ELISA Techniques PDF

Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2...

Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is a biochemical technique used to detect the presence of an antibody or antigen in a sample. Enzyme-Linked Immunosorbent Assay (ELISA) is also known as an enzyme immunoassay (EIA). Components of ELISA: 1. Antibody: IgG or IgM commonly. 2. Enzyme: Horse Radish Peroxidase (HRP), Alkaline phosphatase. 3. Substrate: TMB (tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. 4. Stop Solution: H₂SO₄, converts the blue Principle: The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding. The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. Types of ELISA 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA 4. Competitive ELISA Direct ELISA: A direct ELISA test which is considered to be the simplest type of ELISA the antigen is adsorbed to a plastic plate, then an excess of another protein is added to block all the other binding sites. 1  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. 1.Apply a sample of known antigen to a surface. 2.Enzyme linked primary antibody is applied to the plate. 3.Washed, after this wash, only the antibody-antigen complexes remain attached. 4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal. The enzyme activity measured is proportional to amount of specific antibody in the original serum. 5 Advantages of direct detection 1. Quick methodology since only one antibody is used. 2. Cross-reactivity of secondary antibody is eliminated. Disadvantages of direct detection 1. Immunoreactivity of the primary antibody may be reduced as a result of labeling. 2  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. 2. Labeling of every primary antibody is time-consuming and expensive. 3. No flexibility in choice of primary antibody label from one 4. experiment to another. 5. Little signal amplification. Indirect ELISA: Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that the secondary antibody may bring about. 1. Antigen is added to plate. 2. Added Blocking buffer. 3. Primary antibody is added. 4. Secondar antibody- HRP is then added which binds to primary antibody. 5. TMB substrate is added, is converted to detectable form. ❖ The enzyme activity measured is proportional to amount of specific antibody in the original serum. 3  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. Advantages Of indirect ELISA 1. Wide variety of labeled secondary antibodies are available commercially. 2. Versatile, since many primary antibodies can be made in one species and the Same labeled secondary antibody can be used for detection. 3. Immunoreactivity of the primary antibody is not affected by labeling. 4. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Disadvantages of indirect ELISA 1. Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. 2. An extra incubation step is required in the procedure Sandwich ELISA: Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection. Moreover, many commercial ELISA pair sets are built on this sandwich ELISA. 1. a. Plate is coated with suitable antibody. b. Blocking buffer is added. 2. Sample is added to plate so antigen is bounded by capture antibody. 3.A suitable biotin labeled detection antibody is added to plate. 4. Enzyme HRPO is added and binds the biotin labeled detection Ab. 5. TMB substrate is added and converted by HRP to colored product. 6. The enzyme activity measured is proportional to the Amount of specific antigen in the original serum. 4  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. Sandwich ELISA advantages: 1. High specificity, since two antibodies are used, and the antigen is specifically captured and detected 2. Suitable for complex samples, since the antigen does not require purification prior to measurement 3. Flexibility and sensitivity, since both direct and indirect detection methods can be used. Competitive ELISA competitive ELISA is a competitive binding process executed by original antigen (sample antigen) and add-in antigen. Solid phase coated with antibody Add unknown amount of unlabeled antigen and known amount of labeled antigen Free and labeled antigen are captured Color formation by oxidation of substrate into a colored compound 5  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. Advantages: Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement. Disadvantages Each antigen may require a different method to couple it to the enzyme. Materials Needed For Elisa Kit 1. ELISA Plate 2. Positive control 3. Negative control 4. Dilution Buffer 5. Conjugate 6. TMB Substrate 7. Stop Solution Before addition of Stop solution After addition of Stop solution 6  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. ELISA results: Quantitative: ELISA data can be interpreted in comparison to a standard curve in order to precisely calculate the concentrations of antigen in various samples. Qualitative: ELISAs can be used to achieve a positive or negative results indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen. Non competitive ELISA curve competitive ELISA curve APPLICATIONS Screening donated blood for evidence of viral contamination by: 1. HIV-1 and HIV-2 (presence of anti-HIV antibodies) 2. Hepatitis C (presence of antibodies) 3. Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels: 1. HCG (as a test for pregnancy) 2. LH (determining the time of ovulation) 3. TSH, T3 and T4 (for thyroid function) Detecting infections: 7  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 2 Dr. Hayder A. 1. Sexually-transmitted agents like HIV, syphilis and chlamydia 2. Hepatitis B and C 3. Toxoplasma gondii Detecting illicit drugs. Detecting allergens in food and house dust 8