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

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.

[1]  R. Booth,et al.  Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis. , 1989, Nucleic acids research.

[2]  R. Finkelstein,et al.  Checkerboard immunoblotting (CBIB): an efficient, rapid, and sensitive method of assaying multiple antigen/antibody cross-reactivities. , 1990, Journal of immunological methods.

[3]  T. Yamada,et al.  Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG , 1989, Infection and immunity.

[4]  T. Yamada,et al.  Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen , 1988, Journal of bacteriology.

[5]  S. Bhaskar,et al.  A single-step immunochromatographic test for the detection of Entamoeba histolytica antigen in stool samples. , 1996, Journal of immunological methods.

[6]  S. Challacombe,et al.  ELISA and other solid phase immunoassys : theoretical and practical aspects , 1988 .

[7]  V. Sharma,et al.  A rapid immunochromatographic test (ICT) for diagnosis of Plasmodium falciparum. , 1998, Diagnostic microbiology and infectious disease.

[8]  A. Rendon,et al.  Dot Blot Assay for Detection of Antidiacyltrehalose Antibodies in Tuberculous Patients , 1999, Clinical Diagnostic Laboratory Immunology.

[9]  S. Cole,et al.  Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex , 1995, Infection and immunity.

[10]  M. Alric,et al.  Cross-blot and cross-dot system: a high-performance system for the detection of antigen-antibody complexes on nitrocellulose. , 1986, Analytical biochemistry.

[11]  C. Prantera,et al.  Antibodies to Mycobacterium paratuberculosis‐specific protein antigens in Crohn's disease , 1992, Clinical and experimental immunology.

[12]  N. Ohara,et al.  Cloning and Sequencing of a Unique Antigen MPT70 from Mycobacterium tuberculosis H37Rv and Expression in BCG Using E. coli‐Mycobacteria Shuttle Vector , 1995, Scandinavian journal of immunology.

[13]  Å. Andersen,et al.  Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis , 1989, Infection and immunity.

[14]  N. Ohara,et al.  Characterization of the Gene Encoding the MPB51, One of the Major Secreted Protein Antigens of Mycobacterium bovis BCG, and Identification of the Secreted Protein Closely Related to the Fibronectin Binding 85 Complex , 1995, Scandinavian journal of immunology.

[15]  B. Alarcón de Noya,et al.  The multiple antigen blot assay (MABA): a simple immunoenzymatic technique for simultaneous screening of multiple antigens. , 1998, Immunology letters.

[16]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[17]  M. Gennaro,et al.  Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis. , 1998, Journal of chromatography. B, Biomedical sciences and applications.

[18]  S T Cole,et al.  Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis , 1993, Journal of bacteriology.

[19]  R. Lathigra,et al.  The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins , 1992, Journal of bacteriology.

[20]  M. Gennaro,et al.  Heterogeneous Antibody Responses in Tuberculosis , 1998, Infection and Immunity.

[21]  J. Touchstone.  Practice of Thin Layer Chromatography , 1978 .

[22]  R. Cole,et al.  Detection of pulmonary and extrapulmonary tuberculosis patients with the 38-kilodalton antigen from Mycobacterium tuberculosis in a rapid membrane-based assay , 1996, Clinical and diagnostic laboratory immunology.

[23]  R. Rosengarten,et al.  Development of a Recombinant Antigen for Antibody-Based Diagnosis of Mycoplasma bovis Infection in Cattle , 1999, Clinical Diagnostic Laboratory Immunology.

[24]  E. Engvall,et al.  Enzyme immunoassay ELISA and EMIT. , 1980, Methods in enzymology.

[25]  A. Essig,et al.  Analysis of the Humoral Immune Response toChlamydia pneumoniae by Immunoblotting and Immunoprecipitation , 1999, Clinical Diagnostic Laboratory Immunology.

[26]  A. Casadevall,et al.  Antibody Response to Cryptococcus neoformans Proteins in Rodents and Humans , 1999, Infection and Immunity.

[27]  M. Gennaro,et al.  Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis , 1997, Infection and immunity.

[28]  A. L. Sørensen,et al.  Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis , 1995, Infection and immunity.

[29]  M. Gennaro,et al.  MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex , 1997, Infection and immunity.