Generation of monoclonal antibodies against 17α-hydroxyprogesterone for newborn screening of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.

Uso de un péptido sintético de la glucoproteína 36 del VIH 2 en mezclas de antígenos para el inmunodiagnóstico de la infección por VIH tipo 1 y 2 / Use of a synthetic peptide of HIV-2 glycoprotein 36 in antigen mixtures for the immunodiagnosis of HIV types 1 and 2

INTRODUCCIÓN: La glucoproteína gp36 del VIH2 es muy utilizada en los ELISA. Pretendimos evaluar los índices de diagnóstico de mezclas de antígenos con el péptido sintético del VIH 2 gp36 (5). MÉTODOS: Se prepararon 5 mezclas con gp36 (5) y proteínas recombinantes del VIH 1/2. Se evaluaron 1.306 muestras con el UMELISA HIV 1 + 2 RECOMBINANT como referencia. RESULTADOS: La mezcla (V-1) mostró muy buena concordancia respecto a la referencia. CONCLUSIÓN: La variante V-1 demostró elevada eficacia en el inmunodiagnóstico del VIH ½

INTRODUCTION: The HIV-2 glycoprotein 36 (gp36) is often used in ELISA. An evaluation of the diagnostic indexes of antigen mixtures with a synthetic peptide of HIV 2 gp36 (5) is performed in this study. METHODS: Five mixtures of gp36 (5) and the recombinant proteins of HIV 1/2 were prepared. A total of 1306 samples were evaluated with UMELISA HIV 1 + 2 RECOMBINANT used as reference. RESULTS: The variant (V-1) showed very good agreement as regards the reference method. CONCLUSION: The V-1 variant was shown to be highly effective in the immunodiagnosis of HIV 1/2

Desarrollo de un ensayo sandwich de doble antígeno para la detección de anticuerpos contra el VIH-2 empleando un péptido sintético biotinilado de la proteína gp36 / Development of an antigen ‘sandwich’ enzyme immunoassay for the detection of antibodies against HIV-2 by using a biotinylated synthetic peptide of gp36 protein

INTRODUCCIÓN: Existen varios métodos para la detección de anticuerpos contra el virus de inmunodeficiencia humana (VIH), y entre estos se encuentra el ELISA tipo sandwich de doble antígeno, muy utilizado en la actualidad. El objetivo de este trabajo es evaluar un péptido sintético monomérico biotinilado de la glucoproteína de transmembrana gp36 del VIH-2, en un ensayo de sandwich, para la detección de anticuerpos contra la esta proteína del VIH-2. MATERIALES Y MÉTODOS: Para desarrollar el ensayo se utilizaron placas recubiertas con la proteína recombinante gp36 a 0,5μg/ml y con el péptido sintético gp36(5) a 1μg/ml; la concentración del péptido sintético gp36(5) biotinilado (gp36(5)-B) utilizada fue 0,1μg/ml, preparada con una solución regula- dora Tris-BSA-NaCl y el conjugado Estreptavidina-Fosfatasa Alcalina diluido 1:30.000 preparado con la solución PBS-Sacarosa-BSA. Se evaluaron muestras de suero positivas a anticuerpos contra los virus VIH- 1 y VIH-2 (88 y 34, respectivamente), 483 muestras negativas procedentes de donantes de sangre y 96 muestras de suero para evaluar la especificidad analítica. Todas las muestras fueron evaluadas en el UMELISA HIV1+2 RECOMBINANT, las que resultaron reactivas se confirmaron por el ensayo confirmatorio DAVIH-BLOT. RESULTADOS: Las 34 muestras con anticuerpos contra el VIH-2 fueron evaluadas como positivas en ambas variantes de recubrimiento; la mayor especificidad se obtuvo con la variante que empleó el péptido sintético gp36(5) en el recubrimiento. El ensayo sandwich de doble antígeno desarrollado empleando el gp36 (5)-B permite la detección de anticuerpos contra la proteína gp36 del VIH-2

INTRODUCTION: Among the several existing methods for the detection of antibodies to HIV, the 'sandwich' ELISA is currently the most used. This study aims to assess a biotinylated monomeric synthetic peptide of the glycoprotein transmembrane gp36 from HIV-2, in a sandwich assay, for the detection of antibodies against this HIV-2 protein. MATERIALS AND METHODS: To perform the assay, plates coated with recombinant protein gp36 at 0.5 μg/mL and synthetic peptide gp36(5) at 1 μg/mL were used. The concentration of the biotinylated synthetic pep- tide (gp36(5)-B) used was 0.1 μg/mL prepared with a Tris-BSA-NaCl buffer solution and the Streptavidin- Alkaline Phosphatase conjugate diluted 1:30000 prepared with a PBS-Sucrose-BSA solution. Positive serum samples to antibodies against HIV-1 and HIV-2 viruses (88 and 34, respectively) were tested, with 483 negative samples from blood donors and 96 serum samples to assess the analytical specificity. All the samples were tested using the UMELISA HIV 1+2 RECOMBINANT assay, and all positives were confirmed using a DAHIV-BLOT assay. RESULTS: Thirty four samples with antibodies against HIV-2 were assessed as positive for both coating variants. The highest specificity was obtained with the variant using the synthetic peptide gp36(5) in its coating. The antigen 'sandwich' assay developed by using gp36(5)-B enables the detection of antibodies against gp36 protein of HIV-2

[Use of a synthetic peptide of HIV-2 glycoprotein 36 in antigen mixtures for the immunodiagnosis of HIV types 1 and 2]. / Uso de un péptido sintético de la glucoproteína 36 del VIH 2 en mezclas de antígenos para el inmunodiagnóstico de la infección por VIH tipo 1 y 2.

INTRODUCTION: The HIV-2 glycoprotein 36 (gp36) is often used in ELISA. An evaluation of the diagnostic indexes of antigen mixtures with a synthetic peptide of HIV2 gp36 (5) is performed in this study. METHODS: Five mixtures of gp36 (5) and the recombinant proteins of HIV1/2 were prepared. A total of 1306 samples were evaluated with UMELISA HIV1+2 RECOMBINANT used as reference. RESULTS: The variant (V-1) showed very good agreement as regards the reference method. CONCLUSION: The V-1 variant was shown to be highly effective in the immunodiagnosis of HIV 1/2.

[Development of an antigen 'sandwich' enzyme immunoassay for the detection of antibodies against HIV-2 by using a biotinylated synthetic peptide of gp36 protein]. / Desarrollo de un ensayo sandwich de doble antígeno para la detección de anticuerpos contra el VIH-2 empleando un péptido sintético biotinilado de la proteína gp36.

INTRODUCTION: Among the several existing methods for the detection of antibodies to HIV, the 'sandwich' ELISA is currently the most used. This study aims to assess a biotinylated monomeric synthetic peptide of the glycoprotein trans-membrane gp36 from HIV-2, in a sandwich assay, for the detection of antibodies against this HIV-2 protein. MATERIALS AND METHODS: To perform the assay, plates coated with recombinant protein gp36 at 0.5µg/mL and synthetic peptide gp36(5) at 1µg/mL were used. The concentration of the biotinylated synthetic peptide (gp36(5)-B) used was 0.1µg/mL prepared with a Tris-BSA-NaCl buffer solution and the Streptavidin- Alkaline Phosphatase conjugate diluted 1:30000 prepared with a PBS-Sucrose-BSA solution. Positive serum samples to antibodies against HIV-1 and HIV-2 viruses (88 and 34, respectively) were tested, with 483 negative samples from blood donors and 96 serum samples to assess the analytical specificity. All the samples were tested using the UMELISA HIV 1+2 RECOMBINANT assay, and all positives were confirmed using a DAHIV-BLOT assay. RESULTS: Thirty four samples with antibodies against HIV-2 were assessed as positive for both coating variants. The highest specificity was obtained with the variant using the synthetic peptide gp36(5) in its coating. The antigen 'sandwich' assay developed by using gp36(5)-B enables the detection of antibodies against gp36 protein of HIV-2.

Chimeric synthetic peptides as antigens for detection of antibodies to Trypanosoma cruzi.

Six chimeric synthetic peptides (QCha-1, QCha-2, QCha-3, QCha-4, QCha-5, and QCha-6) incorporating antigenic sequences of two immunodominant repeat B-cell epitopes of Trypanosoma cruzi were synthesized by conventional solid-phase peptide synthesis. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of positive Chagasic sera (n=82), while specificity was evaluated with samples from healthy blood donors (n=44) and patients with other infectious diseases (n=86). The antigenicity of the chimeric peptides in solid-phase immunoassays was compared with that of the monomeric peptides. Data demonstrated that the chimeric peptide QCha-5 was the most reactive because it detected antibodies to parasite efficiently. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of Chagas' disease.

Use of a chimeric synthetic peptide from the core p19 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection.

A chimeric synthetic peptide incorporating immunodominant epitope of the p19 gag protein (116-134) and the gp46 env protein (178-200) of HTLV-II virus, separated by two glycine residues, was synthesized by conventional solid-phase peptide synthesis. The antigenic activity of this peptide was evaluated by Ultramicro Enzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-II positive sera (n = 9), anti-HTLV-I/II positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 1),and anti-HTLV-I positive sera (n = 14), while specificity was evaluated with samples from healthy blood donors (n = 20). The efficacy of the chimeric peptide in solid-phase immunoassays was compared with the monomeric peptides. Data demonstrated that the chimeric peptide was the most reactive because it detected antibodies to virus efficiently. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of HTLV-II infection.