A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases

doi:10.1016/S0022-1759(00)00241-6

Journal of Immunological Methods

Volume 242, Issues 1–2, 28 August 2000, Pages 91-100

https://doi.org/10.1016/S0022-1759(00)00241-6 Get rights and content

Abstract

Serological diagnosis of infectious diseases that generate a highly heterogeneous antibody repertoire, such as tuberculosis, requires tests based on cocktails of antigens. We describe a new method called multi-antigen print immunoassay (MAPIA) for cocktail-based serological diagnosis. The assay entails the application of antigen to nitrocellulose membranes by micro-aerosolization (printing), followed by antibody detection using standard chromogenic immunodevelopment. Cocktails of protein antigens of Mycobacterium tuberculosis tested by MAPIA were found to maintain the serological activity of each of their components. In contrast, the same cocktails tested by enzyme-linked immunosorbent assay (ELISA) had a serological activity that was lower than the sum of the activities of their components. Consequently, cocktail-based MAPIA attained the diagnostic sensitivity expected on the basis of single antigen results, while a significant loss of diagnostic sensitivity was observed with cocktail-based ELISA. Thus, the MAPIA format is superior to conventional ELISA for the serological diagnosis of infectious diseases characterized by heterogeneous antibody responses.

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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¹: Present address: Division of Infectious Diseases, Department of Medicine, Montefiore Medical Center, Bronx, New York, NY 10467, USA.

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A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases

Abstract

Introduction

Section snippets

Sera

Antigens

Choice of solid phase

Discussion

Human subjects

Acknowledgements

Anal. Biochem.

J. Immunol. Methods

J. Chromatogr. B

J. Immunol. Methods

Immunol. Lett.

Diagn. Microbiol. Infect. Dis.

Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis

Infect. Immun.

Nucleotide sequence of the 19kDa antigen gene from Mycobacterium tuberculosis

Nucleic Acids Res.

Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle

Clin. Diagn. Lab. Immunol.

Antibody response to Cryptococcus neoformans proteins in rodents and humans

Infect. Immun.

Humoral immune responses to multiple antigens of Mycobacterium tuberculosis in tuberculosis patients co-infected with human immunodeficiency virus

Int. J. Tuberc. Lung Dis.

Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn’s disease

Clin. Exp. Immunol.

Enzyme immunoassay ELISA and EMIT

Analysis of the humoral immune response to Chlamydia pneumoniae by immunoblotting and immunoprecipitation

Clin. Diagn. Lab. Immunol.

Characterization of the katG gene encoding a catalase–peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis

J. Bacteriol.