| Autor: |
Nasim FU; 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., Ejaz S, Ashraf M, Ahmad G |
| Jazyk: |
angličtina |
| Zdroj: |
Applied immunohistochemistry & molecular morphology : AIMM [Appl Immunohistochem Mol Morphol] 2016 Jan; Vol. 24 (1), pp. 64-70. |
| DOI: |
10.1097/PAI.0000000000000150 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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