Indian Journal of Medical Microbiology, (2002) 20 (4):200-205 Original Article EVALUATION OF AN INDIGENOUS WESTERN BLOT KIT FOR HUMAN IMMUNODEFICIENCY VIRUS *V Lakshmi, SPD Ponamgi Abstract Purpose: The Western Blot test is considered a gold standard test for the confirmation of an ELISA and/ or rapid assay screened reactive sample in the diagnosis of HIV infection, especially in the low risk population. In this study, an indigenously developed HIV W. Blot kit (J.Mitra & Co., New Delhi, India) was compared for its performance characteristics with a widely used Western Blot kit, HIV Blot 2.2 (Genelabs, Singapore). Antigens of both HIV-1 and the indicator antigen gp36 of HIV-2 are included in the strips. Methods: A panel of 150 clinical serum samples was used in the evaluation. All the sera were tested simultaneously by both the kits. Results: The HIV W. Blot kit had high performance characteristics (100% sensitivity and 100% specificity), like the HIV Blot 2.2. The test procedure was easy to perform. There was clear delineation of the bands. Conclusions: The interpretation of the results on the HIV W. Blot was less prone to subjective errors. The test gave positive bands at even very high serum dilutions in the test kit. This fact indicates that HIV W. Blot probably has a potential application in early phases of infection, when the antibody concentrations are still very low. Key Words: HIV W. Blot, indeterminate, intensity, serum dilution Laboratory assessment and serological diagnosis and confirmation is the only method of identifying Human Immunodeficiency Virus (HIV) infected patient. A well established, reference gold standard test must be available to the laboratory, the results of which should correlate with the actual status of the patient. 1 Essentially, any repeatedly positive result by Enzyme Linked Immuno Sorbent Assay (ELISA) or another rapid screening method for HIV must be confirmed by a more specific assay such as Western Blot (WB) test. The current strategy for a serological confirmation of infection with HIV is the WB assay 2 especially in low risk groups. The WB detects antibodies to specific denatured HIV-1 proteins including core (p17, p24, p39 and p55), polymerase (p31, p51, p66), and envelope (gp41, gp120 and gp160) proteins using the principle of an enzyme immunoassay. 3-5 At present, there are several commercially available WB kits in the international market, that can confirm an HIV infection (HIV 1 &/or 2). The objective of the present study was to evaluate the performance characteristics of an indigenously developed WB assay for HIV-1 (HIV W. Blot) from *Corresponding author Department of Microbiology, Nizam s Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Andhra Pradesh, India. Received : 22-06-2002 Accepted : 25-07-2002 J.Mitra and Co. Ltd., New Delhi with the FDA approved kit (HIV Blot 2.2) of Genelabs, Singapore. The authors have no financial interest in either of the kits. Materials and Methods 150-serum specimens (serum panel) from the Department of Microbiology, Nizam s Institute of Medical Sciences were included in the study. The serum panel included is shown in Table 1. The sera were stored at - 20 0 C until tested. Table 1 : Description of the serum panel used in the study Panel type ELISA result No. of samples HIV 1 Reactive 50 Reactive HIV 2 Reactive 10 HIV 1 & 2 Reactive 10 Non Reactive HIV 1 & 2 Non Reactive 50 Indeterminate HIV ELISA Reactive & WB (Blot 2.2) Indeterminate 10 HIV ELISA Non Reactive & WB (Blot 2.2) Indeterminate 13 Weakly HIV ELISA Reactive Reactive with low OD 7 Differentiation between HIV-1 & 2 was done by DOT blot assay (HIV TRIDOT, J.Mitra & Co. Ltd., New Delhi).
October, 2002 Lakshmi & Ponamgi - Indigenous Western Blot Kit for HIV 201 The WB kits used in the study were essentially manufactured by a similar process, using partially purified HIV-1 viral antigen. The antigen used in the W. Blot was produced from HIV-1 cell line, which was developed from an Indian strain, HIV-1 subtype C. The antigens were then electrophoresed and electrotransblotted on to a nitrocellulose paper. The Synthetic antigen of HIV-2 (gp36) was applied as a line blot on the strip. 6,7 The antigen band patterns in each type of kit is shown in (Table 2). An internal or inbuilt control band (serum control) was also incorporated in each of the strips. This was to ensure quality of the test procedure. The conjugate used in both kits is an antihuman IgG (raised in goat in HIV Blot 2.2 and rabbit in HIV W. Blot) conjugated with alkaline phosphatase, with its specific substrate 5 bromo, 4 chloro, 3 indolyl phosphate + nitroblue tetrazolium (BCIP / NBT). The test duration for the kits is either 1 hour (HIV Blot 2.2) or 2 hours (HIV W. Blot) or overnight (both kits) for sample incubation, 1-hour conjugate and 5-15 minutes of substrate incubation in both the test kits. Depending on color development of the bands the last step should be performed. 6,7 Table 2 : Antigen profile in the Western Blot kits HIV Gene Molecular HIV Blot 2.2 HIV W. Blot Weight (Gene labs) (J.Mitra & Co.) HIV 1 Gp160 3 3 Envelope (env) Gp120 3 3 Gp41 3 3 P66 3 3 Polymerase (pol) P51 3 3 P31 3 3 P55 3 3 P39 3 5 Core (gag) P24 3 3 P17 3 5 HIV 2 Gp36 Envelope (synthetic 3 3 (indicator) peptide) Serum Anti IgG 3 3 Control Band band Both kits provide for the detection of HIV Sub group O All the 150 sera were tested by both the WB kits. The tests were performed as per the manufacturers instructions 6,7 and were performed under identical conditions by the same investigator, using the overnight procedure. The readings were taken visually and the reactive indices were calculated as given in the kit literature of HIV Blot 2.2. Results All the tests were validated by the presence of the serum control band. Positive WB Positive WB was considered by the presence of atleast two of the three bands representing the envelope bands (gp160, gp120, and gp41 of HIV-1 and gp36 of HIV-2) and gag gene p24. 8 The WB results of 47/50 of the ELISA HIV-1 reactive samples were similar by both the kits. Three sera had discrepant results at the minor band positions of p31, p24, and p51/55. Since the reactive indices for these sera were >2.5 in both the strips, they were considered positive. The HIV W. Blot was able to clearly detect the HIV- 2 indicator band in all the 10 sera as detected by HIV BLOT 2.2. HIV W. Blot detected 10 HIV 1 + 2 (dual reactive) samples detected by Blot 2.2. The antigen band patterns were correlating in both the kits in all these samples. Negative WB Absence of all the bands except control band was considered as negative test. Presence of any nonspecific band or isolated p17 in HIV Blot 2.2 and p55 in HIV W. Blot was considered negative. 6,7 Fifty out of 63 ELISA HIV-1 & 2 non-reactive samples were negative by both the WB kits. These blots had no bands corresponding to the HIV antigens. Indeterminate WB Westernblot results that could not be classified as negative or positive were categorized as indeterminate. 8 The HIV W. Blot interpreted 10 ELISA HIV-1 and 2 reactive sera that gave indeterminate result in HIV Blot 2.2 also as indeterminate. The bands obtained were also the same. WB patterns of the remaining 13/63 ELISA HIV-1 and 2 non-reactive panel showed an indeterminate result (Table 3). Eight of these samples were from patients with autoimmune disorders and the rest five were from
202 Indian Journal of Medical Microbiology Vol.20, No.4 patients with other viral infections. While 4/13 of the ELISA non-reactive sera were indeterminate in HIV W. Blot, all (13/13) these sera showed indeterminate results with HIV Blot 2.2 (reactive indices in between 0.25 and 2.5) in our study. Isolated positive band at antigen sites like gp160 (n=2), gp120 (n=1), gp41 (n=2), p31 (n=3) and p24 (n=3) and gp41 + p31 bands were observed (n=2) in HIV Blot 2.2. Further the three samples that showed a p24 band were tested for p24 antigen by ELISA (J&J) and were found negative. Table 3 : Results of ELISA non reactive indeterminate panel Sample Test Core antigens Endonuclease Envelope antigens Reactive Index no Kit (GAG) polymerase antigens (POL) (ENV) Value P 24 P 55 P 31 P 51 P 66 Gp 41 Gp 120 Gp 160 1* Blot 2.2 - - - - - + - - 1.00 2* Blot 2.2 - - - - - - - + 1.00 3* Blot 2.2 - - + - - + - - 1.50 4* Blot 2.2 - - - - - - + - 1.00 5* Blot 2.2 - - + - - - - - 0.50 6* Blot 2.2 - - + - - - - - 0.50 7# Blot 2.2 - - + - - + - - 1.50 8* Blot 2.2 - - - - - - - + 1.00 9* Blot 2.2 - - + - - - - - 0.50 10# Blot 2.2 - - - - - + - - 1.00 11# Blot 2.2 + - - - - - - - 0.50 12# Blot 2.2 + - - - - - - - 0.50 13# Blot 2.2 + - - - - - - - 0.50 Samples * - from patients with autoimmune disorders; Sample # - from patients with other viral infections. Staining Intensity To evaluate the intensity of the staining of the antigen bands, seven reactive serum samples that gave a weak OD (within the gray zone of the test run) in the ELISA at 1:100 dilution were used. The two WBs were then performed using 1:100 and 1:1000 dilutions of these sera. In the WB procedure these sera underwent a further 1000 fold dilution. Thus at a final dilution of 1:10 5 and 1:10 6 in the WB, all the seven sera gave a positive result by the HIV W. Blot while in the HIV Blot 2.2 they gave an indeterminate result (figure).
October, 2002 Lakshmi & Ponamgi - Indigenous Western Blot Kit for HIV 203 gp 160 +120 1:10 5 1:10 6 centers interpret the results as per the recommendations of the CDC, ASTPHLD or WHO (Table 4). The same guidelines have been recommended by NACO, India. 11 We have applied the same recommendations in the interpretation of the WB results in this study. The results were also interpreted based on the reactive indices as indicated in the kit literature of Genelabs HIV Blot 2.2. gp 41 p 24 Figure : Comparison of HIV Blot 2.2 and HIV W. Blot. Lanes 1, 3 - HIV Blot 2.2; lanes 2, 4 - HIV W. Blot Discussion The development, introduction, and use of the newly arriving HIV antibody detection assays are a dynamic process. It is essential that these assays are thoroughly evaluated before use at different levels and conditions. The sensitivity, specificity, predictive values, false positive ratio, ease of performance and interpretation, suitability for use in small collection centers and the test efficiency need to be evaluated using different combinations of assays. 9 The recommended HIV-1 testing algorithm comprises of an initial screening with an approved enzyme linked immunoassay (ELISA). The repeatedly reactive ELISAs are confirmed with a licensed supplemental test. 5 Because of the distinct staining patterns of polypeptides, WB test is considered a gold standard test for the confirmation of an HIV reactive sample. 11 Although the overall sensitivity and specificity of the WB for detection of antibodies to the various viral proteins are high, there has been substantial debate regarding the interpretive criteria. This is because, the final result and the interpretation depend on the several types of antigen ligands (lysate, recombinant or synthetic) that are used for blotting. As a result, the definition of what should constitute a positive WB has been subjected to a considerable debate. Various regulatory bodies have given the specific criteria for the interpretation of a positive WB result. Majority of the Table 4 : Criteria for interpretation of Western Blot 6,7 Regulatory bodies CRTS (Center Nationale Transfusion Sanguine) CDC (Center for Disease Control) ASTPHLD (Association of State and Territorial Public Health Laboratory Directors) WHO (World Health Organisation) NACO (National Aids Control Organisation) Antigen bands Two ENV (2) with GAG or POL. At least one ENV(gp41 & gp120/160) & p24 At least one ENV (GP41 & GP120/160) & p24 Two ENV bands with or without GAG or POL Two ENV bands with or without GAG or POL The sensitivity of the HIV W. Blot as compared with the ELISA or the HIV Blot 2.2 was 100%, as all the 50 HIV-1 ELISA reactives, were positive by both kits. A further measure of the sensitivity of the kit under evaluation would be to use a weak reactive serum. However, no sero-conversion panels were available with us nor did we get any opportunity to identify a weak reactive sample. The criteria for a negative WB interpretation specify absence of antigen bands of diagnostic significance. This interpretation is essential because some observed bands may reflect the presence of antibodies to HIV regulatory proteins or may indicate partially processed or degraded viral structural proteins. Furthermore, different WBs (commercial, as well as in-house preparations) and different virus-antigen preparations used to prepare WBs may contain different numbers and concentrations of both viral-specific and contaminating cellular proteins that may have unpredictable molecular weights. Presence of such non-specific bands is not taken into consideration and the result is reported negative. The indeterminate WB results are observed in healthy persons with antibodies that cross-react with 12, 13 specific HIV-1 peptides or recombinant antigens. The proportion of indeterminate results also varies according to the immunoblots used, the prevalence of
204 Indian Journal of Medical Microbiology Vol.20, No.4 HIV infection in the population tested, and the interpretive criteria used. 14 While 4/13 of the ELISA non-reactive sera were indeterminate in HIV W. Blot (Table 3), all (13/13) these sera showed indeterminate results with HIV Blot 2.2 (reactive indices in between 0.25 and 2.5) in our study. The bands were probably due to cross-reactive antibodies 2 as all these samples were from patients with autoimmune diseases and other viral infections. As they were ELISA non reactive all these sera were interpreted as negative for HIV-1 and 2 antibodies. Thus, the specificity of the HIV W. Blot against the HIV Blot 2.2 was found to be 100% with no false positives (All the 63 ELISA non reactive were interpreted as negative). This strategy of using both ELISA and supplementary tests further increases the accuracy of results and diagnosis. In principle, any WB kit that gives a high frequency of indeterminate reactivity (the overwhelming preponderance of which represents nonspecific binding) is not appropriate as a primary screening tool for the population at large. Its strength is only as a confirmatory assay in the setting of a positive or indeterminate HIV-1 ELISA or initial screening test. 15 The same has been shown in our results using ELISA non reactive serum samples where we obtained indeterminate results on WB. Hence, like any other HIV WB test, the HIV W. Blot is not to be used for primary screening for HIV antibodies. The intensity of staining of the bands is a valuable measure of standardization of any WB. 16 Since in majority of the laboratories the results of the WB are read visually, there is always a high probability of subjective errors. Higher the intensity of the bands lesser is the visual error. 16 We attempted to evaluate the same feature in both the WB kits by using serial dilution of ELISA HIV-1 reactive serum samples. The results indicate that the HIV W. Blot has a higher staining intensity than the HIV Blot 2.2 at higher dilutions of the antibodies. Hence there is a lesser probability of false negative interpretation with the HIV W. Blot even at lower concentrations of the antibodies. Charles T Hardy, using a scanning densitometry in the reflectance mode, did objective analysis of such a finding. 16 Based on the results in this study, we conclude that the indigenously developed HIV W. Blot kit had high performance characteristics like the widely used HIV Blot 2.2 of Genelabs. The test procedure is easy to adopt. With clear delineation of the bands, the interpretation of the results on the HIV W. Blot is less prone to subjective errors. The fact that the HIV W. Blot gave a positive result even at very high serum dilutions indicates that it probably has a potential application in early phases of infection, when the antibody concentrations are still very low. References 1. Baveja U, Chattopadhya D, Aggarwal S. Evaluation of HIV kits and preparation of serum panels, Chapter 11. In: HIV testing manual, laboratory diagnosis, biosafety and quality control. National Institute of Communicable Diseases and National AIDS Control Organisation, Ministry of Health and Family Welfare, Govt. of India New Delhi, 107-113. 2. Kline RL, McNairn D, Holodniy M, Mole L, Margolis D, Blattner W, Quinn T C. Evaluation of Chiron HIV-1/HIV-2 recombinant Immunoblots assay. J Clin Microbiol 1996;34:2650-2653. 3. Carlson JR, Bryant ML, Hinrichs SH, et al. AIDS serology testing in low and high risk groups. JAMA 1985;253:3105-3108. 4. Schwartz JS, Dans PE, Einosian BP. Human Immunodeffiency virus test evaluation, performance, and use. Proposals to make good tests better. JAMA 1988;259:2574-2579. 5. CDC Recommendations. Interpretation and use of the Western blot assay for serodiagnosis of Human immunodeficiency virus type 1 infections. MMWR 1989;38:1-7. 6. Instruction manual, Genelabs Diagnostics Pvt. Ltd., 85, Science Park, Drive #04-01, Singapore Science Park, Singapore 118259. 7. Instruction manual, J.Mitra & Co. Ltd., A-180, Okhla Ind. Area, Ph-1, New Delhi- 20 India. 8. Edward B, Susan WB. Human Immunodeficiency Virus. Chapter 97 In: Manual of Clinical Microbiology, 6th ed. Murray PR, Baron ES, Pfaller MA, Tenover FC, Yolken RH, Eds. (ASM Press, Washington DC) 1995:1098-1114. 9. Tegbaru B, Meles H, Fisseha B, Mekonnen Y, Haile H. Evaluation of five commercial assays for detecting HIV 1 and 2 antibodies, Addis Ababa Ethiopia. J Health Dev 1999;13(3):175-180.
October, 2002 Lakshmi & Ponamgi - Indigenous Western Blot Kit for HIV 205 10. Hardy CT. Quantitation of Antibody Reactivity to Human Immunodeffiency Virus (HIV-1) Proteins and glycoproteins on Western Blots by Reflectance Densitometry (Dissertation), Chapter 1. Ph.D. Thesis, University of Washington, Seattle, Washington, 1993. 11. U Baveja. Antibody testing with special reference to HIV-1, Chapter 6. In: HIV testing manual, laboratory diagnosis, biosafety and quality control. National Institute of Communicable Diseases and National AIDS Control Organisation, Ministry of Health and Family Welfare, Govt. of India, New Delhi, 107-113. 12. Bush MP, Amad Z, McHugh TM, et al. Reliable confirmation and quantitation of Human Immunodeficiency Virus type-1 antibody using a recombinant-antigen immunoblot assay. Transfusion 1991;31:129-137. 13. Blomberg J, Vincic E, Cetal J. Identification of regions of HIV-1 p24 reactive with sera which give indeterminate results in electrophoretic immunoblots with the help of long synthetic peptides. AIDS Res Hum Retrovirus 1990;6:1362-1372. 14. Celum CL, Coombs, et al. Risk factors for repeatedly reactive HIV-1 EIA and indeterminate western blots. A population based case control study. Arch Intern Med 1994;154:1129-1137. 15. Julia AM, Richard TD Jr, Clafford HL. Acquired Immunological Syndrome: serological and Virological Tests. Chapter 11. In: AIDS: Biology, Diagnosis Treatment and Prevention. Vincent T Devita, Jr. Samuel Hellman and Steven A. Eds. 4th Ed. (Rosenberg Lippincott Publishers, Philadelphia, USA) 1997:117-195. ooooooooooooooooooooooooooooooooooo oooooooooooooooooooooo INDIAN COUNCIL OF MEDICAL RESEARCH AWARDS AND PRIZES 2001 The Indian Council of Medical Research invites nominations / applications from Indian Scientists for its various prizes and awards in the field of Biomedical Sciences including Dr. B.R. Ambedkar Centenary Award for Excellence in Biomedical Research. Details of the Awards/Prizes and also the format for application may be obtained from the Division of International Health, Indian Council of Medical Research, V. Ramalingaswami Bhawan, Ansari Nagar, Post Box 4911, New Delhi - 110 029. Last date of issue of applications is 30th November, 2002. Last date of submission of completed applications/nominations is 31st December, 2002. Incomplete applications may be summarily rejected. ooooooooooooooooooooooooooooooooooo oooooooooooooooooooooo