 10 0,10

I NEGATIVE PANEL 5 L o 10 0,10 j 0,10 _ 0,10 NEGATIVE PANEL 6 j 0,10 0, 10 0, 10 0,10 I NEGATIVE PANEL 7 0,10 0,10 0, 10 0,10

I NEGATIVE PANEL 8 0,10 _0,10 0, 10 0,10

I NEGATIVE PANEL 9 0,10 0,10 0, 10 0,10

rSAMPLES FROM SKI,OND PANEL MEMB RS - NEGATIVE S RUM SAMPLES j 9705-0863735 1 +,10 0,10 1 3, 10 0,10 9705-086-3759 _ 2,10 0,10 1 2,10 0,10

1 9705-086-3801 F,10 _ 0,10 1 1,10 0,10

j 97 -086-3784 0,10 0,10 F,10 0,10 L 9711-086-2647 _ F,10 0,10 F. 10_ 0, 10

SAMPLES FROM THIRD PANEL MEMBERS - NEGATIVE PLASMA SAMPLES_

1 1F. 10 j 0,10 1 1, 10 1 0,10 j 17 _ 0,10 j 0,10 _ i_ 010 1 0,10 1 28 1F+ ,10 1 0,10 1+, 10 1 0,10

j 69 11,10 1 0,10 1+, 10 j 0,10 77 10,10 i 0,10 0,10 1 0,10 i 106 _ _; __ 1,10 1 0,10,+, 1G _ 0,10 1 117 0,10 i 0,10 11 0,10 0,10 it 130 1 1 10 i F. 10 2, 10 1 F,10 1 131 0 10 1 0 10 1 0,10 0,10 i 1 132 1 F. 10 1 0 10 1 1, 10 1 0, 10

190 1 0 10 1 0 10 F. 10 j 0,10 SAMPLES FROM FOURTH PANEL MEMBERS NEGATIVE SERA I

---_ 1 0.10 1 0,10 1 F. 10 1 0, 10

I _ 70 1 0 10 1 0 10 1 F. 10 1 0,10

1 74 11 0 10_! 0 10 1 F. 10 1 0,10

5 Note: Each sample was graded on a visual internal interpretative scale which relates to the colour intensity of the lines observed. Testing was recorded in the form (X, Y). X records the test line colour intensity and Y records the control line intensity.

When O was recorded in the X position no test line was visible, so this result is HIV 10 negative. When F (Faint) was recorded the test line was barely visible, this result was HIV positive. Where a 1 to 10 is recorded the result was also HIV positive; the lower the number allocated the weaker the colour intensity of the test line and vice versa.

The control line, the Y position, utilised the same grading system as the test line.

Results showed that citraconylated human serum albumin was effective in eliminating the false positive reaction in the majority of cases. Positive samples from the members of the first panel were also included to demonstrate that the signal intensity of HIV positive samples was not diminished by the inclusion of s citraconylated human serum albumin. Furthermore the majority of negative samples were shown to give a stable negative result up to 20 minutes when tested on devices containing citraconylated human serum albumin.

An additional benefit of the modified reagent was in maintaining the stability of the 10 test signal over the time frame of the test (20 minutes). It was observed that some negative samples which had given a negative result at 10 minutes and showed a positive result at 20 minutes, now remained negative at 20 minutes when tested with devices containing the modified reagent (samples #190, #38, #70 and #74 in Table 1). A titration was carried out to determine the optimal concentration of citraconylated human serum albumin for use in the HIV test. Citraconylated human serum albumin was incorporated into HIV devices at the following concentrations; 0.5, 1.0, 1.5 and 2.0 mg/ml. As before results were compared at 10 and 20 minutes. Positive 2() samples from the members of the first panel and negative plasma sample were tested and citraconylated human serum albumin was shown to be optimally reactive in the range 0.5 -1.0 mg/ml.

The extent of modification of amine groups of human serum albumin by citraconic

anhydride was assessed using an test utilising 2,4,6 trinitrobenzene sulfonic acid.

The results showed that approximately 75 % of amine groups had been modified.

Evidence for effectiveness-extended testing 5 Large scale testing of devices containing citraconylated human serum albumin compared with control devices was carried out. The following samples were tested: 3 HIV positive panel members 50 HIV-1 positive serum samples 23 HIV-2 positive plasma samples 10 138 HIV negative serum samples 500 HIV negative plasma samples On testing HIV positive samples from the members of the first panel, all positive samples were within specifications on both control devices and devices containing

15 citraconylated human serum albumin.

Table 2: Testing Positive Panel Members ,..,_.._..

10 Minute Result 20 Minute Result Sample!. _ _ Number. Citra HSA Citra HSA _ Control Devece Devices De vnitcre l Devices 1 1, 10 2, 10 1, 10 3, 10

2 1+, 10 1,10 2, 10 1+,10

3 8,5 8,5 9,6 9,5+

The results obtained on testing 50 HIV-1 positive serum samples are outlined below 20 (Table 3). 100% sensitivity was obtained on testing both control devices and devices

containing citraconylated human serum albumin.

Table 3: Testing HIV-1 Positive Serum Samples (n=50) r - I --.

10 Minute Result 20 Minute Result Citra HSA | Control Device | Devices l _ j. -

| 'hi Sensitivity 100 /0 | 100 % 100 % 100 h On testing 23 HIV-2 positive plasma samples (Table 4), 100% sensitivity was obtained on testing control devices and those containing citraconylated human serum albumin.

to Tabio 4: Testing H!''-2 Positive Plasma Samples (n=23) -.. ..

10 Minute Result | 20 Minute Result L._. i... _ Control Device Citra HSA | Control Device Citra HSA _ S -. 100% 100% 100%

Therefore from the results of the test set out in the above tables it can be seen that sensitivity was not affected by the inclusion of citraconylated human serum albumin in the HIV assay.

In total 638 negative samples, serum and plasma, were tested on both control devices and those containing the test according to the invention. A summary of the

results obtained are outlined in Table 5. On reading at 10 minutes 95.3% specificity

was recorded on testing control devices which decreased to 92.63% at 20 minutes.

On comparing this result to that obtained on testing devices according to the invention containing citraconylated human serum albumin, 99.4% at 10 minutes and 98.9% at 20 minutes, it can be clearly seen that the addition of citraconylated human 5 serum albumin greatly increased the specificity of the HIV assay.

Table 5: Testing HIV Negative Samples | 10 Minute Result 0 Minute Result LControl Device | Citra HSA Control Device Citra HSA 1 -.... .

% Specificity I 95.3% 99.4% 92.63% 98.9% _, 11. _. __

Accordingly, the addition of citraconylated human serum albumin has been shown to I0 increase specificity while at the same time conserving sensitivity of the HIV test. An additional benefit is the increase in the stability of a negative signal up to 20 minutes compared with the control HIV test.

It has been found that reacting the citraconic anhydride with the amine groups of the is human serum albumin at high pH results in the formation of amide linkages with a terminal carboxylate. This results in a strong negative charge being imparted to the human serum albumin as each positively charged amino group becomes negatively charged when modified. The efficiency of amine modification was measured which indicated that 75% of amine groups in human serum albumin had been modified.

Claims

Claims

1. A test for the detection of antibodies to HIV comprising recombinant HIV antigens and albumin modified by reacting the albumin with citraconic anhydride.

5 2. A test as claimed in Claim 1 in which the albumin is human serum albumin.

3. A test as claimed in Claim 1 or 2 in which the recombinant HIV antigen comprises a modified recombinant protein.

1o 4. A test as claimed in Claim 2 or 3 in which the modified protein of the recombinant HIV antigen is modified by reacting the protein with citraconic anhydride. 5. A test as claimed in any of Claims 2 to 4 in which the human serum albumin 15 is modified by reacting the human serum albumin with dicarboxylic anhydride.

6. A test as claimed in Claim 5 in which the human serum albumin is modified at amine groups with the dicarboxylic anhydride.

20 7. A test as claimed in any preceding claim in which the reaction is carried out at a pH sufficient for the formation of amide linkages with a terminal carboxylate.

8. A test as claimed in Claim 7 in which the pH of the initial reaction buffer is 11.5 before addition of citraconic anhydride.

9. A test as claimed in any preceding claim in which the test the reagents are impregnated on a sheet of material.

s 10. A test as claimed in Claim 9 in which the sheet of material is a glass fibre sheet. 11. A test as claimed in any preceding claim in which the recombinant HIV antigen comprises a modified recombinant protein, which has been modified with lo citraconic anhydride.

12. A test as claimed in any preceding claim in which the modified human serum albumin is mixed with the recombinant HIV gold conjugate in the ratio 0.5mg modified human serum albumin to 1 ml of impregnation solution.

13. A test for the detection of antibodies to HIV, the test being substantially as described herein with reference to the examples.

14. A method for preparing a test for detecting antibodies to HIV, the method 20 comprising modifying albumin by reacting the albumin with citraconic anhydride and mixing the modified albumin with a recombinant HIV antigen to form the test.

15. A method as claimed in Claim 14 in which the albumin is human serum albumin and is modified by reacting protein of the albumin with a dicarboxylic

anhydride. 16. A method as claimed in Claim 15 in which the reaction of the protein with the dicarboxylic anhydride is carried out at a pH sufficient to form amide linkages with a s terminal carboxylate.

17. A method as claimed in any of Claims 14 to 16 in which the pH of the reaction mixture is adjusted to pH 11.5 before the addition of citraconic anhydride.

In 18. A method as claimed in any of Claims 14 to 17 in which the reacted solution is dialysed.

19. A method as claimed in any of Claims 14 to 18 in which the test is impregnated in a sheet of material.

20. A method as claimed in Claim 19 in which the test is impregnated in a sheet of fibre glass material.

21. A method for preparing a test for detecting antibodies to HIV, the method 20 being substantially as described herein with reference to the accompanying examples.