ELISA TESTS

ClinPro International Co. LLC ELISA tests include solid phase sandwich ELISA kits for measurement of antigen and antibody as well as competitive ELISA kits for measurement of small molecule antigens.
 
 

SANDWICH ELISA

Immunological Reaction

Antigens such as tumor markers, hormones and serum proteins may be determined by ELISA sandwich methodology. Sample is incubated in a microwell precoated with capture antibody. Antigen in the sample binds with the capture antibody and becomes immobilized. The microwell is washed to remove unbound antigen and enzyme conjugate is added. The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody-antigen-antibody/enzyme bound to the microwell.
 
 

                                         Solid Phase   Antigen   Enzyme  Conjugate        Sandwich Complex

Sandwich ELISA methodology may also be used to determine serum antibodies specific to certain disease states (i.e. infectious disease). Sample is incubated in a microwell precoated with capture antigen. Antibody present in the sample binds with the capture antigen and becomes immobilized. The microwell is washed to remove unbound antibody and enzyme-labeled anti-human antibody conjugate is added to form the sandwich complex. In both cases the amount of enzyme bound to the microwell is directly proportional to the amount of antigen or antibody in the sample.
 

Enzymatic Reaction and Quantitation

After unbound antigen or antibody and free conjugate are washed off, a chromogenic enzyme substrate is added. The chromogenic enzyme substrate reacts with the bound enzyme to produce a color reaction (blue to yellow color). A quantitative determination of antigen or antibody concentration is obtained by absorbance measurement of the colored reaction product using a spectrophotometric microwell reader.
 
 

                                        Sandwich Complex   Blue Color - Substrate  Yellow Color - Reaction Product

COMPETITIVE ELISA

Immunological Reaction

Competitive ELISA methodology is used to determine small molecule antigens (i.e. T3, T4, Progesterone, etc.). In competitive ELISA a carefully titrated concentration of antibody is coated onto the inside wall of the microwell. In a single reaction, antigen from the test sample and the enzyme-labeled antigen conjugate compete for a limited number of immobilized antibody-binding sites. The amount of antibody-antigen-enzyme complex bound to the solid phase (microwell) is inversely related to the concentration of antigen present in the sample.
 
 

                                         Solid Phase    Antigen   Enzyme-labeled Antigen     Reaction Product

Enzymatic Reaction and Quantitation

After unbound enzyme antigen conjugate is washed off, chromogenic substrate is added. The bound enzyme conjugate reacts with the chromogenic substrate to produce a color reaction (blue to yellow color). A quantitative determination of antigen concentration is obtained by absorbance measurement of the colored reaction product using a spectrophotometric microwell reader. Increased serum antigen results in reduced binding of the antigen-enzyme conjugate with the capture antibody producing less enzyme activity and color (yellow) formation.
 
 

                                       Reaction Complex   Blue Color - Substrate   Yellow Color - Reaction Product

Principle of Operation

The membrane of the Test Card or Strip is pre-coated with anti-X antibodies (i.e. X can be or any hormone, antigen or antibody of interest) at the test line region and anti-mouse antibodies at the control line region. During testing, the urine sample reacts with the dye conjugate (mouse anti-X-antibody-colloidal gold conjugate) which has been pre-coated in the Test Card or Strip. The mixture then migrates upward on the membrane chromatographically by capillary action to react with anti-X-antibodies on the membrane and generate a red line. Presence of this red line indicates a positive result, while its absence indicates a negative result. Regardless of the presence of X, as the mixture continues to migrate across the membrane to the immobilized goat anti-mouse region, a red line at the control line region will always appear. The presence of this red line serves as verification for sufficient sample volume and proper flow and as a control for the reagents.