title: Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.
abstract: Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI.
authors.affiliation: Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775. Bloco C, CIC Curitiba, PR. Brazil. 81350-010.
authors.forename: Sandra
authors.initials: S
authors.lastname: Crestani
authors.affiliation: Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775. Bloco C, CIC Curitiba, PR. Brazil. 81350-010.
authors.forename: Amanda
authors.initials: A
authors.lastname: Leitolis
authors.affiliation: Instituto Carlos Chagas-FIOCRUZ, Rua Professor Algacyr Munhoz Mader, 3775. Bloco B, CIC Curitiba, PR. Brazil. 81350-010; Instituto de Tecnologia do Paraná, Rua Professor Algacyr Munhoz Mader, 3775. Bloco A, CIC Curitiba, PR. Brazil. 81350-010.
authors.forename: Lucianna Freitas Oliveira
authors.initials: LF
authors.lastname: Lima
authors.affiliation: Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775. Bloco C, CIC Curitiba, PR. Brazil. 81350-010; Instituto Carlos Chagas-FIOCRUZ, Rua Professor Algacyr Munhoz Mader, 3775. Bloco B, CIC Curitiba, PR. Brazil. 81350-010.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Krieger
authors.affiliation: Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775. Bloco C, CIC Curitiba, PR. Brazil. 81350-010; Instituto Carlos Chagas-FIOCRUZ, Rua Professor Algacyr Munhoz Mader, 3775. Bloco B, CIC Curitiba, PR. Brazil. 81350-010. Electronic address: leo.foti@fiocruz.br.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Foti
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D000913:Antibodies, Protozoan / Q000097:blood; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D046228:Microarray Analysis; D008863:Microspheres; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D014210:Treponema pallidum / Q000737:chemistry / Q000276:immunology; D014349:Trypanosoma cruzi / Q000276:immunology; D001618:beta-Lactamases / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000913:Antibodies, Protozoan; D000942:Antigens, Bacterial; D018937:Hepatitis C Antibodies; D011994:Recombinant Proteins; C485221:Tp47 protein, Treponema pallidum; D001618:beta-Lactamases
keywords: Blocking; Escherichia coli lysate; Immunoassay; Liquid microarray; Recombinant-antigen