Lab 1: ELISA Flashcards
Monoclonal antibodies
Produced from a cultured population of cells from a single B cell line. Produced by immunization with antigen (usually a mouse). Ab producing cells from spleen are fused with an immortalized cell line to produce hybridomas which are then cloned. Recognize one epitope of the antigen.
Polyclonal antibodies
Collected from serum of animals after immunization with antigen. Small antigens are often attached to carrier proteins (keyhole limpet hemocyanin) to increase immune response. These antibodies come from a population of B-lymphocytes and recognize different epitopes of the antigen. Derived from several B cell lines.
ELISA
Enzyme-linked immunosorbent assay. Enzyme linked to an antibody is used to detect the binding of antibody to specific antigen by converting a colorless substrate to a colored product. Can be used to detect presence of antibodies or antigens in a sample.
Substrate for horseradish peroxidase
OPD or ABTS
Substrate for alkaline phosphatase and p-nitrophenyl phosphate
pNPP (colorimetric detection) , 4-methyllumbelliferyl phosphate (fluorimetric detection)), 5-bromo-4-chloro-3-imdolyl phosphate/ nitroblue tetrazolium (western blot). Alkaline phosphatase is more stable than peroxidases and is not influenced by false positives due to endogenous peroxidase activity in the sample. ABTS was used in this experiment. OPD can be used for higher sensitivity.
Direct immunodetection versus indirect
Direct involves an enzyme conjugated to the primary antibody. Indirect involves the enzyme conjugated to a secondary antibody that is specific for the primary antibody.
Detecting antibody using indirect ELISA
Antigen is attached to well surface. Antibody being tested is added, incubated, washed. Secondary antibody covalently linked to enzyme is added, incubated, washed. Substrate added, if enzyme-linked antibody binds to the test antibodies on the surface then the enzyme modifies the substrate to a colored product. Used for antibody screening and epitope mapping. Does not require the use of preexisting specific antibodies; requires relatively large amounts of antigen.
To detect antigen using antibody sandwich ELISA
Coat well with primary antibody. Incubate with antigen standards or unknown. Add a different primary antibody which sandwiches the antigen. Sometimes this antibody is directly linked to the enzyme, if not, add the secondary antibody. Add substrate and measure color change, more antigen will give a higher absorbance. Used for antigen screening and to detect soluble antigen. Most sensitive antigen assay; requires large amount of pure or semi-pure specific antibody (capture antibody). When monoclonal antibodies are used, it’s important that the detection antibody and capture antibody recognize different epitopes of the antigen.
Competitive ELISA to detect antigen
Coat wells with antigen, incubate with antigen standards or unknown sample together with primary antibody conjugated to enzyme, add substrate and measure color change. More antigen in the sample means less of the enzyme linked antibody will bind to the antigen on the walls and gives less absorbance. Used for antigen screening and to detect soluble antigen. Rapid assay with only two steps; excellent for measuring antigenic cross-reactivity. Can be quantitated with inhibition curve of serial dilutions of standard antigen solution.
Capture antibody? Detection antibody?
Capture antibody was goat anti-rabbit IgG. Detection was goat anti-rabbit IgG with horseradish peroxidase conjugated to it. The antigen was rabbit IgG. Substrate was ABTS.
What was the negative control?
Rows C and D which had been treated with blocker alone and had no capture antibody.
What was the blocker?
5% w/v non-fat dried milk in 1x PBS.
Max and min of pipettor
Max = 100%, min = 10% (for accurate results )
Standard deviation
Square root of the variance; measures the normal spread of values in the data set.
Standard error of the mean
Standard deviation divided by the square root of the number of data points; measure of the uncertainty in the mean.
