2. What is immunoassay? The term “immunoassay” is a combined term of “immuno” (= antigen-antibody-reaction) and “assay” (= determination of the amount of any particular constituent of a mixture ). So, immunoassay means a method to measure any particular substance in a mixture using its specific-binding antibody, for example, one constituent of blood without any purification process.

3. Applications of immunoassays in Endocrinology Immunoassays are used for diagnosis, quantification (example: TSH in screening of the newborn) and monitoring (example: IGF- 1 measurement before and after growth hormone therapy) of endocrine diseases

4. Most immunoassay configurations can be divided into two large groups: – Competitive immunoassays: "limited reagent" methods – Noncompetitive immunoassays: "reagent excess“ methods Both can be divided into heterogenous and homogenous Immunoassays Noncompetitivecompetitive Homogenous Heterogenous RIA Compitive ELISA Heterogenous ELISA (sandwich and indirect) Homogenous EMIT

6. In competitive immunoassays, the analyte and the labeled analyte (tracer) are mixed with a limited amount of anti-analyte antibody. After incubation for a certain period, the bound or the free fraction of the tracer is measured and related to the concentration of the analyte in the sample. In noncompetitive immunoassays, an excess of immunoreactant (antibody or antigen) is added, so that all the analyte is practically in the form of an immunocomplex. Then, the immunocomplex is quantified and related to the analyte concentration in the sample.

7. Heterogeneous immunoassays require the separation of the immunocomplex from the free immunoreactants. – Solid phase is the most common way for separation. But in homogeneous immunoassays, a modulation of the signal occurs as a result of the immunoreaction. Therefore the immunocomplex formation can be monitored directly without prior separation of the bound and free tracer.

9. Brief history…. Berson and Yalow’s discovery of the radioimmunoassay (RIA) for measuring insulin in 1959. The enzyme immunoassay (EIA) was introduced in the 1960s, bringing with it significant advantages, including the replacement of radioisotopes with enzymes, faster reaction times, higher specificities, and longer shelf-life than RIA. Other immunoassays were developed, they all have the same principle, but differ in details.

11. Important components in immunoassay Antibody (antiserum) – In immunoasay, we use antibody as a binding reagent of high specificity and high binding ability, that is, high affinity to the substance to be measured. – We can get antibody by immunizing an animal with such “immunogen” Antibody appears after the first inoculation is IgM-type which is a pentamer of the basic component of antibody. This type of antibody changes to IgG-type after the repeated inoculations. This is called “class switch”, and mostly IgG type of antibodies are used in immunoassay.

12. Antigen and hapten – substances that can produce antibody and can bind to it is called “antigens”, they have large molecular size > 1000 Dalton. – Substances with smaller molecular sizes cannot produce antibody by itself, but they can bind antibody if produced. Those substances are called “haptens”. – In order to get antibody for hapten, it has to be coupled with some carrier proteins of large molecular size.

13. Standard preparation Standard preparation is necessary for immunoassay. Using a standard preparation, we draw a standard curve from graded reaction results of various standard concentrations, and by comparison of a sample reaction result with the standard curve, we get assay value of the sample

14. Labeling materials In immunoassay, it is necessary to use any marker to know the antigen-antibody binding. For such purpose, we label either antigen or antibody with some materials that do not interefere with the binding. We use radioisotopes, enzyme, fluorescent substance.

15. An analyteis anything measured by a laboratory test. In immunoassay testing, the analytemay be either an antibody, or an antigen.

16. ELISA ELISA (Enzyme-linked immunosorbent assay), The ELISA is a rapid test used for detecting and quantifying antibodies or Antigens This assay method utilizes enzyme as a labeling material.  Has many configurations: – Sandwich – Indirect – competitive

17. In ELISA technology, a solid phase consists of a 96-well polystyrene plate, although other materials can be used. The function of the solid phase is to immobilize either antigens or antibodies to which the analyte in the sample will bind, After incubation, the plates are washed to remove any unbound material, finally the reaction is detected using a conjugate. The conjugate consists of either an antigen or antibody that has been labeled with an enzyme.

18. The most common enzymes used as labels for ELISA are: – horseradish peroxidase – calf intestine alkaline phosphatase – E. coli ß-D-galactosidase. These enzymes are typically used because they each meet most, if not all, of the criteria a necessary to produce a sensitive, inexpensive, and easily performed assay.

19. These criteria include: – stability at typical assay temperatures: 4°C, 25°C, and 37°C, – greater than six months shelf life when stored at 4°C, – commercially available, – capable of being conjugated to an antigen or antibody, – inexpensive, – easily measurable activity, – high substrate turnover number, – unaffected by biological components of the assay.

20. Turnover number has two different meanings: In enzymology, turnover number (also termed k cat ) is defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration.enzymologysubstratecatalytic siteenzyme For example, carbonic anhydrase has a turnover number of 400,000 to 600,000 s −1, which means that each carbonic anhydrase molecule can produce up to 600,000 molecules of product (bicarbonate ions) per secondcarbonic anhydrasebicarbonate

21. Some Enzymes Used as Immunoassay Labels: SourceEnzyme Electrophorus electricusAcetylcholinesterase Calf intestineAlkaline phosphatase LiverCatalase Escherichia coli/3-o-Galactosidase AspergiUus nigerGlucose oxidase Leuconostoc MesenteroidesGlucose-6-phosphate dehydrogenase Bacillus species/3-Lactamase HorseradishPeroxidase Escherichia coliPyrophosphatase Jack beanUrease

22. ELISA KIT components

23. ELISA Kit Components Coated Plates The 96-well plates are made of polystyrene and coated with either inactivated antigen or antibody. This coating is the binding site for the antibodies or antigens in the sample. Sample Diluent Most assays require a specific dilution of the sample. Samples are added to the sample diluent and mixed prior to putting them onto the coated plates.

24. Controls The positive control is a solution that contains antibody or antigen. The negative control is a solution without antibody or antigen. The controls help to normalize or standardize each plate. controls are also used to validate the assay and to calculate sample results. In most tests, the controls are prediluted and ready to use. Conjugate ELISA conjugates are enzyme-labeled antibodies or antigens that react specifically to plate-bound sample analytes. unbound conjugate is washed away after incubation and before the addition of substrate.

25. Standareds For calibration curve construction, thought quantitative measurement Substrate For peroxidase conjugates, the substrate is a mixture of hydrogen peroxide and a chromogen that reacts with the enzyme portion of the conjugate to produce color. Wash Concentrate The wash concentrate is a buffered solution containing detergent used to wash away unbound materials from the plates. Stop Solution The stop solution is a strong acid or base that stops the enzyme-substrate reaction and, thereby, the color development.

26. Each enzyme has its coressponding substrate and stop solution The two most common ELISA enzymes:

27. Antigen-Capture (Direct) ELISA In this format, the antigen in the sample is sandwiched between antibodies coated on the plate and an enzyme-labeled conjugate. The antibody conjugate can be either monoclonal or polyclonal. The addition of an enzyme substrate-chromogen reagent causes color to develop,This color is directly proportional to the amount of the target antigen present in the sample.

28. A basic procedure of direct ELISA (sandwich) 1)Standard solutions or assay samples are added to the antibody-coated wells, and incubated for several hours so as to the antigen molecules are captured by “capture antibody”.

29. 2) After this binding reaction, the reaction mixture is discarded, and wells are washed to remove excessive materials. 3) The second antibody which recognizes another epitope in antigen is added. This second antibody has been labeled with an enzyme such as horseradish peroxidase (HRP)

30. 4) The enzyme-labeled second antibody will bind to the antigen which is bound to the capture antibody on the bottom area of wells. This means that the enzyme (HRP) is also fixed on the bottom of wells. The amount of the antigen captured is proportional to fixed enzyme.

31. 5) Enzyme activity is measured by adding a chromogenic substrate of this enzyme. In the case of HRP, tetramethylbenzidine (TMB) is often used. After incubation for some period, the chromogenic substrate is changed to a colored product. The reaction is stopped by adding a reaction stopper “stop solution”, e.g. diluted sulfuric acid, and absorbance is measured using a plate reader.

32. 6) The standard curve is prepared from the concentration of standard solutions and their absorbance. And the sample assay values are obtained from the absorbance using the standard curve (calibration curve). – It is done by plotting standard concentration on X-axis and absorbance on Y-axis, both in normal scale, looks like a linear line signal concentration

33. When both X- and Y-axes are transformed to logarithmic scales, the standard curve looks nearly linear, though still curve linear strictly. By such plotting all the standard points are located with enough intervals with regard to both axes, and also third order regression fits very well due to the slight curve. Manual reading is also easy.

34. ELISA for measurement of antibody ELISA system can be also set up for antibody measurement, called “ indirect ELISA”. In this system, antigen molecule, specific to the antibody to be measured, is adsorbed on the bottom of wells, and samples containing antibody are added to the wells. For estimation of the captured antibody, the second antibody* labeled with enzyme is added and washed out after the binding reaction. Then by incubation with chromogenic substrate, the coloration is measured from the absorbance.

36. L L S P Enzym. reaction Product measurement L IncubationL L Coating

37. Competitive ELISA Sandwich binding principle cannot be applied to small molecular substances, haptens such as steroids some hormons, olgo-peptides, neurotransmitters, etc. Small molecular substances cannot have plural antigenic determinants to form sandwich. For these substances, competitive assay with an enzyme is applied. Sometime it is called competitive ELISA which has competitive binding principle like RIA (radioimmunoassay).

38. Competitive ELISA In this assay system an enzyme-labelled antigen is used which competes with the unlabelled antigen in the sample for binding to the immobilised antibody. The greater the concentration of the antigen in the sample the lower the amount of labelled antigen binding to the antibody.

39. Competitive ELISA steps 1.Antibody to the antigen of interest is immobilised on to the solid phase. This may be a plastic tube, microtitre plate, plastic strip, etc. 2.Washing is carried out to remove unbound antibody. 3.A mixture of labelled antigen and sample solution, containing the antigen of interest, is added. Both labelled and unlabelled antigen compete for binding to the antibody. Controls containing labelled antigen only and a set of standards containing known amounts of unlabelled antigen are also used. Suitable blanks are also included.

40. 1.The plate is washed to remove all unbound antigen. 2.Enzyme substrate is added and after a suitable incubation period the levels of enzyme activity are measured. 3.A graph of enzyme activity versus antigen concentration is drawn and the levels of antigen in the sample determined. The amount of enzyme activity is inversely proportional to the concentration of unlabelled antigen in the sample