Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo

INTRODUCTION AND OBJECTIVES: This study aimed to compare the effects of different biotinylation methods on the performance characteristics of allergen-specific IgE detection. MATERIALS AND METHODS: The Gal d 6 gene was cloned into the pAN6/pAC6 vector, resulting in rGal d 6-Bio/Bio-rGal d 6 vector. The fusion protein was expressed in Escherichia coli AVB101 and simultaneously biotinylated in a site-specific manner. The Gal d 6 gene was amplified via PCR and cloned into the pET-28a vector and transformed into E. coli BL21 and purified via Ni-NTA, followed by chemical biotinylation using Sulfo-NHS-LC-Biotin. Twenty-eight patients allergic to hen's egg white were examined for sensitization against egg yolk. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed to detect allergen-specific IgE. RESULTS: rGal d 6, Bio-rGal d 6, and rGal d 6 were prepared using different biotin binding modes to detect allergen-specific IgE. rGal d 6-Bio (Kd = 0.6154) and Bio-rGal d 6 (Kd = 0.6698) had a markedly better detection performance than rGal d 6 (Kd = 28.93), and the rGal d 6-Bio had a better detection performance in small-volume serum samples. CONCLUSIONS: rGal d 6-Bio improved the sensitivity for the detection of allergen-specific IgE

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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