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Immunoassay systems

Brief review on immunoassays.

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Immunoassay systems

  1. 1. Presentation on Immunoassay Systems T.Manoj Kumar
  2. 2. INTRODUCTION:- • An immunoassay is a test that uses antibody and antigen complexes as a means of generating a measurable result. • An antibody:antigen complex is also known as an immuno-complex. • “Immuno” refers to an immune response that causes the body to generate antibodies, and “assay” refers to a test. Thus, an immunoassay is a test that utilizes immunocomplexing when antibodies and antigens are brought together.
  3. 3. TYPES:- • Competative immunoassays. • Non-competative immunoassays. • Homogenous immunoassays. • Heterogenous immunoassays.
  4. 4. Competitive Format • In competitive formats, unlabelled analyte (usually antigen) in the test sample is measured by its ability to compete with labeled antigen in the immunoassay. • The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied.
  5. 5. • Thus, in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format.
  6. 6. One step competitive format • In the one step competitive format (see Figure 1-8), both the labeled antigen reagent (Ag*) and the unlabeled specimen (or test sample analyte) compete for a limited amount of antibody.
  7. 7. Two step competitive format • In the two step competitive format, the antibody concentration of the reaction solution is present in excess in comparison to the concentration of antigen. • Antibody reagent is first incubated with specimen containing antigens of interest; then in the second step, labeled antigen is added. • Remember that in the competitive format, less bound labeled antigen indicates more antigen present in the test sample. assay formats provide several fold improved assay sensitivity compared to one step assay formats.
  8. 8. Noncompetitive (Sandwich) Method • Noncompetitive assay formats generally provide the highest level of assay sensitivity and specificity and are applied to the measurement of critical analytes such as cardiac and hepatitis markers. This format is referred to as a “sandwich” assay because analyte is bound (sandwiched) between two highly specific antibody reagents.
  9. 9. • Noncompetitive assay formats can also utilize either one step or two step methods, as with the competitive assay. • The two step assay format employs wash steps in which the sandwich binding complex is isolated and washed to remove excess unbound labeled reagent and any other interfering substances. • The two step noncompetitive format usually offers the highest specificity and sensitivity of all the assay formats discussed here.
  10. 10. Homogeneous and Heterogeneous Immunoassay Methods • Immunoassay methods that require separation of bound Ab-Ag* complex are referred to as heterogeneous immunoassays. Those that do not require separation are referred to as homogeneous immunoassays. • Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs. Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform.
  11. 11. Heterogeneous Technique principle Non specific adsorbents Low molecular weight ligands are adsorbed by particles such as charcoal and removed by centrifugation Specific adorbents Antibodies to the ligand or to the ligand binding antibody are immobilized on the surface of a Solid matrix such as glass fibres,latex micro particles. The immobilized antibody ligand complex are separated from unbound ligand by washing,filtration,diffusion chromatography The protein bound ligand moves at a rate through the chromatographic medium different from that of free ligand Precipitation by ammonium sulfate The antibody bound ligand is ppt. by ammonium sulfate
  12. 12. HETEROGENOUS IMMUNOASSAYS • It can measure both small and large molecules. • Heterogeneous immunoassay such as the popular EMIT assay on the other hand measures only small molecules such as drugs.
  13. 13. EMIT-homogeneous As [Test antigen] is increased it occupies more of the antibody molecules, so fewer of the enzyme labels are blocked. Substrate ---> product response increases
  14. 14. ELISA-heterogeneous
  15. 15. • The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or antibody (anti-HIV in the screening test for HIV infection) in body fluids or tissue culture supernatents.
  16. 16. • Purified antigen (to detect or quantify antibody). • Purified antibody (detect or quantify antigen). • Standard solutions (positive and negative controls). • Sample to be tested. • Microtiter dishes: plastic trays with small wells in which the assay is done. • Wash fluid (buffer). • Enzyme-labeled antibody and enzyme substrate. • ELISA reader (spectrophotometer) for quantitative measurements.
  17. 17. To detect antibody • Coat the microtiter plate with purified antigen by letting an antigen solution sit in the wells for 30-60 minutes. Wash away unbound antigen with buffer and cover any sites that might nonspecifically bind antibody with unrelated protein (such as solution of powdered milk), again washing away unbound protein. • Add serum sample to be tested for specific antibody to plate and allow specific antibody to bind to the antigen. Wash off unbound antibody.
  18. 18. • Add anti-Ig that will bind to Fc region of specific antibody (for example, anti-human gamma chain that will bind human IgG). The Fc region of the anti-Ig is covalently linked with enzyme. Wash off unbound antibody-enzyme complex. • Add chromogenic substrate: colorless substrate that the enzyme will convert to a colored product. Incubate until color develops; measure color in a spectrophotometer. • The more color that is detected, the more specific antibody is present in the unknown sample.
  19. 19. • Negative controls include • omit the antigen • omit the test antiserum or substitute with an antibody that will not bind the antigen. • Positive control substitutes known positive serum for unknown serum.
  20. 20. TO DETECT ANTIGEN • Coat the microtiter plate with purified antibody to the antigen. Wash away unbound antibody and cover any sites that might nonspecifically bind with unrelated protein. • Add sample to be tested for antigen to plate and allow antigen to bind antibody.Wash off unbound antigen. • Add enzyme-labeled specific antibody to a different epitope of the antigen to make a "sandwich"; wash away unbound antibody.
  21. 21. • Add chromogenic substrate for enzyme that will be converted to a colored product. • Negative control omits unknown antigen; positive control uses known antigen.
  22. 22. How to interpret the results • The amount of colored product is proportional to the amount of enzyme-linked antibody that binds, which is directly related to the amount of antibody that was present to bind antigen or antigen that was present to bind antibody. • If known amounts of antigen or antibody are added, a standard curve can be constructed which will allow the amount of unknown antigen or antibody to be determined.
  23. 23. Fluorescence Immunoassay: • In fluorescence, a photon of an appropriate energy excites the molecule from its ground state to a higher electronic state. • When the molecule returns to the ground state, energy is released as light emitted at a longer wavelength. • The difference between the excitation wavelength and the emission wavelength is the stocks shift. • A large stokes shift in nanometer means that there is a large difference between the excitation and emission wavelengths.
  24. 24. • For example, fluorescein, a common flurophore has small stocks shift at 30 min. It has a maximal absorption at 490nm and emission at 520 nm. • In contrast a rare earth chelate such as europium has a large stocks shift of 270 nm. • It has a maximal absorption at 340 nm and an emission at 610 nm. • In principle, a fluoroimmunoassay using flurometry has an advantage over an enzyme immunoassay using colorimetry.
  25. 25. • In colorimetry, the light absorbed by a sample is related directly to the concentration of the absorbing molecule and is independent of the intensity. • In fluorometry the intensity of the fluorescence emission is directly proportional to the intensity of the incident light. • There are problems associated with fluorescence measurements.
  26. 26. 1) Endogenous fluorophores , such as bilirubin and proteins, can increase the non specific background fluorescence and reduce the sensitivity of FIA. 2) Light scattering by high concentrations of protein, lipid and other particles in serum will reduce the fluorescence signal. 3) The inner filter effect of hemoglobin and albumin will absorb part of the excitation or emission beam. 4) Quenching duo to the non specific binding of albumin and interaction with other specific quenching species may change the quantum yield of the fluorescence.
  27. 27. • An important function of this step is the removal of endogenous fluorescent compounds and interfering substances from the sample prior to the detection step. • This also allows large sample sizes to be used and improves sensitivity as well as sensitivity. • Solid phase FIA is a convenient approach either as competitive FIA or sandwich immunometric assay.
  28. 28. Fluorescence Polarization Assay • Fluorescence Polarization (FP) assays are homogeneous, single-step assays ideally suited for high-throughput screening (HTS) of large numbers of samples. • All FP assays employ a large molecular species, or binding partner (BP), in conjunction with a small, low molecular weight fluorophore-labeled analyte (FA). • When the large BP molecule is an antibody, the assay is referred to as a fluorescence polarization immunoassay (FPIA).
  29. 29. • FPIAs are based on the competition of FA with free (i.e. unlabeled) analyte in the samples or standards for the high affinity binding site an antibody. • A microplate well filled with the FA:antibody complex will give a high FP reading. • Addition of a increasing amounts of unlabeled analyte will result in a competition between the unlabeled FA for the antibody.
  30. 30. • As the competition happens, some of the FA will be released from the antibody, and will resume its intrinsic, rapid rate of rotation. This will cause a detectable loss of FP in the well. • The addition of large amount of analyte will result in a much larger reduction in the mP of the well (well Plotting mP versus analyte concentration allows the construction of a standard curve with a broad dynamic range. This is similar to, but not strictly analagous to, the sigmoidal dose-response curve in a traditional solid phase EIA.
  31. 31. RIA-heterogeneous
  32. 32. • A mixture is prepared of radioactive antigen • Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used. • Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
  33. 33. • At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. • The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured from these data, a standard binding curve.
  34. 34. • The samples to be assayed (the unknowns) are run in parallel. • After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.
  35. 35. Summary • Immunoassays can be either competitive or noncompetitive. • In competitive immunoassays, the amount of antigen is indirectly proportional to the amount of signal. • In noncompetitive immunoassays, the amount of antigen is directly proportional to the amount of signal.
  36. 36. • Homogeneous immunoassays do not require separation of unbound complexes from the bound complexes, and thus are faster and easier to perform then heterogeneous immunoassays. • Heterogeneous immunoassays require the separation of unbound complexes, often utilizing a solid phase reagent such as a magnetic particle or plastic bead.
  37. 37. REFERENCES:- • Wild, David (Ed.). (2005).The Immunoassay Handbook. Kidlington, Oxford: Elsevier. • Evans, Susan (2004, June 15). Retrieved January 19, 2008, from SACB OnlineWeb site: • Bell, Suzanne (2006). Forensic Chemistry. Upper Saddle River, New Jersey: Pearson Prentice Hall. • Moody, David E. (2006).Immunoassay in Forensic Toxicology. Encyclopedia of Analytical Chemistry.

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Brief review on immunoassays.


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