|Year : 2002 | Volume
| Issue : 2 | Page : 102-104
Evaluation of two commercially available anti human immunodeficiency virus antibody Elisa Kits using clinical samples
K Anuradha, S PD Ponamgi, V Lakshmi
Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, India
Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, India
Laboratory diagnosis is the mainstay in the diagnosis of HIV infection in an individual. The wide range of diagnostic kits available, for the detection of anti HIV antibodies, makes it mandatory that the kits are evaluated before they are made available in the market. We evaluated the performance of a Microlisa HIV kit (J.Mitra & Co.)† using a panel of HIV reactive and non reactive clinical specimens. The sensitivity and specificity of the kit were found to be 100% as compared to the UBI HIV 1/2 EIA kit. The ease of testing, the assay characteristics including efficiency of the Microlisa favour its use by laboratories as a primary screen for HIV infection.
|How to cite this article:|
Anuradha K, Ponamgi S P, Lakshmi V. Evaluation of two commercially available anti human immunodeficiency virus antibody Elisa Kits using clinical samples. Indian J Med Microbiol 2002;20:102-4
|How to cite this URL:|
Anuradha K, Ponamgi S P, Lakshmi V. Evaluation of two commercially available anti human immunodeficiency virus antibody Elisa Kits using clinical samples. Indian J Med Microbiol [serial online] 2002 [cited 2014 Apr 17];20:102-4. Available from: http://www.ijmm.org/text.asp?2002/20/2/102/8360
Laboratory diagnosis is the only method of determining the status of Human Immunodeficiency Virus (HIV) infection in an individual. The Enzyme Linked Immunosorbant assay (ELISA) continues to be the recommended primary screening method for the detection of antibodies to HIV. High standards and quality must be maintained and regularly monitored by the laboratory in screening an individual for HIV infection.
The first generation ELISAs, using viral lysate as the antigen ligand, have now been replaced by the second and the third generation assays. The antigen used in these kits is recombinant antigen or synthetic peptides of the immunodominant epitopes of the HIV virus, respectively.
Though the later generation kits have a high sensitivity and specificity, these parameters are not absolute and inherent problems of false negative and false positive results are inevitable. Hence, periodic evaluation of HIV kits is a pre-requisite of a good quality management.
A large number of new HIV kits are continuously introduced commercially. It is mandatory that the new kits be evaluated for their performance characters, before they are released into the market. The evaluation must be done with an established and FDA approved kit with high performance standards. The test conditions and the panel of sera used in both the kits must be the same to assess the efficiency of the new test kit. The discordant results, if any, should be further validated by a reference test used as a gold standard.
Recently, M/S J.Mitra & Company Ltd., New Delhi, India, have developed an indigenous HIV 1/2 ELISA (Microlisa) kit to detect anti HIV antibodies. In this study, the performance of this kit was evaluated against the FDA approved UBI HIV 1/2 (UBI HIV 1/2 EIA, Beijing). [Table - 1]
| ~ Materials and Methods|| |
Description of the Kits:
Standard kit: The UBI HIV Kit is a solid-phase indirect immunoassay which employs synthetic HIV peptides for the detection of anti HIV-1 and HIV-2 antibodies in human serum or plasma.
Test kit: Microlisa HIV kit is an indirect enzyme immunoassay. The antigen used is a complex of recombinant proteins, representing immunodominant epitopes, gp 41, C terminus of gp 120 of HIV-1 (including Subgroups O & C) and gp 36 of HIV-2.
In both the assays, the enzyme-conjugate used was horseradish peroxidase - conjugated to goat anti-human IgG antibodies. The substrate solution in UBI was O-Phenylene Diamine (OPD) and in Microlisa was Tetramethyl Benzidine (TMB) containing hydrogen peroxide. The optical density (OD) of the color developed by enzyme substrate reaction was read at a wavelength of 492 nm and 450 nm respectively.
Reference Gold Standard: Western Blot (LAV Blot HIV 1, Bio - Rad, France) using electrophoresed HIV 1 proteins blotted on Nitrocellulose membrane.
Composition of test serum panel:
The serum panel included 100 HIV 1/2 ELISA reactive and 300 HIV 1/2 non reactive serum samples, obtained from patients attending our hospital.
Control serum panel:
Two types of control panels were used, such as (a) Internal Control serum panel, and (b) External control serum panel. Internal Control serum panel consisted of positive and negative sera supplied along with the kits. They were run simultaneously and under the same conditions as the serum panel as per the kit instructions. These were essential to validate the quality of each run and to calculate the cut off points. Four external control sera were included with each test run to monitor consistent technical performance and any variation with each run that may not be detected using internal controls. The four external controls used were:
1. Highly reactive serum: A Western Blot confirmed, UBI HIV 1/2 reactive serum with high OD of > 3 at 492 nm absorbance.
2. Borderline Reactive serum: Western Blot confirmed, UBI HIV 1/2 ELISA reactive serum with an OD of 1.2 - 1.5 at 492 nm absorbance. This control was included to detect any minor variations in the cutoff OD values. Such a variation would influence the interpretation of the results of unknown samples with OD values near the cutoff.
3. Negative serum: HIV 1/2 ELISA Non reactive serum sample.
Standardization of the quality of the External control sera:,
The working dilution of the Western Blot confirmed, borderline reactive control was standardized by intra-run and inter-run reproducibility, as per the NACO protocol. This was done to overcome any false results by over / under dilution of the serum. Both the UBI and the Microlisa assays were carried out simultaneously under identical conditions, by the same person in the same laboratory, using the same pipettes and testing protocols as per the kit insert. In the UBI kit, all the controls were run in duplicate, except the internal negative control, which was run in triplicate. In the Microlisa kit, the positive internal control was run in 4 wells and the negative control in triplicate. The external controls were run in duplicate, as for the UBI kit. A blank well was included in position A1 in the entire assay runs in both the kits.
In all, five test runs were performed using 100 HIV 1/2 reactive and 300 nonreactive sera. In each run 80 test samples (20 reactive and 60 nonreactive) were tested by both the assays. The cutoff values were calculated as per the kit instructions. A test sample with discrepant results was retested in duplicate by another person using new wells of both the assay kits. Also, it was tested by a 3rd ELISA Detect HIV (Biochem ImmunoSystems, Montreal, Canada), HIV Tridot (J.Mitra & Co.Ltd, New Delhi, India) and by the Western Blot assay.
| ~ Results|| |
The cutoff was calculated for each assay and all the test runs met the validation criteria using the ODs of the internal controls. Any test sample with an absorbance OD less than the cutoff was considered nonreactive for HIV. Test samples were considered reactive if the OD was more than the cutoff. The results of the external controls (highly reactive, borderline reactive, nonreactive), 99 reactive and 300 nonreactive test sera matched in both the assays, with a 100% correlation. The ODs of all the controls were consistently same and reproducible.
The remaining serum sample was reactive by UBI and nonreactive by Microlisa. Retesting of this test sample with discrepant results gave a consistently high OD with UBI but was found nonreactive with Detect HIV, Microlisa and HIV Tridot and was also negative by Western Blot. The table shows the comparative test results and the performance characteristics of both the assays. The performance characters were calculated using Western Blot test results as Gold Standard for comparison. The one false reactive result in the UBI kit, was probably due to cross-reacting autoantibodies (patient was a proven case of Systemic Lupus Erythematosus). However, such false positive results are acceptable when large populations are being screened and can be reconfirmed using confirmatory tests.
| ~ Discussion|| |
Infection with human immunodeficiency virus, which causes AIDS, has become a worldwide epidemic since its first documentation in 1981, and it is a major public health concern for all countries. Diagnosis of HIV infection is important for prevention and patient management. Laboratory diagnosis is the mainstay in identifying HIV infection in an individual. The wide range of diagnostic kits available from both the international and local diagnostic manufacturers, for the detection of anti HIV antibodies, makes it mandatory that the kits are evaluated before they are made available in the market and put to routine use by the laboratories.
An ideal assay should be inexpensive, highly sensitive and specific, easy to perform with a short assay performance time, with a long shelf - life of the reagents and not require additional or sophisticated equipment.8 The test performances of both the kits tested in this study were found to be satisfactory and correlated well with the confirmatory Western Blot test.
The indigenous Microlisa kit, developed in India by M/S J.Mitra & Co. Ltd., performed well with sensitivity, specificity and efficiency, using clinical samples, being 100%. The ease of testing, the assay characteristics including efficiency of the Microlisa favor its use by laboratories as a primary screen for HIV infection.
| ~ References|| |
|1.||U Baveja. HIV antibody testing with special reference to HIV-1. In HIV testing manual: Laboratory diagnosis, biosafety and quality control, National AIDS Control Organization, Ministry of Health and Family Welfare, Govt. of India. 45-67. |
|2.||U Baveja, D.Chattopadhyaya and RK Aggarwal. Evaluation of HIV kits and preparation of serum panel. In HIV testing manual: Laboratory diagnosis, biosafety and quality control, National AIDS Control Organization, Ministry of Health and Family Welfare, Govt. of India. 107-113. |
|3.||Direction insert, UBI HIV 1/2 EIA, Beijing United Biomedical Company LTD, Beijing, PR China. |
|4.||Instruction manual, Microlisa, J Mitra & Co. LTD., New Delhi, India. |
|5.||Product literature. New Lav Blot -1 and 2, Bio - Rad, France. |
|6.||P Seth. Quality assurance programme. In HIV testing manual: Laboratory diagnosis, biosafety and quality control, National AIDS Control Organization, Ministry of Health and Family Welfare, Govt. of India. 92-106. |
|7.||R Kannangai, S Ramalingam, SP Kumar, K Damodharan, G Sridharan. Hospital-based evaluation of two rapid human immunodeficiency virus antibody screening tests. Journal of Clinical Microbiology. 2000; 38:3445-3447. |
|8.||AA Raj, R Kannangai, DP Daniel, PG Babu, S Ramalingam. Evaluation of commercial microwell ELISA and microparticle EIA for the detection of HIV antibody. Indian J Med Microbiol 2000; 18 (1): 21-24. |