LABELED IMMUNOASSAYS PART 2 ENZYME LINKED IMMUNOSORBENT ASSAY
LABELED IMMUNOASSAYS PART 2 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Lab. 3
LABELED IMMUNOASSAYS • The basic underlying principles of indicator labeled immunoassays are the same • There are differences with respect to the detail of the protocols • The designation given to each test differs according to the label used to detect the antigen/ antibody complexes
Enzyme Linked Immunosorbent Assay (ELISA) • One of many assays collectively called enzyme immunoassays (EIA) • Can be used to detect both antibody and antigen • Very Sensitive (ng & pg/m. L), • Relies on Monoclonal Abs • An enzyme is used as an indicator molecule • The enzyme does not provide detection directly but through the break-down of a substrate
ENZYMES USED IN ELISA • Enzymes used as labels for immunoassay are chosen according to the following criteria: § Turnover number § Sensitivity § Ease and speed of detection § Stability § Absence of interfering factors in patient samples § Availability and cost of enzyme and substrate
ENZYMES USED IN ELISA • The most commonly used enzymes are: § Horseradish peroxidase (HRP) § Alkaline phosphatase (AP) • Each has a high turnover number (rapid conversion of substrate to a product) resulting in high sensitivity • Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options • These include beta-galactosidase, acetylcholinesterase and catalase
ENZYME SUBSTRATES • A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate • Substrates for AP and HRP, depending upon the plate-reading equipment available and the level of sensitivity required in the ELISA • An array of, and substrates is available for use with either enzyme • Chromogenic Color • Fluorogenic fluorometer Fluoroescence spectrophotometer
ENZYME SUBSTRATES • Chemiluminescent and chemifluorescent substrates provide a stronger signal than Chromogenic substrates • Steadily gaining in popularity because of their: • sensitivity (less than 1 pg/ml “ 10 -12”), • large linear range for detection • and excellent antibody conservation
ENZYME SUBSTRATES 1. Chromogenic substrates • A common ELISA substrate for HRP is: • Tetramethylbenzidine (TMB), • TMB is oxidized to yield a blue product that is water-soluble and absorbs light at 650 nm • The reaction can be halted by addition of acid or another stop reagent • Using sulfuric acid turns TMB yellow color which may be read at 450 nm
ENZYME SUBSTRATES • The most common chromogenic substrate for alkaline phosphatase is: • p-nitrophenyl phosphate (PNPP) • PNPP yields a yellow reaction product that is water-soluble and absorbs light at 405 nm
ENZYME SUBSTRATES 2. Chemiluminescent substrates • Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxide results in creation of an excited state product called 3 -aminophthalate • This product decays to a lower energy state by releasing photons of light • Using chemiluminescence allows multiple exposures to be performed to obtain the best image • Sensitivity: picogram or femtogram level (1. 0 × 10 -15 grams)
ENZYME SUBSTRATES • Fluorogenic substartes • A Fluorogenic Substrate is a nonfluorescent material that is acted upon by an enzyme to produce a fluorescent compound • Different substartes are also available for AP and HRP enzymes • p-Hydroxphenylpropionic acid (HPPA) is used for HRP enzyme
LINKAGE OF ENZYME The enzyme label is linked to antibody or analyte by several means 1. Glutaraldehyde is often used as a crosslinker to join amino groups of the enzyme and the molecule to be labeled 2. Maleimide derivatives are also used to attach the enzyme label § The heterobifunctional cross-linker
LINKAGE OF ENZYME 3. Treatment with periodate • Glycoproteins such as horseradish peroxidase can be activated for conjugation by treatment with periodate • This provides a mild and efficient way of generating reactive aldehyde groups for subsequent conjugation with amine- or hydrazide-containing molecules
LINKAGE OF ENZYME 4. The use of biotinavidin binding • The secondary Ab is labeled with biotin • An enzyme-avidin conjugate is added
COATING OF MICROPLATE • A key feature of the solid-phase ELISA is that antigens or antibodies can be attached to surfaces easily by passive adsorption • This process is commonly called coating • Most proteins adsorb to plastic surfaces, probably as a result of hydrophobic interactions between nonpolar protein substructures and the plastic matrix
COATING OF MICROPLATE • Since most of proteins' hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside • Partial denaturation of some proteins results in exposure of hydrophobic regions and ensures firmer interaction with the plastic • This can be achieved by exposing proteins to low p. H or mild detergent
ELISA PROTOCOL 1 - Coating antibody or antigen onto the microplate § Dilute the protein to be coated in a buffer such as PBS or Carbonate-Bicarbonate and add 100 μl of this solution per well § Incubate for 18 -20 hours at room temperature or 4°C Coated with Antibody when analysing antigen Coated with Antigen when analysing antibody Analyte = antigen Incubate, wash
ELISA PROTOCOL 2 - Blockage of free binding sites § Block the unoccupied sites on the surface of the well to reduce the amount of nonspecific binding of proteins [blocking agent (200 -300 μl/well)] § § A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites The blocking buffer should improve the sensitivity of the assay by reducing the background interference Analyte = antibody Analyte = antigen Incubate, wash
ELISA Protocol 3. Add sample. Incubate, Wash Analyte = antibody Analyte = antigen 4. Add conjugate. Incubate, Wash E E Analyte = antibody E E Analyte = antigen
ELISA Protocol 6. Incubate, stop, measure color change 5. Add substrate ENZYME Colourless OD Concentration
TYPES OF ENZYME IMMUNOASSAY § Three main types: 1. Competitive ELISA 2. Indirect ELISA 3. Sandwich ELISA
1 - Competitive ELISA • Enzyme activity is inversely proportional to the concentration of the test substance -9 • A sensitivity of nanograms (10 g)/ml can be achieved • This method can be used for measurement of small molecules that are relatively pure • such as insulin, and estrogen
1 - Competitive ELISA Competition ELISA to detect Antigens (Antibody-coated plate) Low [analyte] E 1. Anti-analyte E E High [analyte] E 3. Wash 2. Analyte- E E + sample 1. Anti-analyte Low [analyte] High [analyte] 2. Analyte-E + sample 1. Anti-analyte E E High [analyte] 4. Substrate 3. Wash 2. Analyte-E + sample 1. Analyte
• Indirect ELISA is more sensitive than the competitive • Much assays are capable of detecting concentrations of less than 1 pg/ml (10 -12 g)/ml.
• • Screening of hybridoma supernatants Detecting clinically important antibodies (eg. Autoantibodies) E E E 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. Antigen E E 4. Substrate 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. Antigen
3 - Sandwich ELISA • If the antibody is bound to the solid phase, these assays are called sandwich immunoassays • Antigens captured in these assays must have multiple epitopes • Used for detection of hormones, drugs, tumor antigens, cytokines
3 - Sandwich ELISA E E 1. Anti-analyte 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyte 4. Substrate 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyte
Anti HIV 1/2 TEST KIT
HUMAN IMMUNODEFICIENCY VIRUS The causative agent of acquired immunodeficiency syndrome (AIDS). Belong to the lentivirus subfamily of the retroviridae family. Enveloped, icosahedral, RNA containing particles. Have envelope glycoproteins: gp 120 and gp 41. Infect immune system (T helper lymphocyte; CD 4). Incubation period: 3 – 5 years.
TRANSMISSION Sexual contact (primarily). Sharing contaminated intravenous needles. Infected mother to her baby, either across the placenta, at birth, or via breast milk. Blood transfusions and tissue transplantation.
SIGNS AND SYMPTOMS
TEST KIT INTENDED USE SD HIV-1/2 ELISA 3. 0 kit is sandwich ELISA for the qualitative detection of antibodies to all isotypes(Ig. G, Ig. M, Ig. A) specific to HIV -1 including subtype-o and HIV-2 simultaneously in human serum or plasma
PRINCIPLE SD HIV-1/2 ELISA contains a microplate, which is pre-coated with recombinant HIV ½ antigens (gb 41, P 24, gp 36) on well. During first incubation, anti-HIV in patient serum is bound to the recombinant HIV 1/2 antigens. Following this incubation, all unbound materials are removed by washing. The recombinant HIV 1/2 antigens-enzyme conjugate (gp 41, P 24, gp 36) is bound to anti. HIV 1/2, by performing a sandwich.
PROCEDURE 1. Prepare the strip wells for negative control, positive control and sample 2. Pipette 100 ul of sample diluent to each well 3. Add 50 ul of positive and negative controls. 4. Cover the microplate and mix well. 5. Incubate the wells for 30 min at 37 C 6. Wash the wells 5 times with 350 ul of diluted washing solution. 7. Pipette 100 ul of enzyme conjugate to each well 8. Cover the wells 9. Incubate at 37 C for 30 min. 10. Wash 5 times. 11. Add 100 ul of mixed substrates (50 ul A + 50 ul B) 12. Incubate for 10 min 13. Pipette 100 ul of stopping solution 14. Read the absorbance at 450 nm
CALCULATION OF RESULTS Calculate the mean value of the negative controls, then calculate the cut-off value by adding 0. 3. A(neg) + 0. 3 = cut-off Abs. of negative controls = 0. 193 , 0. 167 Abs. of positive controls=1. 216, 1. 260
VALIDATION REQUIREMENT AND QUALITY CONTROL The individual values of the absorbance for the control sera are used to calculate the mean value if 0. 010<A(neg)<0. 200 A (pos)>1. 000 If these specifications are not met, the test must be repeated.
INTERPRETATION OF RESULTS A sample < cut-off anti-HIV 1/2 negative A sample > or equals cut-off anti-HIV 1/2 positive
The End
- Slides: 38