SciELO - Scientific Electronic Library Online

vol.21 issue4Echocardiographic parameters related to pulmonary arterial hypertension in patients with chronic myeloid leukemia under dasatinib treatment author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




  • Have no cited articlesCited by SciELO

Related links

  • Have no similar articlesSimilars in SciELO


Revista de la Facultad de Medicina Humana

Print version ISSN 1814-5469On-line version ISSN 2308-0531

Rev. Fac. Med. Hum. vol.21 no.4 Lima Oct./Dec. 2021 

Original article

Standardization and validation of a Western Blot for the diagnosis of human immunodeficiency virus

Eduardo Miranda-Ulloa1  , Biologist, Master in Microbiology.

Soledad Romero-Ruiz1  , Biologist

Bernardina Amorín-Uscata2  , Biologist Master of Science

Kevin Serrano-Segura1  , Biologist

Ronal Briceño-Espinoza1  , Medical technologist

Fany Cárdenas-Bustamante1  , Biologist

1Laboratorio de Referencia Nacional Virus de Transmisión Sexual VIH/SIDA, Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú.

2Instituto de Medicina Tropical de São Paulo, São Paulo, Brasil.



To standardize and validate a western blot test for the diagnosis of human immunodeficiency virus.


A prospective observational study was carried out during 2017 and 2018. The western blot test was standardized, using the polyacrylamide gel electrophoresis technique with sodium dodecyl sulfate (SDS PAGE), being the nitrocellulose blot strips prepared with an Optimal HIV-1 antigen concentration of 2.71 µg / mm. The western blot was validated in the laboratory against 400 reference samples (300 sera and 100 plasmas): 200 positive and 200 negatives for antibodies against HIV-1, being the reference test the Immunoblot of the Fujirebio brand. Diagnostic performance parameters were estimated using Epidat v3.1 and Excel.


Eight important bands of the HIV-1 antigen were identified: p17, p24, p31, p39, gp41, p55, p66, and gp120. According to the Consortium for the normalization of serology for retroviruse, those that were taken as specific diagnostic bands were: p24, p31, gp41, and gp120. The sensitivity, specificity, positive and negative predictive value and validity index against sera were: 96.7%, 96.0%, 96.0%, 96.6%, 96.3%; and against plasmas: 98.0%, 100.0%, 100.0%, 98.0%, 99.0% respectively. No false positives and negatives were found, but some were undetermined.


The development of this western blot test with proprietary technology presented similar diagnostic performance to the reference test, without showing cross-reactions, being useful for confirming HIV.

Keywords: Western Blotting; HIV; Diagnosis; Sensitivity and Specificity (Source: MeSH NLM).


In Peru, within the strategies for the approach to infection by the human immunodeficiency virus (HIV) is the coverage of the diagnosis1, so much so that there are serological screening tests (ELISA, chemiluminescence and rapid tests) and confirmation: indirect immunofluorescence (IIF) and immunoblot (IB) or western blot (WB)2,3. The IIF is a test with its technology4, relatively inexpensive and constitutes 95% of the confirmations at the national level, however when the result is indeterminate or nonspecific, the IB or WB is used for having superior diagnostic performance3.

The commercial IB of the Fujirebio brand INNO-LIA HIV I / II Score (Online Immunoassay: sensitivity 100.0% and specificity 96.7%) is the kit most used as a second opinion test for samples that did not resolve IIF5, being at the same time applied in numerous studies as a gold standard6-10, but its main disadvantage is that its costs are high, approximately $800 for 20 determinations5.

Trademarks available from WB include MP Diagnostics HIV BLOT 2.2 (sensitivity: 100.0%; specificity: 91.9%)11, Biorad NEW LAV-BLOT I (sensitivity: 87.0%; specificity: 99 , 5%)12and Biokit bioblot HIV-1 Plus (sensitivity: 100.0%; specificity: 91.9%)13. Although these three brands do not report false positive and negative results (only indeterminate), their use is very limited due to the expensive prices attributed to their kits (1,000 to 1,200 dollars for 18 determinations)11-13.

The western blot, immunoblot, or immunoblotting is a useful test in identifying antibodies against HIV. It consists of separating the viral antigens by electrophoresis in polyacrylamide gels, then transferred to a nitrocellulose membrane, which will be subsequently exposed with the antibodies of the serum or problem plasma. When they come into contact with an anti-immunoglobulin labeled with an enzyme, they will react immunologically, giving rise to a pattern of bands, which will be interpreted under some of the criteria described by international organizations14.

It should be said that, in Peru, around 1,200 annual samples are resolved by immunoblot/western blot15, whose expenses reach more than 60,000 dollars per year. Therefore, due to the need for a low-cost western blot with our technology and the high prices of commercial kits, we set ourselves the following objective: Standardize and validate a western blot to diagnose human immunodeficiency virus.


Design and study area

Prospective observational study of standardization and validation of a diagnostic test carried out during 2017 and 2018 at the National Institute of Health (INS) of Peru. Under this approach, the design is appropriate to estimate the capacity of a measure that allows discriminating between people with the disease and without the disease.

Population and sample

The population consisted of sera and plasmas that belonged to the serum and plasma library of the National Reference Laboratory of STV-HIV / AIDS of the INS during the years 2016 and 2017. The estimation of the sample size was defined by convenience, using a non-probabilistic sampling, with 400 reference samples: 150 HIV-1 positive sera; 150 HIV-1 negative sera (30 from healthy individuals and 120 from individuals with the following conditions: twenty with syphilis, twenty from pregnant women, twenty with rheumatoid disease (RD), twenty with hepatitis B, twenty with HTLV-1, ten with cytomegalovirus and ten with dengue); 50 HIV-1 positive and 50 negative plasmas. The Reference or Gold Standard test was the commercial IB (INNO-LIA ™ * HIV I / II Score; Fujirebio, Belgium)5.

Variables and instruments

The standardization and validation of a diagnostic test implied considering the following variables: Sensitivity, Specificity, Validity Index, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Youden Index, and Negative Likelihood Ratio. Three stages were previously carried out to measure these variables.

Optimization in the preparation of the HIV-1 antigen. - The cell cultures of the H9 / HTLV-IIIB line infected with HIV-1 were prepared, following the methodology described by Romero -Ruiz et al.16. The cell supernatant was washed and centrifuged three times with 0.9% physiological saline solution (SSF) at 4,400 rpm for 15 minutes at 2 to 8 ° C. The sediment or pellet was then resuspended with SSF. A sonicator was used to carry out the viral and cell lysis at 6 periods of 60 decibels with 2 minutes per period and one minute of rest between periods, maintaining the cold chain (2 to 8 ° C).

The sonicated product was centrifuged at 1,500 rpm for 10 minutes, and its supernatant was registered as HIV-1 antigen. Finally, the Bradford method17was used to quantify the HIV-1 antigen proteins.

Standardization in the preparation of Western blot strips.- This step was standardized following electrophoresis methodology in polyacrylamide gels with sodium dodecyl sulfate (SDS-PAGE)18. The Mini-Protean vertical electrophoresis chamber (Bio-rad) was used for 1.0 mm gels (gel size: 8.3 by 7.3 cm). The percentage of acrylamide was optimized to prepare the resolving gel, establishing two concentrations (mixed) 9% (3.8 cm) and 15% (2 cm). The antigen treatment, separation, and electrophoretic transfer were carried out following the methodology described by Miranda et al.19. Finally, the optimal concentration of the HIV-1 antigen was established to prepare the WB strips, being 2.71 µg / mm.

Optimization in the Immunoenzymatic reaction of the Western blot test.- The optimum volume that was established in all the steps of this stage was 1 mL. The blot strips were incubated for three hours in PBS / tween-milk (PBS / 0.1M NaCl, 0.05 M Na2 PO4, pH 7.2; Tween 20 / 0.3% tween 20; milk / 5% milk) containing the sera or plasmas of the study at a 1/40 dilution. Next, the nitrocellulose strips were washed five times with PBS / Tween-20 and incubated for one hour in PBS / tween-milk containing the conjugate Anti-human IgG linked to a peroxidase, at a dilution of 1/1000. The strips were again washed three times with PBS / Tween-20 and twice with PBS. Bands were developed against a substrate solution (30% hydrogen peroxide at 1uL / mL and diaminobenzidine at 0.5 mg / mL in PBS pH 7.2). The reaction was stopped by washing the strips with distilled water.


Reading, interpretation, and validation of the Western blot test.- For the positivity criterion, the Consortium guidelines for the normalization of retrovirus serologies (CRSS)11-14,20were considered. POSITIVE: A band of p24 or p31 and an ENV band (gp41 or gp120). NEGATIVE: No specific viral band present. UNDETERMINED: Any specific viral band present, but the pattern does not meet the criteria for positive. With these criteria, the WB was validated, performing the readings and interpretations of the 400 reference samples incorporated into the study.

Statistical analysis

The analysis was estimated using a contingency table, the Epidat v3.1 program, and Excel. The percentages of Sensitivity, Specificity, Validity Index, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Youden Index, and Negative Likelihood Ratio were reported, considering a 95% confidence level (95% CI).

Ethical considerations

The Institutional Research Ethics Committee approved the study protocol of the National Institute of Health of Peru with the code: OI-022-14 and with Directorial Resolution N °: 418-2014-DG-OGITT-OPE / INS. Likewise, the General Office for Research and Technology Transfer approved the final report of this study with MEMORANDUM N ° 027-2019-OGITT / INS.


Among our findings, we were able to show eight important bands corresponding to the HIV-1 viral proteins: p17, p24, p31, p39, gp41, p55, p66, and gp120. Of these, those that were taken as specific diagnostic bands according to the CRSS were: p24, p31, gp41, and gp120 (Figure 1).

Figure 1:  Reactivity pattern of the western blot test for the detection of antibodies: (C +1) HIV positive in serum; (C-1) HIV negative in serum; (C +2) HIV positive in plasma; (C-2) negative for HIV in plasma. 

Among other findings, we highlight that against the serum samples, there were eleven indeterminate results (5 from the positive panel and 6 from the negative panel), and against the plasma samples, there was an indeterminate from the positive panel. Said results of the WB test are shown in the flow diagram of samples incorporated into the study (Figure 2).

Figure 2:  The flow of samples incorporated into the study 

An important highlight of this WB test is that it did not show false positives and negatives. However, only some serum samples had indeterminate results: syphilis (2/20), hepatitis B (2/20), ER (1/20), and HTLV-1 (1/20) (Table 1).

Table 1.  Western blot test validation for detection of antibodies against HIV using positive, negative and interfering samples. 

Western blot VIH
Condition Amount True positives False positives True negatives False negatives Indeterminate
VIH-1 150 145 - 0 0 5
Syphilis 20 - 0 18 - 2
Pregnant 20 - 0 20 - 0
RE* 20 - 0 19 - 1
Hepatitis B 20 - 0 18 - 2
HTLV-1 20 - 0 19 - 1
CMV** 10 - 0 10 - 0
Dengue 10 - 0 10 - 0
Healthy 30 - 0 30 - 0
VIH-1 50 49 - 0 0 1
Negative to VIH-1 50 - 0 50 - 0
Total 400 194 0 194 - 12

Hyphen: data does not apply; *Rheumatoid disease; ** Cytomegalovirus

Among the main achievements, we show that the results of the validation parameters (sensitivity, specificity, PPV, NPV and Validity Index) for our WB test were higher than 96.0%. In the same way, the Youden index with the values of 0.93 and 0.98, confirms a minimal possibility of obtaining false positives or false negatives; as well as a low negative likelihood ratio of 0.03 and 0.02, which is consistent with the other results (Table 2).

Table 2.  Western blot test parameters for the detection of antibodies against HIV against serum and plasma samples 

Western blot VIH
Parameters Serum Plasma
IC Value(95%) IC Value(95%)
Sensitivity(%) 96,7 (93,5 - 99,9) 98,0 (93,1-100,0)
Specificity (%) 96,0 (92,5 - 99,5) 100,0 (99,0 - 100,0)
Validity index (%) 96,3 (94,0 - 98,6) 99,0 (96,6 - 100,0)
Positive predictive value (%) 96,0 (92,6 - 99,5) 100,0 (99,0 - 100,0)
Negative predictive value (%) 96,6 (93,4 - 99,9) 98,0 (93,3 - 100,0)
Youden index 0,93 (0,88 - 0,97) 0,98 (0,94 - 1,02)
Negative likelihooh ratio 0,03 (0,01 - 0,08) 0,02 (0,0 - 0,14)

IC 95%: Confidence interval 95%.


The reference test used in the present study is the most used in Peru2,3,6,15, and other countries5,7-10, the insert reports a sensitivity of 100% and specificities of: 96.7% (blood bank) and 96.1% (clinical samples). This kit incorporates recombinant proteins and synthetic peptides from HIV-1 and HIV-25. The sensitivity and specificity of our WB using antigenic proteins from HIV-1 lysis were higher than 96.0%; even so, we did not have false negatives and positives, which allows us to deduce that our results for the same parameters are similar.

Western blot kits NEW LAV BLOT I (Biorad)12and bioblot HIV-1 plus (Biokit)13using the CRSS positivity criteria, report sensitivities of 99.5% and 94.9% and specificities 87.0% and 91.9%, respectively. At the same time, they report that they did not have false negatives and positives, only indeterminate12,13. Likewise, the Immunoblot Recom Line HIV-1 & HIV-2 IgG kit (Mikrogen diagnostik)21, reports a sensitivity of 100% and specificities of: 99.3% (blood bank), 98.5% (samples clinical) and 96.4% (interfering). Our WB test for the same parameters showed results comparable to the three western blot / Immunoblot brands described.

Due to the indeterminate results obtained, a sensitivity of 100.0% was not achieved; This could be due to the fact that the antibodies against p24 and p31 decrease during the course in the AIDS phase, which causes a displacement of the interpretation from positive to indeterminate11; however, we do not know if the samples came from patients in this phase.

To establish specificity (sera), indeterminate results represented 4.0% (6/150); It should be said that, for the calculations in other validation studies, indeterminates are not considered in the category of false positives22, being this way, we would have a global specificity of 100.0%, in the 144 samples (144/144). In general, the indeterminate (12/400) that we obtained (Table 1) does not affect the diagnostic efficiency of our WB since the Peruvian technical standard indicates to perform the HIV-1 viral RNA PCR test (viral load) on the indeterminate or proviral-HIV-1 DNA PCR(2).

Our standardized and validated test followed a WB design developed for parasites, which had excellent results in Peru19,23-25. The different commercial brands of immunoblot/western blot for HIV use a conjugate containing the enzyme alkaline phosphatase, the substrate 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium5,11-13,21. While in our WB we used a conjugate containing peroxidase, the substrate hydrogen peroxide and diaminobenzidine as a chromogen, similar to these reagents they were used in the WB DAVIH-BLOT kit from Cuba, which showed good diagnostic performance against samples serum, urine and oral fluid26.

In addition, the strip size format and the volume to be used are similar to the Reference test5, while the other commercial brands use larger blot strips and consequently require double the volume of reagents. The aforementioned conditions are important to highlight because our methodology saves, since it uses fewer reagents. At the same time, we prepare our viral antigen, having the advantage that the INS has a Biosafety Level III Laboratory, where it is performing the maintenance and development of cell cultures of the H9 / HTLV-IIIB line infected with HIV-1. Consequently, our WB test would cost up to ten times less than commercial kits.

The WB test was standardized and validated in the laboratory, being a limitation of the study in completing its validation in the field. It can be implemented in the reference laboratories of Peru.


The results of the parameters obtained in the WB test for the detection of antibodies against HIV qualify it as a test of good diagnostic performance and make it useful for serological confirmation. Consequently, we recommend its use in the National Reference Laboratory of the INS Sexually Transmitted Virus Test as an alternative assay to commercial confirmatory tests in addition to significantly reducing costs in HIV confirmation.


1. Garcia-Fernandez L, Novoa Rommy, Huaman Byelca, Benites Carlos. Continuo de la atención de personas que viven con VIH y brechas para el logro de las metas 90-90-90 en Perú. Rev. perú. med. exp. salud pública. 2018 Jul; 35(3): 491-496. Disponible en: [ Links ]

2. Norma técnica de salud de atención integral del adulto con infección por el Virus de la Inmunodeficiencia Humana (VIH). NTS N° 169- MINSA/2020/DGIESP (Resolución Ministerial N°:1024-2020/MINSA) Ministerio de Salud, Perú, [Internet]. 2020. [citado el 8 de junio del 2021]. Disponible en: ]

3. Álvarez-Carrasco RI. Interpretación de las pruebas usadas para diagnosticar la infección por virus de la inmunodeficiencia humana. Acta Med Peru. 2017; 34 (4):309-16. Disponible en: ]

4. Ministerio de Salud. Manual de procedimientos para el diagnóstico del virus de la inmunodeficiencia humana tipo 1 (VIH 1) por inmunofluorescencia indirecta. Serie de Normas Técnicas N° 29. Lima, Perú: Instituto Nacional de Salud; [Internet]. 2001 [citado el 8 de junio del 2021]. Disponible en: ]

5. Fujirebio. INNO-LIA(tm)* HIV I/II Score [Internet]. Gante, Bélgica: INNOGENETICS.N.V. 2017 [citado el 8 de junio del 2021]. Disponible en: ]

6. Miranda-Ulloa E, Romero-Ruiz S, Briceño-Espinoza R, Santos-Anaya D. Evaluación de tres marcas comerciales de pruebas rápidas para la detección de VIH. Rev. perú. med. exp. Salud pública. 2015 Abr; 32(2): 404-405. Disponible en: [ Links ]

7. Mor O, Mileguir, F, Michaeli, M, Levy, I, Mendelson E. Evaluación del ensayo bio-rad geenius HIV 1/2 como alternativa al ensayo INNO-LIA HIV 1/2 para confirmar la infección por VIH. Journal of Clinical Microbiology. 2014; 52 (7), págs. 2677-2679. Disponible en: ]

8. Demir T, Yildiran D, Kiliç S. Experiencia del Centro Nacional de Referencia sobre VIH-SIDA de Turquía, en la transición al nuevo algoritmo de diagnóstico del VIH; Análisis comparativo de la prueba de inmunoensayo en línea y el ensayo confirmatorio de anticuerpos del VIH-1/2 de Bio-Rad Geenius (tm). Mikrobiyol Bul. 2021 Jan; 55(1):17-29. Disponible en: ]

9. Wong, C.C., Lim, S.H., Tan, C.T., Lui, S.Y., Lee, Y.L., Chan, K.P. Performance of the HIV Blot 2.2, INNO-LIA HIV I/II score, and geenius HIV 1/2 confirmatory assay for use in HIV confirmation. 2018; PLoS ONE, 13 (6). Disponible en: ]

10. Barros Liñan, E. Evaluación de las pruebas confirmatorias disponibles en Colombia para el diagnóstico de la infección por VIH-1: Western blot e inmunoblots de peptidos sintéticos y proteínas recombinantes. [Internet]. 2015 [citado el 8 de junio del 2021]; Universidad Nacional de Colombia Sede Bogotá Facultad de Medicina. Disponible en: ]

11. MP Diagnostics. HIV BLOT 2.2. MP Biomedicals Asia Pacific Pte Ltd. Singapore. [Internet]. 2016 [citado el 8 de junio del 2021]. Disponible en: ]

12. Biorad. NEW LAV BLOT I. Equipo de confirmación para la detección de anticuerpos anti-HIV-1 en suero/plasma mediante inmunotransferencia. [Internet]. Francia, 2009. [citado el 8 de junio del 2021]. Disponible en: ]

13. Biokit. bioblot HIV-1 Plus. [Internet]. Barcelona, España. 2012 [citado el 8 de junio del 2021]. Disponible en: ]

14. Sigifredo-ospina O. Diagnóstico de la infección por el virus de la inmunodeficiencia humana. Infectio. 2006; 10(4): 273-278. Disponible en: ]

15. Ministerio de Salud. Instituto Nacional de Salud (INS). Anuario Estadístico 2019. Lima, Perú. Enero, [Internet]. 2020 [citado el 8 de junio del 2021]; 308 p. Disponible en: ]

16. Romero-Ruiz S, Miranda-Ulloa E, Briceño-Espinoza R. Rendimiento diagnóstico de la prueba de inmunofluorescencia indirecta para la detección de anticuerpos contra HTLV-1. Rev. Perú. Med. Exp. Salud Pública. Set 2017; 34 (3): 461-465. Disponible en: ]

17. Sigma Aldrich. Bradford Reagent [Internet]. USA [citado el 8 de junio del 2021]. Disponible en: ]

18. MINSA. Manual de procedimientos de electroforesis para proteínas y ADN, Serie de Normas Técnicas Nº:38- Lima, Perú, [Internet]. 2003 [citado el 8 de junio del 2021]. Disponible en: ]

19. Miranda E, Velarde F, Somocursio J, Ayala E. Evaluación de dos pruebas de inmunoblot con antígeno hidatídico de caprino y ovino para el diagnóstico de equinococosis humana. Rev Peru Med Exp Salud Pública. 2010. 27(2):209-14. Disponible en: ]

20. Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). Diagnóstico microbiológico de la infección por el VIH. Procedimientos en Microbiología Clínica. [Internet]. 2014 [citado el 8 de junio del 2021]. Disponible en: ]

21. Recom Line HIV-1 & HIV-2 IgG. Mikrogen diagnostik. [Internet]. [citado el 8 de junio del 2021]. Disponible en: ]

22. Geenius(tm) HIV 1/2 Confirmatory Assay. Un ensayo cualitativo para la confirmación y diferenciación de anticuerpos individuales contra VIH-1 y VIH-2 en muestras de sangre entera, suero o plasma. Biorad. [internet]. 2013. [citado el 8 de junio del 2021]. Disponible en: ]

23. Ayala-Sulca, E., Miranda-Ulloa, E. Evaluación de electroinmunotransferencia utilizando antígeno mix nativo purificado de líquido de cisticerco de Taenia solium para el diagnóstico de cisticercosis humana. Rev Peru Med Exp Salud Pública. 2015; 32 (3): 485-491. Disponible en: ]

24. Davelois K, Escalante H, Jara C. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Rev Peru Med Exp Salud Pública. 2016 Oct; 33(4): 616-624. Disponible en: ]

25. Escalante H, Davelois K, Ortiz P, Rodríguez H, Díaz E, Jara C. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática. Rev Peru Med Exp Salud Pública. 2011 Jul; 28(3): 454-461. Disponible en: ]

26. Cruz O, Pérez M, Lubián A, Montano T, Silva E. Fluido oral y orina como muestras alternativas en el diagnóstico confirmatorio de la infección por VIH-1. Rev Cubana Med Trop. 2007 Ago; 59(2). Disponible en: [ Links ]

Funding sources: The project was financed by the competitive fund of the Centro Nacional de Salud Pública del Instituto Nacional de Salud de Perú

Received: June 24, 2021; Accepted: July 14, 2021

Correspondence: Eduardo Fernando Miranda Ulloa Address: Defensores del Morro 2268, Chorrillos - Lima, Perú. Telephone: (051) 977783088;

Authorship contributions: The authors participated in the conception and design of the project; collection, analysis and interpretation of the results; writing, critical review and approval of the final version of the article.

Declaration of conflicts of interest: The authors declare that they have no conflicts of interest in the publication of this article.

Creative Commons License Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons