title: Indirect Back-Titration ELISA: A New Format for Estimation of Human Tissue Kallikreins.
abstract: Enzyme-linked immunosorbent assay (ELISA) is either based on sandwich, competitive, or inhibition type of format. However, these formats need 2 or 3 monoclonal antibodies (moAB) to estimate 1 antigen. To get a cost-effective, high throughput, ELISA for estimation of human tissue kallikreins we have now developed an indirect, back-titration style, Time Resolved ImmunoFluorometric (TRIF) ELISA that uses only 1 antigen-specific moAB and a general polyclonal antibody. Polystyrene microtiter plate wells coated with a capture antibody, a mouse moAB prepared against a specific human tissue kallikrein are allowed to interact either with the corresponding pure antigen, as the calibrator, or with the corresponding antigen present in a biological fluid or tissue extract. The detection antibody, anti-mouse IgG conjugated with alkaline phosphatase, is added to find the antigen-free immobilized capture moAB. Conjugated enzyme is allowed to hydrolyze diflunisal phosphate to produce a highly fluorescent complex. The fluorescence measured in TRIF mode corresponds to the antigen-free immobilized capture moAB and is used to quantify antigen-bound capture moAB. The detection antibody binds with the antigen-free capture moAB and strength of the signal correlates inversely with the amount of antigen bound to the capture moAB. With a minimum detection level of 20 ng/L the assay has no cross-reactivity with several test molecules. The method is sensitive, specific, applicable to a variety of biological samples, and cost-effective as it uses only 1 moAB and a polyclonal antibody. Using this assay, a single epitope can be estimated without purification.
authors.affiliation: Departments of *Chemistry †Biochemistry and Biotechnology, BJ Campus, The Islamia University of Bahawalpur ‡Department of Surgery, Quaid-e-Azam Medical College, Bahawal Victoria (BV) Hospital, Bahawalpur, Pakistan.
authors.forename: Faiz-Ul-Hassan
authors.initials: FU
authors.lastname: Nasim
authors.forename: Samina
authors.initials: S
authors.lastname: Ejaz
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ashraf
authors.forename: Gulzar
authors.initials: G
authors.lastname: Ahmad
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000941:Antigens / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D057230:Limit of Detection; D051379:Mice; D011485:Protein Binding; D020840:Tissue Kallikreins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000941:Antigens; D000939:Epitopes; D018796:Immunoconjugates; D007074:Immunoglobulin G; D000469:Alkaline Phosphatase; D020840:Tissue Kallikreins