Grundlagen und Anwendung der Genom- und Proteomforschung

Transcription

1 Grundlagen und Anwendung der Genom- und Proteomforschung Profilmodul (Vorlesung/Seminar WS 2012/13) Philipps-Universität Marburg Fachbereich Biologie Professor Dr. Egon Amann & Dr. Frank Vitzthum Gastbeiträge zum Thema Immunoassays : Dr. Helmut Peters 1

2 Immunoassays, Part I Overview: Introduction to humoral immunology The nature of antigen-antibody interaction Characteristics of antibodies / immunoglobulins Antibodies counterpart: the antigen Immunoassays based on (monoclonal) antibodies as tools for structure elucidation 2

3 Introduction Antigen-antibody interaction is an essential element of the body s first line of defense, the humoral immune response. Antigen-antibody interaction is an example of highly specific ligandreceptor interaction. Due to this property, antibodies may be used for elucidation of complex biological structures. Within the context of this lecture, immunochemical techniques are helpful tools to characterize, to identify or even to purify target antigens. 3

4 Properties of antibodies A group of highly complex glycoproteins produced by the organism in response to invading foreign structures (organisms) Protein molecules (immunoglobulins) with one or more combining sites called paratopes Bind target antigens by highly specific interaction (lock-and-keyprinciple) Bound to the surface of B-Lymphocytes or Circulating in the blood as free immunoglobulins High diversity in antigen recognition, polyclonal in nature 4

5 What is an antigen? A molecule which elicits a specific immune response when introduced into an animal / into man A molecule that triggers antibody generation Generally large molecules (> 10,000 Daltons in size) Chemically and structurally complex Accessible to the immune system Foreign to the host 5

6 Examples of antigens Proteins, including conjugated proteins such as glycoproteins, lipoproteins and nucleoproteins Polysaccharides (e.g. capsular polysaccharides, lipopolysaccharides) Typically presented on the surface of microbes such as viruses, bacteria, parasites or yeasts. Also surface antigens from human cells may elicite an antibody response after contact with the foreign immune system (transfusion, transplantation, pregnancy). Low molecular antigens (e.g. toxins, haptens) become immunogenic when coupled to carrier proteins. 6

7 The nature of antigen - antibody interactions Electrostatic forces => attraction between opposite charges Hydrogen bonds => Hydrogens shared between electronegative atoms Van der Waal s forces => Fluctuations in electron clouds around molecules oppositely polarize neighbouring atoms Hydrophobic forces => Hydrophobic groups pack together to exclude water (involves Van der Waal s forces) 7

8 Antibody structure Composed of 4 chains (2 heavy + 2 light chains) Y-shaped form Bound by -S-S- bridges Containing constant and variable regions => constant regions define structure and immune function 8

9 Structure of immunoglobulin G Generalized structure of an immunoglobulin (IgG) 9

10 Functional parts of antibodies Fab or F(ab ) 2 : Antigen binding fragment (depending on enzyme used to cleave S-Sbridges). Papain => two Fab fragments Pepsin => one F(ab ) 2 fragment Fc : Complement fixing fragment or cell binding fragment Papain => complete dimer of two Fc arms Pepsin => two monomeric Fc fragments 10

11 Antigen recognition site (paratope) The antibodies paratope is complementary to the antigens epitope 11

12 Terminology - Paratope: antibody combining site whose shape is complementary to the antigen epitope (composed of the VH and the VL-region of Ig) - Complementarity defining region (CDR): part of the antigen binding site (paratope) composed of about 50 hypervariable amino acid residues separated by conserved intervals - Epitope: antibody binding site; antigenic determinant - continuous/linear epitopes defined by a single continuous amino acid sequence - discontinuous / structural epitope 12

13 Lock-and-key principle: Hypervariable regions of the antibody or CDRs (Complementarity defining regions) 13

14 The five classes of Ig-antibodies 14

15 Oligomeric Immunoglobulins Monomeric IgA or IgM are covalently fused by joining chain (J-chain) to dimers or pentamers, respectively. 15

16 The five classes ( isotypes ) of human Ig-antibodies (Part 1) IgG (four subclasses IgG1-IgG4) => Gammaglobulin, predominant antibody found in blood and lymph. Can cross placenta; predominant antibody involved in maternal immunity. IgA IgA1 (serum), IgA2 (secretory) => Predominant antibody found in saliva, tears, sweat, milk, intestinal secretions, and colostrum. Primary defense against local infections on mucosal surfaces IgM Macroglobulin => First antibody type produced during a clonal response. Bound to lymphocytes (as a monomer) and circulating in serum (as a pentamer) 16

17 The five classes ( isotypes ) of human Ig-antibodies (Part 2) IgD Surface bound => Bound to the surface of lymphocytes, very low concentrations in serum. Function unclear IgE Parasite Protection => Has a role in the protection from parasites. Low levels in serum 17

18 Properties of major immunoglobulin classes (1) IgG (four subclasses IgG1-IgG4), monomeric form - Enables cell agglutination, virus neutralization; potent complement activator - Molecular weight: ~150,000 D - H-chain type: gamma (53,000 D) - Serum concentration: 10 to 16 mg/ml - Percent of total immunoglobulin: 75% 18

19 Properties of major immunoglobulin classes (2) IgA IgA1 (serum), IgA2 (secretory) - Molecular weight of secretory IgA: 320,000 D - H-chain type: alpha (55,000 D) - Serum concentration: 1 to 4 mg/ml - Percent of total immunoglobulin: 15% 19

20 Properties of major immunoglobulin classes (3) IgM Macroglobulin, pentameric form - B-cell antigen receptor (monomeric form) - Enables cell agglutination; potent complement activator - Molecular weight: 900,000 D - H-chain type: µ (65,000 D) - Serum concentration: 0,5 to 2 mg/ml - Percent of total immunoglobulin: 10% - Major immunoglobulin during primary immune response against specific antigen 20

21 Definition of terms - Affinity: binding force between single antibody binding site (paratope) and corresponding antigen binding site (epitope) - Avidity: strength of binding of a multivalent antibody with a multivalent antigen - Antibody class switch: during differentiation of B-cells, antibody class may change from IgM (primary response) to other classes 21

22 Binding strength of antibodies and their complementary antigens 22

23 IgG is the preferred isotype for immunochemical analysis - Highest concentration in serum/plasma - Antigen-specific titer and avidity can be boostered by further immunization with the target antigen ( immune priming ) - Well defined structure; easy to purify from the mixture of plasma proteins - Established enzymatic techniques to separate Fc from Fab or F(ab ) 2 - Well established conjugation techniques (e.g. FITC, HRPO, Biotin ) - Hybridoma technology offers option to produce taylored antibodies in large scale (Köhler & Milstein, 1975) 23

24 Properties of polyclonal (pabs) and monoclonal antibodies (mabs) pabs - Higher lot-to-lot variation - Broader reactivity; often recognize multiple epitopes - Often more sensitive - More options regarding animal species - Shorter lead times - Lower initial costs - Prefered for therapeutic use of broadly reactive immunoglobulins mabs - High reproducibility - Indefinite supply - High specificity; advantage in differentiation of closely related antigens - Mice as the prefered host organism - Longer lead times - Higher initial costs - Prefered for therapeutic use to battle specific targets such as tumour antigens 24

25 Generation of monoclonal antibodies 25

26 Generation of monoclonal antibodies (part 1) 26

27 Generation of monoclonal antibodies (part 2) From Darwish, I.A.: Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances; I J B S, vol. 2 no. 3 (2006), pp

28 Major steps in generation of monoclonal antibodies Immunization of mice with the antigen (mixture) containing the desired target epitope (native, denatured, complexed /conjugated.) Screening for reactive hybridoma supernatants in appropriate assay format maintaining native binding conditions (e.g. EIA) =>Nota bene : most mabs are poor agglutinins! Selection and propagation of clones; confirmation of initial reactivity or further analysis of fine specificity Characterization of subclass => important for Fab / Fc cleavage efficiency! Large scale production in cell culture 28

29 Surprising experiences in mab screening mab may show target specificity not expected from the initial immunization protocol (e.g. in case that complex immunization antigen has been used) =>final mab specificity may be more interesting than original target antigen Mice may not show an appropriate immune response (e.g. antigen concentration too low, antigen denatured, tolerance against self antigens ) 29

30 Monoclonal antibodies are particularly suited for epitope mapping - Cristallography of antigen/antibody complexes (epitope/paratopepairs) is the gold standard for elucidation of protein or carbohydrate structures - Differentiation of linear / conformational epitopes - Carbohydrates may show repeating epitopes (=> ladder like pattern of LPS in Western Blots) - Size of an epitope some 5 to 15 amino acid or 3 to 4 sugar residues - Binding site on linear epitopes may be identified by overlapping peptides 30

31 Continuous and discontinuous epitopes 31

32 Immunoassay techniques Due to the nature of the antigen-antibody pair, immunoassays are suited to detect or even to quantify either the antigen or the antibody part. Solid phase immunoassays are particularly suited because they offer specific solutions within short term. Techniques are well established and automation allows large scale screening. Assay components may be easily purified by immunoaffinity purification, thus allowing even higher sensitivity and specificity. 32

33 Immunochemical techniques for epitope analysis (1) Agglutination reaction (may be evaluated visually in case of particles, e.g. by hemagglutination, or quantified by Nephelometry in case of soluble antigens, e.g. by antisera against plasma proteins 33

34 Immunochemical techniques for epitope analysis (2a) Immunofluorescence techniques (e.g. Microscopy, FACS) Examples: microbial or cellular antigens Koivunen,

35 Immunochemical techniques for epitope analysis (2b) (Fluorescence activated cell sorting) 35

36 Immunochemical techniques for epitope analysis (3) Enzyme immunoassays (EIA) or Enzyme linked immunosorbent assays (ELISA) are widely established in research laboratories. Typical assay formats are the indirect, competitive, or the antigen capture/ sandwich format. 36

37 Characteristics of Indirect Enzyme Immunoassay (EIA) for identification of antigen Assay principle similar to indirect immunofluorescence assay: - Antigen is fixed to solid phase, e.g. to MTP surface or to polystyrene bead - First (antigen-specific) antibody is incubated ; antibody excess is washed away - Second, labeled antibody is incubated which finally converts chromogen substrate to coloured product which is measured by photometry => Signal generation if specific target antigen is present Potential for false positives depending on purity of solid phase antigens 37

38 Example : Structure analysis of bacterial polysaccharides 38

39 Example, Part 1 In E.coli R1 and R4 core LPS, ECA is covalently bound to the LPS and is copurified by the extraction procedure 39

40 Example for repeating epitopes in carbohydrates: Ladder- like structure of enterobacterial common antigen (ECA) 40

41 Example : fine specificity of mabs against carbohydrate structures 41

42 Competitive EIA principle for antigen detection An ELISA can also be performed as a competitive assay. This is common when the antigen is small and has only one epitope, or antibody binding site. One variation of this method consists of labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody. A decrease in signal from purified antigen indicates the presence of the antigen in samples when compared to assay wells with labeled antigen alone 42

43 Characteristics of Competitive Immunoassays Signal decrease with increasing analyte concentration 43

44 Antigen capture immunoassay (Sandwich immunoassay) Y Y Y Y Y Y Y EY E E E Solid phase coated antibody Sample Antigen PODconjugate directed against analyte wash Chromogen TMB 44

45 Characteristics of Antigen Capture Immunoassays - Signal increase with increasing analyte concentration - No quantification (screening assays with +/- differentiation) - High sensitivity + high specificity (high sample concentration needs sophisticated blocking buffers) Example: Blood donor screening for Hepatitis B surface antigen (HBsAg) in human serum. 45

46 Immunochemical techniques for epitope analysis (4) Western Blot / Dot Blot - Antigens are separated by SDS-PAGE and tranfered to nitrocellulose sheets or - purified antigens are dotted to nitrocellulose sheets => Nitrocellulose membrane is used as solid phase for immunoassays with specific antibodies against target antigens 46

47 Western Blot 47

48 Epitope mapping by immunochemical techniques: fields of application Cancer research Autoimmune diseases / Transplantation Hematology / Hemostasis Vaccine research Infectious disease diagnostics 48

49 Example (Vaccine Research) 49

50 Example (Plasma protein analysis) 50

51 Review article for further studies 51