免疫学2012.ppt

Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Steps in the ELISA Scoring ELISA Compare color to positive and negative controls Indicate the range of dark blue The larger the range of color, the more concentrated the antibodies Highest concentration of antibodies in those samples that were originally “infected” Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate ELISA plate wells already coated with HIV antigen Patient serum added which contains antibodies to HIV antigen Primary (1?) antibody (“body fluid” sample) Unbound antibodies washed away Primary (1?) antibody Variable domain binds antigen; constant domain free e e e e e e e e Secondary (2?) antibody (conjugated to horse radish peroxidase) Antisera added which contains anti-human secondary antibody-enzyme conjugate Variable domain of 2? antibody binds constant domain of 1? antibody e e e e e Secondary (2?) antibody Variable domain of 2? antibody binds constant domain of 1? antibody Unbound antibodies washed away Chromagen or substrate added (tetramethylbenzidine or TMB) Addition of chromagenic substrate results in color reaction Color intensity increases with concentration (titer) of antibodies Darker color = higher titer NEGATIVE REACTION: There are no antibodies present that bind HIV antigen Primary (1?) antibody NEGATIVE REACTION: All primary antibodies washed away e e e e e e e e Secondary (2?) antibody NEGATIVE REACTION: There are no primary antibodies for the secondary antibodies to bind NEGATIVE REACTION: All secondary antibodies washed away NEGATIVE REACTION: There is no enzyme to carry out the color reaction NO COLOR CHANGE Substrate added Monoclonal Antibodies Technique developed by Miller and Kohler (1980); revolutionized immunology; involves development of single antibody secreting immortalized cell Inject mice