Biochemical instrumental analysis - 12 Dr. Maha Al-Sedik 2015 CLS 332

Enzyme Linked Immuno Sorbent Assay Used to detect the presence of Ag, Ab from the Serum. Ag or Ab is fixed to the well. By measuring the enzymatic activity which interacts with substrate to produce color. This Enzyme may be linked to Ag or Ab. The density of color is either directly or indirectly proportional to target according to the type of ELISA.

ELISA Screening test Positive ELISA Test antigens coated the well Patient serum Ab Anti-human immunoglobulin coupled to an enzyme. Chromogen or substrate Negative ELISA Test

1-Non Competitive ELISA to detect Ag Ab labeled with enzyme HBsAg absorbed from serum Specific Ab coating the well

2-Non Competitive ELISA to detect Ab: HCV Ab HCV coating Ags Enzyme labeled anti-Ab

3-Competitive ELISA with labeled Ag

4-Competitive ELISA with labeled Ab.

5- Competitive Neutralizing ELISA ( HBe Ab).

Sample 1 positive Sample 2 negative Antigen-specific antibody is attached to a solid-phase surface

Sample 1Sample 2 Test specimen is added, which may or may not contain the antigen Washing will remove excess or un bounded Ag

Sample 1Sample 2 An enzyme-labeled antibody specific to the antigen is added (conjugate) Washing will remove excess Conjugate

Sample 1Sample 2 Chromogenic substrate is added, which in the presence of the enzyme, changes its color. Stopping solution is added to stop any reaction s s s s s s s s s s s s

The amount of color that develops is directly proportional to the amount of antigen in the test specimen. Optical Density (color) Concentration of Analyte

Sample 1Sample 2 Specific antigen is attached to a solid-phase surface

Sample 1Sample 2 Test specimen is added, which may or may not contain the antibody Washing will remove excess serum with un bounded Ab

Sample 1Sample 2 An enzyme-labeled antibody specific to the serum antibody is added (conjugate) Washing will remove excess or un bounded Labeled Ab

Sample 1Sample 2 Chromogenic substrate is added, which in the presence of the enzyme, changes its color. Stopping solution is added to stop any reaction s s s s s s s s s s s s

Optical Density (color) Concentration of Analyte The amount of color that develops is directly proportional to the amount of antibody in the specimen.

The competition is between the serum Ag and the tracer on the Ab that coat the well.

Sample 1Sample 2 Antigen-specific antibody is attached to a solid-phase surface

Sample 1Sample 2 Test specimen is added, which may or may not contain the antigen

Sample 1Sample 2 An enzyme-labeled antigen( tracer ) is added with serum.Competition on coated Ab. Washing will remove any un bounded Ag

Sample 1Sample 2 Chromogenic substrate is added, which in the presence of the enzyme, change its color. Stopping solution is added to stop any reaction s s s s s s s s s s s s

Optical Density (color) Concentration of Analyte The amount of color that develops is inversely proportional to the amount of antigen in the test specimen.

The competition is between the serum Ab and the tracer on the Ag that coat the well.

Sample 1Sample 2 Antibody -specific antigen is attached to a solid-phase surface

Sample 1Sample 2 Test specimen is added, which may or may not contain the antibody

Sample 1Sample 2 An enzyme-labeled Antibody (conjugate) specific to the attached antigen are added with serum. Washing will remove any un bounded Ag

Sample 1Sample 2 Chromogenic substrate is added, which in the presence of the enzyme, change its color. Stopping solution is added to stop any reaction s s s s s s s s s s s s

Optical Density (color) Concentration of Analyte The color that develops is inversely proportional to the amount of antibody in the test specimen.

The competition is on neutralizing solution between the well’s Ab and the serum Ab.

Sample 1Sample 2 Antibody specific to HBe Ag is attached to a solid-phase surface

Sample 1Sample 2 Neutralizing solution is added, which contains the Ag

Sample 1Sample 2 Serum is added which it may contain Ab to Neutralizing Sol. Competition between the serum Ab and coating Ab to bind to Neutral.Ag Washing will remove any un bounded Ag tracer and serum Ab

Sample 1Sample 2 An enzyme-labeled antibody specific to the antigen is added (conjugate) Washing will remove excess or un bounded Labeled Ab

Sample 1Sample 2 Chromogenic substrate is added, which in the presence of the enzyme, change its color. Stopping solution is added to stop any reaction s s s s s s s s s s s s

Optical Density (color) Concentration of Analyte The color that develops is inversely proportional to the amount of antibody in the test specimen.