Question

1. In a positive result, the second antibody in the indirect ELISA binds to?

   		a. an antigen on the well
   		b. binds to the variable region on the first antibody
   		c. a fluorescent dye giving a color change
   		d. the constant region of the patient’s antibody
   		e. none of the above

Answer 1

Ans.

• ELISA is a bioanalytical assay which is commonly used for the detection of a protein in a liquid sample by using antibodies agains protein of interest.
• It is a commonly used diagnostic tool in medicine, biological research as well as in biotechnology.
• Based on the bonding and use of analyte and antibody, ELISA test can be divided into different categories.
• In the indirect ELISA, quantification of antigen-specific antibody is done instead of antigen.
• This ELISA type can be used to detect antibodies against several pathogenic microbes such as rotavirus and HIV.
• Initial step of indirect ELISA assay involves attaching the antigen of knowledge to the bottom of the wells of microtiter plates, instead of capturing antigen with the use of antibody.
• Addition of patient serum is done after the unbound sites on the plates are blocked.
• If present, then primary antibody will attach to the antigen.
• Any unbound protein is removed by washing and secondary antibody with it's conjugated enzyme is added against the primary antibody.
• Amount of antigen specific antibody present in the patient's serum can be quantified with the help of secondary antibody by determining the intensity of color resulting from the reaction of conjugated enzyme - chromogen.
• In a positive result of indirect ELISA assay, the secondary antibody binds to an antigen on the well.