STANDARD OPERATING PROCEDURE http://proteomics.cancer.gov M-102.1 Page 1 of 5 STANDARD OPERATING PROCEDURE Title: Evaluation using Indirect ELISA SOP#: M-102 Version #: 1 Author: R. Saul / G. Whiteley Date Approved: Feb. 6, 2009 Date Modified: 1. PURPOSE This procedure is to be used for the characterization of purified monoclonal antibody. 2. SCOPE This document describes the procedure for the indirect Enzyme Linked ImmunoSorbant Assay (ELISA) that is used to assess the reactivity of the monoclonal antibodies for the recombinant antigens used as immunogens. It is not intended as a procedure for measurement of native antigens. It is also not to be used as the procedure for assessment of reactivity in unpurified monoclonal antibodies. 3. RESPONSIBILITIES It is the responsibility of the person(s) performing this test to be familiar with lab safety procedures and to have basic laboratory skills. The interpretation of the results must be done by a person trained in the procedure and familiar with such interpretation. 4. EQUIPMENT Plate Shaker; Boekel Scientific Jitterbug model 130000 Plate Washer; BioRad Immunowash model 1575 Plate Reader: BioRad iMark microplate reader (450nm filter) 5. MATERIALS Microtiter plates – medium binding; Costar Cat. #9017 Carbonate Coating Buffer (pre-mixed powder) – prepared according to manufacturer’s directions; Pierce-Thermo Cat. #28382 http://proteomics.cancer.gov M-102.1 Page 2 of 5 Phosphate Buffered Saline (PBS), 10x solution (Fisher Scientific Cat. #BP399-1) diluted to 1x with deionized water to give 11.9 mM phosphate, 137mM NaCl, 2.7mM KCl, pH 7.4 PBS – 0.05% Tween (PBST) prepared by addition of 500uL Tween 20; (ACROS Cat. #23336-2500) into 1 liter of PBS PBS – 0.05% Tween, 0.1% BSA (PBST-BSA) prepared by addition of 1 gram of BSA (Sigma Cat. # A7030) into 1 liter of PBST 1-Step TMB Turbo Substrate, Pierce-Thermo catalog # 34022 0.