A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases
Introduction
Detection of antibodies is one of the approaches most commonly used to diagnose infectious diseases (Rose, 1998). Serological techniques are simple, usually rapid, relatively non-invasive, and do not require isolation or culturing of the pathogen. In many infections, most, if not all, infected persons produce antibodies against the same, immunodominant antigen(s). In other cases, most notably in infections caused by certain intracellular pathogens, the antibody repertoire is highly diverse, with sera from different persons reacting with different antigens. Examples include tuberculosis (Lyashchenko et al., 1998), Crohn’s disease (Elsaghier et al., 1992), chlamydial infections (Essig et al., 1999), and cryptococcosis (Chen et al., 1999). Accurate serodiagnosis of such infectious diseases requires cocktails of multiple antigens to cover the breadth of the antibody response.
To develop a serodiagnostic test that utilizes a multi-antigen cocktail, it is necessary to have an immunoassay that gives optimal cocktail performance. Below, we describe a new, cocktail-based method for antibody detection. The method, which we call multi-antigen print immunoassay (MAPIA), is based on immobilization of antigens onto nitrocellulose membranes by semi-automated micro-spraying, followed by standard chromogenic immunodevelopment. As a test of the method, we used tuberculosis. The serological activity in MAPIA of a cocktail containing multiple antigens of Mycobacterium tuberculosis was equal to that expected from single antigen results and it was much greater than that observed with an enzyme-linked immunosorbent assay (ELISA) in polystyrene microtiter plates. Thus, MAPIA constitutes a significant step towards developing a cocktail-based, serodiagnostic test for tuberculosis and other infectious diseases.
Section snippets
Sera
Sera were obtained from 75 patients with culture-confirmed active pulmonary tuberculosis and from 67 healthy, control individuals. All study subjects were negative for infection with human immunodeficiency virus.
Antigens
In this study, we used twelve proteins of M. tuberculosis (Table 1) that had been previously shown to generate antibody responses during tuberculosis (Lyashchenko et al., 1998, Colangeli et al., 1999). Proteins were purified to near-homogeneity from cultures of recombinant Escherichia
Choice of solid phase
The formulation of a multi-antigen cocktail for serodiagnosis is a two-stage process. First, antigens are selected on the basis of seroreactivity. Second, the serological activity of a cocktail of the selected antigens is assessed. We found that ELISA using polystyrene microtiter plates was inadequate because multi-antigen cocktails tended to perform suboptimally: for a given serum, absorbance values measured with an antigen cocktail were often 10 to 50% lower than those obtained with single
Discussion
In the present work, we describe MAPIA, a novel assay for antibody detection that entails the application of antigen onto nitrocellulose membranes by micro-aerosolization. In MAPIA, a cocktail of multiple antigens retains the serological activity of its components. Thus, MAPIA is well suited for developing cocktail-based serological assays for the diagnosis of tuberculosis and other infectious diseases that are characterized by a heterogeneous antibody repertoire (Lyashchenko et al., 1998,
Human subjects
Research described in this manuscript is IRB-exempt, because human sera were obtained through a commercial supplier and had no personal identifiers.
Acknowledgements
We thank Karl Drlica, Sam Kayman and Tomas Haendler for comments on the manuscript. This work was supported by NIH grants AI-43781 (K.P.L.) and AI-36989 (M.L.G.).
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Present address: Division of Infectious Diseases, Department of Medicine, Montefiore Medical Center, Bronx, New York, NY 10467, USA.