RADIOIMMUNOASSAY (RIA)

Transcription

1 RADIOIMMUNOASSAY (RIA) József Németh Department of Farmacology and Farmacoterapy University of Debrecen

2 Fig. 1: Introduction and history of RIA Development: S. Berson R. Yallow (insulin, 1960) Principle: the method is based on a reversible competition of labelled and non-labelled antigen molecules for the limited binding sites of the antibody Detection limit: 0.1 fmol/ml Advantage: the radioimmunoassay combines the specificity of antigenantibody reaction and the high sensitivity of radiation measurement techniques Nobel Prize award: 1977

3 Fig. 2: Necessities of RIA 1. Antigen 2. Radiolabelled antigen 3. Antigen specific antibody 4. Optimal conditions of assay 5. Adequate separation technique Sample preparation

4 Fig. 3: Antigen Antigen is a foreign molecule (for example: protein, hormone, drug, peptide, steroid) that is capable inducing an immune response in vertebrates Categories: - complete antigens (immunogens), Mw > 5,000 - incomplete antigens (haptens), Mw < 5,000 Application: - immunization - RIA standard (high purity)

5 Fig. 4: Radionuclides 3 H 125 I 14 C 35 S 32 P Half life: 12,2 a 60,1 d 5730 a 88 d 14,3 d Type of radiation: β- γ, rtg β- β- β- Energy of radiation: 18 35, (kev)

6 Fig. 5: 125 I-labelling of tyrosine Oxidation methods: chloramine-t iodogen hydrogen peroxide I - Oxidáció 125 I I I - O O O N H H C C N H H C C N H H C C CH 2 CH 2 CH 2 I 2 ph 7,5 + I I I OH OH OH

7 Fig. 6: Chloramine-T Iodination process: - 10 nmol synthetic hg-17/20 μl 0.25 mol/l (ph:7.5) phosphate buffer MBq carrier-free Na 125 I/5 μl 0.04 mol/l NaOH - 10 μg chloramine-t/10 μl 0.25 mol/l (ph:7.5) phosphate buffer - reaction time: 20 sec (room temperature) - 20 μg sodium metabisulphite/10 μl 0.25 mol/l (ph:7.5) phosphate buffer - HPLC purification

8 Fig. 7: Iodogen Iodination process: - 80 μg Iodogen/100 μl dichloromethane - dryness μl 0.25 mol/l (ph:7.5) phosphate buffer - 15 nmol Tyr (0) -thrittene/30 μl 0.25 mol/l (ph:7.5) phosphate buffer MBq carrier-free Na 125 I/5 μl 0.04 mol/l NaOH - reaction time: 5 min (room temperature) μl 0.1% (v/v) TFA - HPLC purification

9 Fig. 8: Purification methods Procedures: - dialysis - gel filtration (Sephadex) - paper electrophoresis - strach gel electrophoresis - ion-exchange chromatography - high performace liquid chromatography (HPLC)

10 9. Fig: HPLC radio-chromatogram of 125 I-gastrin-17

11 Fig. 10: HPLC radio-chromatogram of 125 I-Tyr (0) -Thrittene

12 Fig. 11: Antiserum, Antibody Definitions: Antiserum is a serum from the immunized animal, containing antibodies Antibodies are immunoglobulins produced by lymphoid cells in response to antigens by immunization Immunoglobulins are classified into five groups: IgG (80 %), IgM, IgA, IgD, IgE Property of immunoglobulins is that react specifically with theirs antigen in vivo and in vitro

13 Fig. 12: Produce of antibody Substances (Mw > 5,000) are good immunogens themselves Haptens (Mw < 5,000) must be coupled to a carrier protein Couplig methods: (haptens + protein: BSA, TRG) - mixed anhydride (-COOH) - glutaraldehyde (-NH2) - carbodiimide (-COOH, -NH2) Production: - immunization polyclonal antibodies - hybridization monoclonal antibodies

14 Fig. 13: Characterization of antiserum 1. Titre is a dilution of the antiserum which binds 50 % of labelled antigen in RIA measuring circumstances in the absence of nonlabelled antigen 2. Specificity of the antiserum gives information about the selectivity of binding between the antibody and the antigen 3. Affinity of the antiserum gives information about the strength of the interaction between the antigen and the antibody

15 Fig. 14: Titration curve of TH3/6 thrittene antiserum

16 Fig. 15: Cross-reaction data of the TH3/6 thrittene antiserum Thrittene 100,00 (%) Tyr (0) -thrittene 96,49 Somatostatin-28 1,12 Somatostatin-28(1-14) 0,28 Somatostatin-28(1-12) 0,00 Somatostatin-14 0,00 Somatostatin-14(3-14) 0,00 Cortistatin-14 (rat) 0,00 Cortistatin-29 (rat) 0,00 Cortistatin-17 (human) 0,00 Cortistatin-29 (human) 0,00

17 Fig. 16: Antigen-antibody reaction k 1 k 2 Ag + Ab AgAb AgAb Ag + Ab (association) Ag: free antigen Ab: free antibody AgAb: bound complex (dissociation) k 1 : rate constant of association k 2 : rate constant of dissociation Law of mass action: [AgAb] k 1 K = = [Ag] [Ab] k 2 (High affinity means a sensitive method.)

18 Fig. 17: Optimal conditions of RIA Test tube: Volume and material of test tube (5 ml ; glass, polypropylene, polystyrene) Buffer: - composition - ion-concentration - ph (Requirement is all the reagents must be intact in the buffer until end of incubation.) Incubation: - temperature - time

19 Fig. 18: Separation techniques Separation allows the examination of equlibrium state 1. Adsorption method: Separation of the free antigen Adsorbents: dextran coated charcoal, cellulose, glass powder After centrifugation, the free antigen fraction appears in the pellet, the bound remains in the supernatant 2. Precipitation method: Isolation of the bound antigen - Precipitation (organic solvent, salt) - Double antibody precipitation - Double antibody + Organic solvent precipitation After centrifugation, the free antigen fraction remains in the supernatant, the bound appears in the pellet

20 Fig. 19: Process of radioimmunoassay Order of reagents are added in a RIA tube 1. RIA buffer 2. antiserum 3. RIA standard or unknown sample 4. radiolabelled antigen Incubation Separation Measurement of radioactivity Evaluation

21 Fig. 20: Principle of RIA

22 Fig. 21: Calibration curve of thrittene RIA

23 Fig. 22: Sample preparation (plasma) 1. Direct measurement: - high antigen concentration - high assay sensitivity 2. Extraction techique: - low antigen concentration - middle/low assay sensitivtiy Extraction: isolation of the substance from other biological materials and it is used to concentrate it Extraction methods: - organic solvent precipitation (alcohol, acetone) - adsorption on a solid phase (glass powder, reversephase silica gel, immunosorbent)

24 Fig. 23: Sample preparation (tissue) Examined substance must be extracted from the tissue Steps of the procedure: - sample collection - freezing (stops degradation) - weighing - homogenization in distilled water (10 %, w/v) - centrifugation (10,000 rpm, 4 o C, 10 min) Supernatants are used for radioimmunoassay determination. The antigen concentrations of tissues are expressed as fmol/mg wet tissue weight.

25 Fig. 24: RIA methods developed in our laboratory D 50 (fmol/ml) Plasma Tissue Gastrin 1.81 ± 0.11 Gastrin ± 1.09 Pro-gastrin ± 1.23 Gly-gastrin ± 4.02 Flanking peptide ± 1.96 Glucagon 8.89 ± 1.56 Substance-P ± 3.56 CGRP 4.81 ± 0.35 Somatostatin ± 3.92 PACAP ± 3.47 PACAP ± 3.68 VIP 6.26 ± 0.59 Thrittene 8.61 ± 1.22 GLP ± 2.02