Task 1 Immunoprecipitation curve of human albumin and estimation of albumin concentration by means of immunoturbidimetry

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1 ÚSTAV LÉKAŘSKÉ BIOCHEMIE 1. LF UK Selected Immunochemical Methods Block course of practical lessons on medical biochemistry General Medicine Lenka Fialová (translated by Jan Pláteník) 2010/2011

2 In this block of practical lessons we will get familiar with basic principles of selected immunochemical methods. An immunoprecipitation curve will be made, simultaneously serving as an example of immunoturbidimetry, one of the methods where the immmunoprecipitation takes place in a solution. Next, two techniques employing immunoprecipitation in gel will be presented: double radial immunodiffusion according to Ouchterlony, which will be both practically performed and evaluated, and single radial immunodiffusion according to Mancini, which will be only evaluated. Finally, an ELISA (enzyme-linked immunosorbent assay) will be included, representing a group of highly sensitive methods employing labelled reactants. Task 1 Immunoprecipitation curve of human albumin and estimation of albumin concentration by means of immunoturbidimetry 1. Principle: Immunoprecipitation curve shows amount of the formed immunoprecipitate at different amounts of soluble antigen and constant amount of the corresponding antibody. It is obtained if to a series of test tubes with the same amount of antibody in each tube we add increasing amounts of antigen (Fig. 1). Fig. 1 Immunoprecipitation curve After incubation, during which antigen reacts with the antibody, the amount of immunoprecipitate is measured in each tube. The immunoprecipitate consists of a space lattice formed by interconnected epitopes of antigens and the corresponding paratopes of antibodies. When the aggregate exceeds certain size, it loses its solubility and precipitates in the solution. The prerequisite for immunoprecipitation is a reaction of antigen possessing several epitopes (polyvalent antigen) with antibodies that recognize these epitopes. If this requirement is not met, the immunoprecipitation does not occur. That is why haptens, which are equipped only with one epitope, cannot precipitate. Likewise, monoclonal antibodies that are directed against a single epitope are not suitable for immunoprecipitation. In our experiment human albumin and ram antibody against the human albumin will be used for construction of the immunoprecipitation curve. The albumin is an important protein present in plasma at high concentration. 2

3 We will use concentrations of albumin that cover all parts of the precipitation curve: the area of antibody excess, the zone of equivalence and the area of antigen excess. Cloudiness resulting form the reaction of albumin with the corresponding antibody can be estimated by means of immunoturbidimetry. Using a common spectrophotometer we can measure intensity of light that passes through the cloudy solution in forward direction. For the estimation of albumin concentration in an unknown sample the linear, ascending part of the precipitation curve (the area of antibody excess) will be used. It is important to stress that if the immunoprecipitation is to be used for measurement of concentration of some substance, the reaction mixture must contain the antibody in excess. If this condition is not kept, for high antigen concentrations we would get false low values. The sample must always be prediluted to a concentration where the antibody is in excess, and multiply the result with the dilution factor used. Fig. 2 shows an example. Fig. 2 Evaluation of immunoturbidimetry The same values of absorbance can be found for sample 1 with concentration of the estimated substance that lies in region of antibody excess, as well as for sample 2 with a high amount of the estimated substance, which lies in region of antigen excess. In case of sample 2 a false low value would be obtained under this condition. Immunoturbidimetric methods are widely used for estimation of various analytes in biological fluids (e.g. in serum, urine, cerebrospinal fluid). 2. Procedure: Reagents: a) Stock solution of human albumin 400 mg/l b) Ram antiserum against human albumin c) Phosphate buffer, ph mol/l d) Unknown sample 1. Predilution of albumin Stock solution of human albumin at concentration 400 mg/l is successively diluted with phosphate buffer (0.01 mol/l). The first two dilutions (test tubes 1-3) are geometric. Then we continue by performing an arithmetic dilution of the solution in test tube 3. Tube No. Buffer (ml) Albumin solution (ml) Final albumin concentration (mg/l) (from stock solution) (from tube 1) (from tube 2) 100 Geometric dilution (from tube 3) (from tube 3) (from tube 3) 25 Arithmetic dilution 3

4 2. Actual immunoprecipitation reaction The prediluted albumin solutions are pipetted to new test tubes 1 6 (the tube numbers correspond to the ones in the previous table). To other two tubes 7 8 the original undiluted unknown sample and the same sample diluted with phosphate buffer 1 : 1 (0.2 ml of the sample and 0.2 ml of buffer) are measured, respectively. The tube no. 9 serves as the blank sample. Then the antiserum against human albumin is given to all tubes according to the table: Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Measure Albumin solution (mg/l) Unknown sample Blank in ml undil. dil. Albumin prediluted Sample Buffer Antiserum The tubes are mixed and allowed to incubate 30 minutes at room temperature. After the incubation absorbance of all the solutions is measured against the blank in 1 cm cuvette at wavelength 310 nm. 3. Evaluation: a) Plot the measured absorbances of mixtures with prediluted albumin against the corresponding albumin concentrations, construct the precipitation curve and describe it. b) Read the concentration of albumin in the unknown sample from the linear ascendent part of the curve. The value for the diluted sample must be multiplied with two. Compare the results obtained for the diluted and undiluted sample and explain the difference. Task 2 Double radial immunodiffusion according to Ouchterlony 1. Principle: Double radial immunodiffusion is an example of imunoprecipitation method that takes place in a gel medium. In this technique, antigen and antibody are spotted to vicinal wells and diffuse radially against each other. During diffusion a concentration gradient arises: the concentration declines with distance from the well. After certain period of time the antigen and the the responsive antibody meet each other and can react. In the area where the concentrations of antigen and antibody reach the zone of equivalence (see the immunoprecipitation curve), a precipitation line results (Fig. 3). Fig. 3 Course of diffusion of antigen and antibody in double radial immunodiffusion 4

5 If more different antigens are present in the sample and the antiserum contains antibodies against them, several precipitation lines result. Position of the precipitation line between wells depends on concentration of the reactants. The line will be closer to the well where the reactant concentration is lower (Fig. 4). Molecular weights of the reactants have an influence as well. Proteins having higher molecular weight diffuse more slowly. Fig. 4 Position of the precipitation line in relation to reactant concentration Successively diluted antigen Undiluted 1:2 1:4 1:8 1:16 In modification according to Ouchterlony the wells that are cut to the gel form a rosette. An antigen can be applied to the central well whereas antibodies would go to the peripheral ones. Such arrangement can be used e.g. for determination of antibody titre. A sample of tested antibody is diluted and various dilutions are applied to the wells around antigen. When diffusion is completed, we can observe changes in position of the precipitation line, which gets farther from the central well with dilution. If antibody concentration is low, the line can even disappear because the zone of equivalence has not been reached and precipitation has not occurred. Alternatively, an antibody can be applied to the center and antigen to the surrounding wells. In this way an antigen in unknown samples can be demonstrated with a known antibody, or similarity of antigens can be tested. In the latter case we evaluate shape of precipitation lines that can display immunochemical identity, non-identity or partial identity (explained in detail in the text Immunochemical Techniques). In our experiment we will use a human serum and a polyspecific antiserum against it. The antiserum will be placed to the central well and to the peripheral wells a successively diluted human serum will be applied (Fig. 5). Fig. 5 Scheme of applications in Ouchterlony double radial immunodiffusion Human serum in various dilutions We will take notice of the number of lines and shifts in their positions in relation to the decreasing concentration of human serum. 5

6 2. Procedure: Reagents and tools: a) boiled agarose in barbital buffer, ph 8.6 (3.68 g of 5,5 -diethylbarbituric acid is dissolved in distilled water with heating, after cooling 20.6 g of sodium 5,5 -dietylbarbiturate is added, volume adjusted to 1,000 ml with distilled water) POISON b) horse antiserum against human serum c) human serum INFECTIOUS MATERIAL d) physiological solution (9 g/l NaCl) e) staining solution (0.3 g of Amidoblack 10 B and 30 ml of glacial acetic acid is moderately boiled with constant stirring, after cooling 270 ml of methanol p.a. is added and the solution is filtered) POISON f) destaining solution (250 ml methanol, 50 ml glacial acetic acid and 200 ml distilled water) POISON g) glass plates 5 x 5 cm h) punch for cutting the wells i) needle with bended tip Dilution of sample Sample of human serum is diluted geometrically with physiological solution according to the following table. Dilution is performed in microtubes: Microtube No. Physiological solution (ml) Serum (ml) Resulting serum dilution (from stock solution) concentrated (from Tube 1) 1 : (from Tube 2) 1 : (from Tube 3) 1 : (from Tube 4) 1 : (from Tube 5) 1 : 32 Preparation of agarose slab a) Find a clean and degreased glass plate 5 x 5 cm. b) Bring agarose on an electromagnetic stirrer to boiling and cool to about 55 C. c) Using a macropipette with cut tip carefully pour about 5 ml of liquid agarose on the glass plate. The agarose should cover the whole plate. However, prevent flow of agarose over the glass edges. Remove any eventual bubbles in the gel by pricking with needle. Leave the agarose to solidify about 20 minutes in wet chamber. d) Press the punch carefully on the solid agarose gel to cut the wells in hexagonal arrangement with one central well. Remove the agarose from the wells with a needle with bended tip. e) Cut one more well in the upper right corner of the gel slab. It will serve to proper orientation of the gel during evaluation of the results. Application of samples a) According to the provided scheme (Fig. 5) fill the peripheral wells with about 8 µl of the samples of diluted serum. Avoid overflow of the wells (the volume can be less than 8 µl). Use an automatic pipette with a new tip for each dilution. Finally, apply about 30 µl of the antiserum to the central well; again, take care to prevent overflow of the well. 6

7 Immunodiffusion and further processing of the plate a) Place the gel slab in horizontal position to the wet chamber and allow to diffuse for 48 hours. b) Remove the unreacted components of serum and antiserum by washing in physiological saline for about 1.5 hours. In this phase, white precipitation lines can be observed against a dark background. c) 30 minutes of washing in distilled water follows. d) Sprinkle the plate using a squirt bottle and fill the wells with distilled water. Wrap the plate with a pre-wet filter paper so that the paper adheres to the whole surface of the agarose gel. Push any eventual air bubbles to the plate edges. e) The plate wrapped in filter paper is left to dry in the oven set to 60 C. f) After drying wash the filter paper with distilled water and remove it. Wash out any remnants of paper with a squirt bottle. g) Stain the plate by dipping it to the staining solution for about 10 minutes. Then wash with water and destain about 10 minutes in destaining solution. The background should finally be colorless. h) The stained plate can be dried at room temperature, or alternatively in the oven for about 10 minutes. 3. Evaluation: Number of lines and their positions among wells are assessed. Task 3 Evaluation of single radial immunodiffusion for estimation of IgG and IgM 1. Principle: Antigen (in this case human IgG or IgM), which is applied into a round start, radially diffuses into agarose gel that contains a specific (animal) antibody. Reaction of antigen with the specific antibody produces a precipitation ring whose area is proportional to the amount of antigen. 2. Procedure and evaluation: 1. Construction of calibration curve Measure diameters of the precipitation rings for the standard solutions marked S 1 S 8 and by means of a special ruler read the second power d 2. Fill the standard d 2 in the table: Standard No. d 2 (mm 2 ) Standard concentration Standard concentration for IgM (g/l) for IgG (g/l) Plot the second powers of standard diameters and the corresponding concentration values on a graph paper, d 2 on the y-axis and concentration on the x-axis. 2. Estimation of IgG or IgM concentration in unknown samples Measure diameters of the rings of 5 unknown samples, read the second powers and then the concentrations from the calibration curve. Normal values: IgG: 8 18 g/l IgM: g/l 7

8 Task 4 Estimation of antibodies in unknown samples by means of ELISA test 1. Principle of method: The enzyme immunoanalysis (EIA) methods represent a large group of immunoanalytical techniques that employ an enzyme as a label. Technique called ELISA (enzyme-linked immunosorbent assay) is one of the EIA. A typical feature of ELISA is that either antigen or antibody is firmly anchored to a solid phase, usually a surface of the wells in microtitration strips. The ELISA methods are divided to competitive and non-competitive (sandwich). They can be used for estimation of antigens or antibodies. Our model ELISA kit is an example of a sandwich enzyme immunoanalysis designed for estimation of specific antibodies, such as the antibodies against various infectious agents (bacteria, viruses, etc.), autoantibodies directed against the body s own structures or IgE antibodies against particular allergens. In this kit a specific antigen, which can be e.g. some virus or allergen, is attached on a surface of the wells in microtitration strips. In the unknown samples that will be added to the wells, presence of antibodies against this antigen will be examined. If antibodies against the antigen are present in the sample, they will bind to it and form immunocomplexes (Fig. 6, the first step). After washing out the unbound sample components the immunocomplexes are detected with the conjugate of animal antibody against human antibody (antiimunoglobulin anti-ig) with enzyme peroxidase. A sandwich originates, consisting ot the antigen anchored to the well surface, the human specific antibody and the conjugate (Fig. 6, the second step). The unbound conjugates must be removed by washing. The amount of attached labelled antibodies is visualized with an enzymatic reaction, catalyzed by peroxidase. After removal of the unbound molecules of conjugate by washing we add a substrate that is converted to a colored product by the action of peroxidase. The enzymatic reaction is stopped by addition of an acid (stop solution). Presence or absence of antibodies in tested samples is evaluated according to the color intensity (Fig. 6, the third step). A quantitative or qualitative way of evaluation can be employed. Reaction conditions of non-competitive ELISA The non-competitive ELISA for estimation of antibodies requires the amount of antigen bound to the well surface is in excess over the expected amount of antibodies. In case of insufficient amount of the antigen some of the antibodies in tested samples could not bind and would be removed in the washing step. The resulting concentration of antibodies in the sample would be false low. Likewise, the amount of added conjugate must be in excess. A lack of conjugate would again lead to a false low result, because the requirement that all the immunocomplexes made of the antigen and the antibody get the conjugate bound, would not be met. Explanation of some terms 1. Washing: between particular steps of the ELISA procedure the unbound components of the serum (the first step) or the unbound enzyme-labelled antibody (the second step) must be removed. It is done by filling the wells with washing solution followed by its removal. This treatment is repeated several times. If the enzyme-labelled antibodies were not washed out, the resulting color in the wells would be the same regardless of the amount of specific antibodies in the samples. 2. Conjugate: denotes a junction of two or more substances by means of chemical or another reaction. In the EIA methods usually an animal antibody against a human immunoglobulin (anti-ig) or antigen is conjugated with a label, e.g. an enzyme, which enables detection of the immunocomplexes. In our ELISA method the conjugate is anti-ig with peroxidase (anti-ig/px). 3. Substrate solution: depends on the enzyme present in the conjugate. For peroxidase a benzidine derivative tetramethylbenzidine is used, whose reduced form is colorless. By oxidation with hydrogen peroxide in the presence of peroxidase it is converted to a colored product (Fig. 7). If the peroxidase conjugate is absent from the wells, the color conversion of tetramethylbenzidine does not occur and the contents of the wells remains colorless. 8

9 Fig. 6 Procedure of the non-competitive ELISA method for estimation of antibodies First step: binding of specific antibodies from sample on antigen anchored to well surface Second step: addition of conjugate (enzyme-labelled antiimmunoglobulin) to immunocomplexes anchored to well surface and formation of sandwich Third step: visualization of amount of bound conjugates by means of enzymatic reaction 9

10 4. Stop solution is a weak solution of acid that terminates the enzymatic reaction. In order to produce a mutually comparable results, the reaction is stopped after certain period of time. Simultaneously the color changes from blue to yellow, which is measurable with a spectrophotometer at wavelength 450 nm. In case of positive samples the yellow coloration is visible even by a bare eye. Fig. 7 Principle of the chemical reaction catalyzed by peroxidase of the conjugate 2. Procedure: Reagents: 1. ELISA doublestrip with 16 wells 2. standard 3. unknown sample No unknown sample No unknown sample No conjugate anti-ig/px 7. washing solution 8. diluting solution 9. substrate solution (TMB - 3,3,5,5 tetramethylbenzidine and hydrogen peroxide) 10. stopping solution CORROSIVE A. Dilution of samples and standards a. Dilution of samples Every sample will be examined in dilution 21-times. Prepare 3 microtubes and mark them with SA 1, SA2 and SA 3. Use the diluting solution for dilution. Proceed according to the table. Test tube 1 Test tube 2 Test tube 3 Diluting solution ml ml ml SA ml - - SA ml - SA ml Mix the contents in microtubes using vortex. 10

11 b. Dilution of standard The standard is diluted geometrically: Diluting solution (ml) Standard (ml) Final concentration Standard 1 - Stock solution 100 % Standard (from Tube 1) 50 % Standard (from Tube 2) 25 % Standard (from Tube 3) 12.5 % Mix the contents in microtubes using vortex. B. Actual ELISA procedure (Fig. 8) 1. Application of samples and standards According to the provided layout add in doublets (to vertical pairs of wells) 0.1 ml samples and standards. Pipette carefully to the bottom of the wells. The first pair of wells will serve as a blank pipette the diluting solution (DS) instead of the samples. Layout: 1 2 A DS Standard 4 B DS Standard 4 C Standard 1 SA 1 D Standard 1 SA 1 E Standard 2 SA 2 F Standard 2 SA 2 G Standard 3 SA 3 H Standard 3 SA 3 Close the strip with the provided lid and allow to incubate for 15 minutes at room temperature. During this incubation the antibodies present in the sample react with the antigen immobilized to the strip wells. 2. Removal of unbound serum components After the incubation turn the strip upside down above the sink and with a moderate swipe empty its contents to the sink. Then pipette 0.3 ml of washing solution to each well. Gently shake the strip and remove the contents by aspiration or by emptying to the sink as above. Repeat the washing step 5-times in total. Finally dry the strip by turning upside down and pressing onto a layer of tissue. 3. Addition of conjugate Add 0.1 ml of conjugate (anti-ig/px) to each well and incubate the strip 30 minutes at room temperature. During this incubation the second antibody against human immunoglobulin labelled with peroxidase binds onto the immunocomplexes formed in the wells during the previous steps. 11

12 4. Removal of unbound molecules of conjugate After the incubation wash the wells as described above in the step 2. This time a redundant unbound conjugate is removed. 5. Addition of substrate Add 0.1 ml of the substrate solution (TMB) to each well and incubate about 5 minutes at room temperature. In this step the substrate is converted to a product by the action of peroxidase present in the conjugate. The wells with samples containing antibodies against the immobilised antigen now turn blue. 6. Termination of enzymatic reaction To each well add 0.1 ml of stopping solution, which terminates the enzymatic reaction. The blue color in the wells changes to yellow. Fig. 8 Overview of the ELISA procedure ADDITION OF SAMPLES AND STANDARDS Incubation 15 minutes Washing ADDITION OF CONJUGATE Incubation 30 minutes Washing ADDITION OF SUBSTRATE Incubation 5 minutes TERMINATION OF ENZYMATIC REACTION 12

13 3. Evaluation Performance check of ELISA method In the wells with blank sample (A1, B1) no visible color should develop, because instead of sample they received the diluting solution, which does not contain any antibodies, the conjugate has nothing to bind to and is removed during the washing steps. Weakly yellow color can be caused by the remnants of conjugate due to poor washing. Evaluation of samples If the tested samples contain antibodies against the antigen immobilised to the well surface, after the addition of stop solution the well contents is colored yellow. The intensity of yellow coloration can be evaluated qualitatively or quantitatively. a. Qualitative evaluation Yellow colored samples are considered positive, while the colorless samples are negative. The extent of sample positivity can be assessed by means of diluting (titrating) the sample. In low dilution all the wells with positive samples are strongly colored. If diluted more, the strongly positive samples remain intensely colored, while the weakly positive samples display a color of reduced intensity. 1 Negative samples do not contain any specific antibodies and the unbound conjugate is removed by washing. Therefore, the wells remain colorless. Likewise, antibodies against other antigens are not bound by the antigen in the wells and are also washed out in the subsequent step. b. Quantitative evaluation If a quantitative estimation of antibodies is required, standards in several dilutions must be processed together with the samples. The intensity of yellow color in the wells is proportional to the amount of antibodies present in the sample. The color intensity is estimated by measurement of absorbance using a special spectrophotometer with vertical light beam at wavelength 450 nm. The absorbance values are used for construction of calibration curve. The concentration of antibodies in the unknown samples is then read from the calibration curve. Literature: Ferenčík M.: Imunochémia. Alfa, Bratislava, 1989 Kaplan, L.A., Pesce A.J.: Clinical Chemistry, 3rd edition, Mosby 1996 Příbalový leták k soupravě ELISA-VIDITEST EDUCO-Diagnostic 1 Not employed in this lesson. 13