Enzyme-LinkedImmunosorbentAssay(ELISA)：酶联免疫吸附试验（ELISA）.pptVIP

Enzyme-Linked Immunosorbent Assay (ELISA) Mary Lea Killian USDA APHIS VS National Veterinary Services Laboratories Ames, Iowa Enzyme-linked Immunosorbent Assay ELISA Kits for Antibody Detection Commercial ELISA test kits are available to detect Avian influenza virus antibody in chicken serum Newcastle disease virus antibody in chicken serum Newcastle disease virus antibody in turkey serum ELISA Kits for AIV Ab Detection ELISA Results Results should be recorded by reading the optical densities of the plates in a plate reader at the correct absorbance: IDEXX: 650nm Each manufacturer supplies computer software specific for their test which calculates which samples are negative and the titers of positive samples. ELISA Results The status of a sample are evaluated by the sample to positive ratio (S/P ratio): Sample mean - negative control mean positive control mean - negative control mean (mean of optical absorbance) ELISA Results Example: Sample mean= 0.820 Negative control mean=0.053 Positive control mean=0.563 Valid ranges for the positive and negative controls Negative control = 0.150 or less The difference between the positive and negative control means must be greater than 0.075 Example: if negative control = 0.100, the positive control must be 0.176 or greater. ELISA Laboratory Materials Needed AIV ELISA plate Record Sheet Test samples (same set as AGID) Dilution Tubes Pipets and tips Materials Needed The materials for your kit ELISA plate Positive control Negative control Dilution Buffer (already in dilution tubes) Conjugate (secondary antibody) TMB Substrate Stop solution ELISA Laboratory 1 Label dilution tubes Add 1ml of diluent to dilution tubes (done) Add 2μl of test serum to a dilution tube Do NOT dilute controls ELISA Laboratory 2 Add 100 μl negative control to wells A1 and A2 Add 100 μl positive control to wells A3 and A4 Add 100μl of diluted test serum to the plate according to your record sheet Incubate for 3