Immunoassay Auto Analyzer PDF

Immunoassay auto analyzer Immuno assay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. Immuno assay auto analyzer It is an instrument in the immunology lab which do a...

Immunoassay auto analyzer Immuno assay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. Immuno assay auto analyzer It is an instrument in the immunology lab which do automation to many manual immune assay techniques.  Principle:- 1. Chemiluminescence immunoassay. 2. Fluoroimmunoassay. 3. Enzyme-linked immunosorbent assay. 4. Counting immunoassay. 5. Radio immune assay. ELISA Technique What is an ELISA: ELISA stands for enzyme-linked immunosorbent assay. It is a plate- based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Types of the ELISA assays:  direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA ELISA tests can be classified into four types depending upon the different methods used for binding between antigens and antibodies, namely: Direct ELISA – Antibody is coated on the microtiter well Indirect ELISA – Antigen is coated to the microtiter well Sandwich ELISA – Primary antibody is coated on the microtiter well with a secondary free antibody Competitive ELISA – Antigen-coated Microtiter well is filled with the antigen-antibody mixture. Direct ELISA Sandwich ELISA is a type of direct ELISA that helps to detect the presence of antigens in a sample. The microtitre well is coated by the antibody. The sample containing the antigen is added to the well and washed to remove free antigens. The enzyme-specific substrate is added to the plate to form a colored product, which can be measured. Indirect ELISA Indirect ELISA detects the presence of an antibody in a sample. The antigen is attached to the wells of the microtitre plate. A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen. The free primary antibodies are washed away and the antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody. All the free secondary antibodies are washed away. A specific substrate is added which gives a colored product. The absorbance of the colored product is measured by spectrophotometry. Sandwich ELISA Sandwich ELISA is a type of direct ELISA that has a coated antibody and a free secondary antibody that helps to detect the presence of antigens in a sample. The microtitre well is coated by the antibody. The sample containing the antigen is added to the well and washed to remove free antigens. Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies. The enzyme-specific substrate is added to the plate to form a colored product, which can be measured. Competitive ELISA Competitive ELISA helps to detect antigen concentration in a sample. The microtitre wells are coated with the antigen. Antibodies are incubated in a solution having the antigen. The solution of the antigen-antibody complex is added to the microtitre wells. The well is then washed to remove any unbound antibodies.  More the concentration of antigen in the sample, the lesser the free antibodies available to interact with the antigen, which is coated in the well. The enzyme-linked secondary antibody is added to detect the number of primary antibodies present in the well. The concentration is then determined by spectrophotometry The principle of ELISA: It depends on the highly specific antigen-antibody interaction. Requirements for ELISA test: Purified antigen or Purified antibody Standard solutions(positive and negative controls). Sample to be tested. Microtiter plates. Wash fluid (buffer). Enzyme labeled antibody and enzyme substrate.  Enzymes used in ELISA: The most commonly used enzyme labels in ELISA are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Complete, ready-to-use ELISA kits: Coated ELISA kits. Uncoated ELISA kits. Specimen sample for ELISA: (Serum _ CSF _ Sputum _ Urine _ Semen _Supernatant of culture _ Stool ) Results of ELISA test: Quantitative Qualitative Semi-quantitative  What is an ELISA reader? An ELISA reader is a highly sophisticated instrument whose job is to detect the presence of a protein in a liquid sample. The ELISA reader is one of the most popular versions of a device called a plate reader. components of the system: ELISA plate Light source Optical system Optic fiber ELISA results