03/13/2025 ELISA Lab

1, antigen, binding, unbound, detergent, detergent, blocking, nonspecific

During step _, a(n) __ is added to the wells of the microplate strip and incubated to allow __, after which __ antigen is washed from the cell walls with __. The __ also serves as a __ agent, binding to all unused protein binding sites in the wells and preventing __ binding of antibody

2, primary antibody, antibody, antigen, unbound primary antibody

During step _, __ __ solution is added to the wells and incubated to allow the __ to bind to the __. Then __ __ __ is washed from the wells.

3, enzyme-labeled secondary antibody, secondary antibody, primary antibody,

During step _, __ __ __ solution is added to the wells and incubated to allow the __ __ to bind to the __ __. Then __ __ __ is washed from the wells.

4, chromogenic (color-producing) enzyme substrate, color, negative, blue

During step _, __ __ __ is added to the wells and incubated to allow __ to develop. Results of the assay are evaluated. Wells that remain colorless are __ and wells that turn __ are positive.

hydrophobic interaction

The microplate strips are made of polystyrene which absorbs (binds) proteins by __ __.

antigen, antigen

In this kit, the __ is chicken gamma globulin purified from egg yolks, which serves as a generic representative of any hypothetical __

incubation temperature, concentrations, reagents, 5, room, color intensity, negative controls

The rate of binding depends on the __ __ and the __ of the __. This kit has been optimized so that each incubation can be performed for __ at __ temperature. Exceeding this time or temperature will cause an increase in __ __ and possibly some background in the __ __.

blocking agent, prevent, false positive

__ __ are added after antigen adsorption to __ nonspecific binding of antibodies to the plastic, which would produce __ __ results. The blocking agent may be a __or a __, or both. Common blocking agents include Tween 20, nonfat dry milk, gelatin, and bovine serum albumin (BSA).

primary antibodies, primary antibody,

__ __ recognize and bind to the antigen in an immunoassay. In this kit the __ __ is a polyclonal rabbit antibody raised against chicken gamma globulin. In the ELISA antibody test starting on page 55, this primary antibody stimulates human antibodies in a sample of human serum.

secondary antibodies, primary, primary antibody

__ __ recognize and bind to __ antibodies. They are made in animals of different species than that used to make the __ __. For this kit, goats were immunized with rabbit IgG to make the secondary antibodies.

Enzyme-Linked Immunosorbent Assay

ELISA stands for __

nonspecific color, TMB precipitates

If the microplate strips sit longer, __ __ may develop. __ that develops after the 5-minute incubation should not be considered in the assay results. After 20-30 minutes, the blue color may begin to fade as __ __ out of solution,

omitted, replaced, bind, specifically, target antigen, antibody, false positive, false negative

For the negative control, the antigen or primary antibody is either __ (as in this kit) or the antigen is __ by a factor that will not __ __ to the antibody. The positive control always contains __ or __. A negative sample that gives a positive assay result is called a __ __. A positive sample that gives a negative assay result is called a __ __.

negative control, antigen omitted, nonspecific binding, antibodies, experimental error, antigen,

In the ELISA we are doing today, __ __ is the experimental variable. An appropriate negative control would be wells with __ __. Any color product in those wells would be the result of either 1) __ __ of the __, or 2) __ __. An appropriate positive control would be a sample known to contain the __.

strong, weak, ambiguous, no detectable, colored substrate, standard curve

An ELISA can give quantitative and qualitative information. Qualitative results can be determined visually. Quantitative can also be estimated visually and scored symbolically as follows: (++) for __ signal. (+) for __ signal (+,-) for __ signal, and (-) for __ __signal. For accurate and precise determination of concentrations, a __ __ is required. These quantitate the absorbance of light by the __ __ in each well of a microplate. They use the negative control wells to set a baseline and then read the absorbance of each well at a specified wavelength. For example, the peak absorbance for TMB is at 655 nm. Quantitative ELISA controls include a dilution series of known concentrations that is used to create a __ __. This allows the concentration of antigen in a sample to be quantitated, which in turn may help a researcher, clinician, or physician determine the infection level of a particular disease.

sequence, added into the wells, immobilized antigen, secondary antibody, horseradish peroxidase, HRP, substrate, TMB, blue

ELISA’s are performed so routinely in both clinical and research laboratories that assays for many antigens are available in kit form. A number of different ELISA methods have been developed that differ primarily in the __ in which antigens and antibodies are __ __ __ __. In an antibody capture assay (as used in this kit), __ is bound in the plastic wells and the __ __ binds to (or is captured by) the __ __. A __ __ is linked to the enzyme __ __ (_), which oxidizes its __ (__), turning the assay solution __.

antigen capture assay, primary antibody, antigen, immobilized primary antibody, antigen, secondary antibody, HRP, TMB

In an __ __ __, __ __is bound in the plastic wells, __is captured by the __ __ __, and the captured __ is detected by a __ __, also linked to __, so that the solution turns blue on reaction with __.