RESUMEN
Recently a new fourth generation microELISA for large scale blood screening has been described in which HIV p24 Ag detection was integrated in an anti-HIV-1/-2 and anti-HIV-1 group O assay based on a direct assay format: (Vironostika HIV Uni-Form II Ag/Ab (Van Binsbergen et al., (1998)). When compared to the third generation a-HIV assay (Vironostika HIV Uni-Form II plus O), the seroconversion window was narrowed with more than one week. A more precise window estimation based on seroconversion series with short sampling time intervals of up to 7 days, is described in parallel with that for the single HIV p24 Ag assay. It was found with 10 relevant seroconversion series that the HIV p24 Ag assay closes the seroconversion window with 6.2 days as compared to the 3rd generation a-HIV assay, while a window reduction of 4 days was found with Vironostika HIV Uni-Form II Ag/Ab. These seroconversion data show that with the new assay two-thirds of the current p24 Ag window is closed. There was no statistical difference in seroconversion sensitivity between the HIV p24 Ag and the new assay format. An extended evaluation of the new assay with subtype HIV-1 M and group O p24 Ag strains and with anti-HIV antibodies obtained from individuals infected with different HIV-1 subtypes showed that all subtypes of HIV-1 M and HIV-1 group O p24 antigen were detected as well as of HIV antibody.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Anti-VIH/análisis , Proteína p24 del Núcleo del VIH/análisis , VIH-1/inmunología , VIH-2/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Seropositividad para VIH , HumanosRESUMEN
The clinical sensitivity of the current anti-HIV assays is based for an important part on their reactivity with seroconversion panels. The most sensitive assay closes the seroconversion window as much as possible, thereby reducing the risk of transmitting false negative donations obtained from individuals infected recently. Because of the absence of anti-HIV antibodies during the early phase of infection, the seroconversion window can be narrowed partially by detection of HIV p24 Ag. To achieve this, the highest affinity anti-p24 binding antibodies were selected with BlAcore and applied in a direct assay format. To achieve optimal conditions for the anti-HIV part of the assay the HIV specific antigens viral HIV-1 gp160, HIV-2 gp36 and HIV-1 group O gp41 peptides were used. These antigens and antibodies were applied for microELISA coating as well as in the conjugate pearl, which is present in the well of the microELISA plate. The (analytical) anti-HIV-1/-2 and anti-HIV-1 group O sensitivity of this new assay, Vironostika HIV Uni-Form II Ag/Ab, is at least at the level of the current Vironostika HIV Uni-Form II plus O. When compared to the Vironostika HIV Uni-Form II plus O, the seroconversion window is narrowed by 1-2 weeks due to the incorporation of HIV p24 Ag detection. The level of reactivity of the anti-HIV and HIV Ag detection part can be improved by about a factor 2 by applying continuous shaking during sample incubation. Initial studies suggested that the specificity of the assay is identical to that of the Vironostika HIV Uni-Form II plus O, namely > 99.9%. Monitoring of proper execution of the assay handling steps was facilitated by implementing a purple dye in the conjugate pearl. Colourless specimen diluent changes into a green fluid upon dissolving of the conjugate pearl and turns subsequently into blue/purple upon sample addition. These visual changes can also be determined by spectrophotometric measurement at 620 nm.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/inmunología , VIH-2/inmunología , Productos del Gen env/inmunología , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/virología , Seropositividad para VIH , Humanos , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
It was shown previously that about 97% of the anti-HIV-1 group O strain-positive samples were detected by crossreaction with native HIV-1 gp160 (Van Binsbergen et al., Evaluation of a new third generation anti-HIV-1/anti-HIV-2 assay with increased sensitivity for HIV-1 group O, J. Virol. Methods 60 (1996) 131-137). Fourteen out of 17 new anti-HIV-1 group O positive samples, selected with the Enzygnost HIV-1/2 plus assay, were already reactive when tested with HIV-1 gp160. When tested by the Vironostika HIV Uni-Form II plus O microELISA all 17 samples were reactive, demonstrating the necessity to implement an HIV-1 group O-specific antigen in the assay. On the other hand, it was surprisingly found that 40 out of 43 (93%) of anti-HIV-1 group M-positive samples, belonging to strain A, B, C, D, E or F, were detected by crossreaction with the HIV-1 group O (strain ANT70) synthetic peptide incorporated in the Vironostika HIV Uni-Form II plus O. Only HIV-1 subtype D-positive samples did not react with this peptide, presumably because of the presence of a histidine residue in the immunodominant region of HIV-1 subtype D gp41. Both crossreactions make the Vironostika HIV Uni-Form II plus O microELISA also sensitive for anti-HIV-1-positive samples originating from different geographical regions and resulting from different HIV-1 subtype infections. With an unusual seroconversion panel in which p24 Ag was present persistently, many anti-HIV-1/-2 assays produce alternating positive/negative results in anti-HIV antibody-positive bleeds. It was shown that the use of viral p24 and gp160 in a direct sandwich, allowing detection of anti-HIV IgG and IgM, explains the identification of all anti-HIV-positive bleeds by the Vironostika HIV Uni-Form II plus O. The high sensitivity of the plus O assay was confirmed with clinical samples of a so-called anti-HIV-1 low titer panel. The specificity of the Vironostika HIV Uni-Form II plus O determined in five blood transfusion centers, based on 135070 tests, was 99.97%.
Asunto(s)
Serodiagnóstico del SIDA , Anticuerpos Anti-VIH/sangre , Antígenos VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Secuencia de Consenso , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Seropositividad para VIH , VIH-1/clasificación , VIH-1/genética , VIH-2/inmunología , Humanos , Epítopos Inmunodominantes , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Datos de Secuencia Molecular , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
First generation ELISA screening assays for antibodies to HTLV-III (HIV) generated between 0.1 and 1.0% false positive results. Western blot analysis in specialized reference centers is almost uniformly used as a method to confirm the specificity of the ELISA results. Yet, the high cost, time delay and lack of standardization in these systems cause a growing demand for tests that can be performed on site and that can at least reduce the number of sera that have to be sent to reference centers. Such tests thus should primarily be aimed at the detection of false positive results. Ancillary to the Vironostika anti-HTLV-III screening test, we developed a set of reagents (VERIFY) which can be used for the verification of initially or repeatedly positive screening results. The test employs a reagent specifically blocking true HTLV-III-anti HTLV-III reactions, a reagent blocking HLA-anti HLA reactions and a control reagent. Use of this test may reduce the number of sera found false positive by reference methods by more than 90%. The introduction of improved versions and second generation screening assays obviously will reduce the number of false positive results. Yet the significant results of this verification assay and the ease with which it can be integrated in the screening procedures will make it a valuable tool in the blood bank screening program.