Principles and methods of elisa, fpia,
- 1. PREPARED BY: ASMA MA FIRST M.PHARM
- 3. ELISA (Enzyme-linked Immunosorbent Assay) is a quantitative immunological procedure in which the Ag-Ab reaction is monitored by enzyme measurements. It is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones.
- 4. The term ELISA was first used by Engvall and Perlma in 1971. The basic steps in ELISA includes: 1. Immobilization of Antigen to a solid surface, 2. Complexation of Antigen with specific antibody (primary), 3. Attachment of primary antibody to enzyme linked secondary antibody, 4. Reaction of substrate with conjugated enzyme to produce a coloured product, 5. Measurement of coloured product.
- 5. ELISA is a popular format of "wet-lab" type assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. It involves detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed). The analyte is also called the ligand and it will specifically bind to a detection reagent.
- 7. The ligand-specific binding reagent is "immobilized” onto a stationary solid phase /solid substrate known as the "ELISA plate". It is then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate which form a coloured product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
- 10. ELISAs begin with a coating step, where the first layer, either an antigen or an antibody, is adsorbed to a well in an ELISA plate. Coating is followed by blocking and detection steps as shown in the simple schematic diagram. Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signal.
- 12. ▪ ELISA plates ▪ Buffers ▪ Antibodies ▪ Enzymes ▪ Sample handling and preparation ▪ Substrate
- 13. Plate format Flat-bottomed, 96-well plates, made from polystyrene or polyvinyl chloride, are coated with either inactivated Ag/Ab. The coating act as the binding site for the Ag/Ab present in the sample. Further variants are 384-well and 1536-well plates.
- 14. Plate characteristics It maintain consistency, minimizing edge effects and providing optimal optical conditions for data collection. Low to medium binding type (100–200 ng of IgG/cm2). High-binding plates (400–500 ng of IgG/cm2). Antigen or antibody pre-coated plates are also commercially available.
- 15. Standard buffers Several different buffers are used during an ELISA: one for coating, another for blocking, another for washing, and perhaps another for sample and antibody dilution. Buffers can be produced in house or sourced from a variety of commercial antibody and reagent suppliers.
- 16. Coating buffers Coating is the process where a suitably diluted antigen or antibody is incubated until adsorbed to the surface of the well. Adsorption occurs passively as the result of hydrophobic interactions between the amino acids side chains on the antibody or antigen used for coating, and the plastic surface. It is dependent upon time, temperature, and the pH of the coating buffer, as well as the concentration of the coating agent.
- 17. Typical coating conditions involve adding 50-100 μl of coating buffer, containing antigen or antibody at a concentration of 1-10 μg/ml, and incubating overnight at 4°C or for 1-3 hours at 37°C. Coating buffers stabilize the antigen or antibody which is used to coat the ELISA multiwell plate, maximizing adsorption to the plate and optimizing interactions with the detection antibody. The two most common coating buffers are bicarbonate buffer at pH 9.6 or PBS.
- 18. Blocking buffers To prevent the non-specific binding of detection antibodies. There are two main types of blocking agents, proteins and detergents. Proteins-permanent blocking agents and hence added after the capture antibody has adsorbed to the well surface. Detergents-block temporarily, blocking function disappears during washing steps. The most basic blocking buffer contains 1% BSA or milk proteins dissolved in PBS. Usually 150 μl of blocking buffer is added to the well to incubate for 1 hour at 37°C in order to fully block the plate.
- 19. Washing buffers To remove unbound materials. Usually PBS, with a small concentration of a non- ionic detergent such as Tween-20 is used. Washing is typically repeated 3-5 times between each step in the ELISA to thoroughly remove unbound material. Excess wash solution must be removed in the final wash step to prevent the dilution of the reagents added in the subsequent stage.
- 20. The antibodies used in ELISA assays can be monoclonal, polyclonal, or a combination of both. The interaction between antibodies and their antigens is described in three ways: specificity, affinity, and avidity. Specificity is an indication of whether an antibody binds solely to a unique antigen. Affinity describes the strength of binding of an antibody to a single antigen. Avidity accounts for the total stability of the antibody- antigen interaction.
- 21. Monoclonal antibodies Monoclonal antibodies can be used for all antibody- containing steps in all types of ELISAs. Monoclonal antibodies are homogeneous by definition, with specificity for a single epitope or small region of a protein. Polyclonal antibodies Polyclonal antibodies are complex antibody pools which represent a collection of specificities to various epitopes found in a single antigen. They can also be used as capture and detection antibodies.
- 22. The most commonly used enzyme labels are ▪ Horseradish peroxidase (HRP) ▪ Alkaline phosphatase (AP) ▪ β-galactosidase, ▪ Acetylcholinesterase, ▪ Catalase
- 23. The specimen samples for ELISA includes: ▪ Serum ▪ CSF ▪ Sputum ▪ Urine ▪ Semen ▪ Supernatant of culture ▪ Stool In the simplest case, ELISA samples are diluted in PBS, wash buffer, or other specialty buffers and applied in a final volume of 100 μl.
- 24. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. HRP- ABTS (2,2-azinobis {3-ethylbenzothiazoline-6-sulfonic acid}-diammonium salt), OPD (o-phenylenediamine dihydrochloride), TMB (3,3,5,5-tetramethylbenzidine). AP- PNPP (p- nitrophenyl phosphate), Disodium salt. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer, etc).
- 25. 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA 4. Competition/Inhibition ELISA
- 26. An antigen is immobilized in the well of an ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP.
- 27. Advantages: Direct ELISA detection is much faster than other ELISA techniques. The assay is also less prone to error since fewer reagents and steps are needed. Best for analyzing the immune response to an antigen. Disadvantages: Antigen immobilization is not specific. Less flexible. No signal amplification- reduces assay sensitivity.
- 28. Antigen is adsorbed to a well in an ELISA plate. Detection is a two-step process. First, an unlabeled primary antibody binds to the specific antigen. Second, an enzyme conjugated secondary antibody that is directed against the host species of the primary antibody is applied.
- 29. Advantages: Economical High sensitivity Greater flexibility Best for determining total antibody concentration in samples. Disadvantages: Possibility of background noise Longer procedure than direct ELISA technique. Additional incubation step for secondary antibody needed.
- 30. Sandwich ELISAs require the use of matched antibody pairs (capture and detection antibodies). Each antibody is therefore specific for a different and non- overlapping region or epitope of the antigen. The capture antibody, binds the antigen that can then be detected in a direct ELISA or in an indirect ELISA configuration.
- 31. Advantages: High sensitivity - 2-5 times more sensitive than direct or indirect ELISA. High specificity - two antibodies are involved in capture and detection. Flexibility - both direct and indirect detection can be used. Best for analysis of complex samples, since the antigen does not need to be purified prior to measurement. Disadvantages: Antibody optimization can be difficult-cross- reactivity may occur between the capture and detection antibodies.
- 32. The competition/inhibition ELISA, also known as a blocking ELISA, is the most complex of all the ELISA techniques. Used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Sample antigen or antibody competes with a reference for binding to a limited amount of labeled antibody or antigen, respectively. The higher the sample antigen concentration, the weaker the output signal, indicating that the signal output inversely correlates with the amount of antigen in the sample.
- 34. Advantages: No sample processing is required and crude or impure samples can be used. More robust - less sensitive to sample dilution and sample matrix effects than the sandwich ELISA. More consistent - less variability between duplicate samples and assays. Maximum flexibility - it can be based on direct, indirect or sandwich ELISA. Commonly used when only one antibody is available for the antigen of interest. It is also suitable for detecting small antigens that cannot be bound by two different antibodies such as in the sandwich ELISA technique.
- 36. Used for determining serum antibody concentrations. Monoclonal Antibody Screening. For Virus test (HIV, West Nile Virus, NDV). For Home Pregnancy Test. In Food industry for detecting potential food allergens E.g. Milk, Peanuts, Almonds, Eggs and Walnuts. Disease diagnosis E.g. HIV, bird flu, common, colds, cholera, STD etc. Detection of enterotoxin of E. coli in feces. To study drug toxicity.
- 38. Fluorescence polarization immunoassay (FPIA) is a class of in-vitro biochemical test used for rapid detection of antibody or antigen in sample. It is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps. The basis of the assay is fluorescence anisotropy, also known as fluorescence polarization.
- 39. If a fluorescent molecule is stationary and exposed to plane-polarized light, it will become excited and consequently emit radiation back to the polarized-plane. If the excited fluorescent molecule is in motion (rotational or translational) during the fluorescent lifetime, it will emit light in a different direction than the excitation plane.
- 40. The rate at which a molecule rotates is indicative of its size. When a fluorescent-labelled molecule (tracer) binds to another molecule the rotational motion will change, resulting in an altered intensity of plane-polarized light, which results in altered fluorescence polarization. Fluorescence polarization immunoassays employ a fluorophore bound antigen that when bound to the antibody of interest, will increase fluorescence polarization. The change in polarization is proportional to the amount of antigen in sample, and is measured by a fluorescence polarization analyzer.
- 42. FPIA quantifies the change in fluorescence polarization of reaction mixtures of fluorescent-labelled tracer, sample antigen, and defined antibody. Operating under fixed temperature and viscosity allows for the fluorescence polarization to be directly proportional to the size of the fluorophore.
- 43. Free tracer in solution has a lower fluorescence polarization than antibody-bound tracer. The tracer and the specific antigen will compete to bind to the antibody and if the antigen is low in concentration, more tracer will be bound to the antibody resulting in a higher fluorescence polarization and vice versa.
- 44. 1. A specific quantity of sample is added to reaction buffer and the solution is allowed to equilibrate at room temperature for approximately 2 minutes. 2. The solution is evaluated in a fluorescence polarization analyzer to gather a baseline measurement. 3. A specific quantity of antigen conjugated with fluorophore is added to the solution and equilibrates for approximately 2 minutes. 4. The fluorescence polarization value for the tracer containing solution is compared to the baseline and magnitude of difference is proportional to quantity of target analyte in sample.
- 45. FPIA has emerged as a viable technique for quantification of small molecules in mixtures, including: Pesticides, Mycotoxins in food, Pharmaceutical compounds in wastewater, Metabolites in urine, Serum indicative of drug use (cannabinoids, amphetamines,barbiturates, cocaine, benzodiazepines, met hadone, opiates) and other small molecule toxins. The analysis of hormone-receptor interactions.