The multiple antigen blot assay (MABA): a simple immunoenzymatic technique for simultaneous screening of multiple antigens

doi:10.1016/S0165-2478(98)00055-8

Immunology Letters

Volume 63, Issue 1, 1 August 1998, Pages 53-56

https://doi.org/10.1016/S0165-2478(98)00055-8 Get rights and content

Abstract

We describe a simple, reliable, reproducible and inexpensive technique termed multiple antigen blot assay (MABA) that permits the simultaneous screening of 28 different antigens based on a dot-blot ELISA methodology. Using an acrylic device (Miniblotter®) containing 28 parallel troughs, multiple antigens are distributed and immobilized onto a nitrocellulose membrane. Strips are then cut perpendicularly and exposed to immune serum. The reaction is detected with secondary antibodies conjugated to horseradish peroxidase, and developed by a chemiluminescent substrate, the results being recorded on film. Positive reactions to the different antigens are seen as small black squares on each strip. We have used this qualitative technique to screen synthetic peptides derived from native schistosomal and malarial proteins, using immune rabbit sera as the detection antibody. This system can also be used in the clinical laboratory according to a dipstick-based diagnostic format for different infectious and non-infectious diseases, and can be designed to detect either antibodies or circulating antigens.

Introduction

Immunoenzymatic assays which include conventional ELISA, dot-ELISA and Western blot have become one of the most powerful tools for the laboratory diagnosis of infectious, autoimmune, metabolic and neoplastic diseases 1, 2. These techniques have some limitations when several antigens need to be evaluated, such as the case of screening a wide range of epitopes of parasite antigens, constructed by molecular engineering or by chemical synthesis. Herein we describe a technique adapted from methodologies published as Cross-Dot System [3]and Checkerboard Immunoblotting (CBIB) [4]. Briefly, antigens, or in this case synthetic peptides, are immobilized on a nitrocellulose membrane in parallel lanes delineated by a template (Multiscreen apparatus). Strips are then cut perpendicularly to the antigen-lanes, individually immersed in immune sera, developed with a secondary antibody conjugated to horseradish peroxidase and exposed to a chemiluminescent substrate. This technique allows the screening, in a single strip, of the reactivity of a serum against 28 different antigens.

Section snippets

Technique

In detail, the technique is as follows:

1.
Nitrocellulose sheets (Schleicher and Schuell, Keene, NH, 0.2 μm, 7.5×10 cm) for the Miniblotter® 28 SL (Immunetics Inc, Cambridge, MA), are cut and labelled with a line on one of the borders for reference, using a fine-tipwater-resistant pen (Fig. 1a).
2.
The nitrocellulose paper is then soaked in distilled water for 5 min, and the sheet is aligned in the upper part of the apparatus, with the reference line parallel to channel number 1 (Fig. 1a). A plastic

Methods

To illustrate this technique, we used 18 synthetic peptides derived from S. mansoni and eight from P. falciparum. Pre- and post-immune sera from seven rabbits immunized with different pools of five to six peptides, were used as the first antibody (Fig. 2). Pre-immune sera and PBS, were used as negative controls for antibodies and antigens, respectively.

In order to validate the sensitivity of the test, MABA was compared to a conventional ELISA test, using three different S. mansoni synthetic

Acknowledgements

We thank Lic. Noraida Zerpa, Lic. Cecilia Colmenares, Lic. Henry Bermúdez, Dr. Hector Arrechedera and Lic. Caridad Vargas for expert technical assistance. We are especially indebted to Dr Neil Lynch and Dr Mark James for constructive comments regarding the manuscript. This work was supported by CONICIT, project RP-IV-130032 and the Corporación Andina de Fomento (CAF), Caracas, Venezuela. Traveling expenses for the presentation of these results in an International Congress was supported by

References (6)

M. Alric et al.
Anal. Biochem.
(1986)
M. Kazemi et al.
J. Immunol. Methods
(1990)
A. Voller et al.
Trans. R. Soc. Trop. Med. Hyg.
(1976)

There are more references available in the full text version of this article.

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For the porcine system, five negative serum samples were used to calculate the cutoff as above. To carry out the multiple antigen blot assay (MABA) (Noya and Alarcon de Noya, 1998), nitrocellulose membrane (Schleicher and Schuell, Germany) was sensitized with cyst fluid, recombinant proteins and synthetic peptides (10 μg/ml in carbonate–bicarbonate buffer, pH 9.5), using an acrylic device Mini-PROTEAN®II Multiscreen apparatus (BioRad, Madrid, Spain). Five hundred microliters of the diluted cyst fluid, recombinant antigens Ts8B2, Ts8B2-NT, Ts8B2-CT, and the six synthetic peptides Ts8B2-1, Ts8B2-2, Ts8B2-3, Ts8B2-4, Ts8B2-5 and Ts8B2-6 were distributed in each channel of the Miniblotter® apparatus (BioRad); and incubated at room temperature on an orbital shaker for 60 min to immobilize the molecules onto a nitrocellulose membrane.
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