1. Immunochemical methods
are based on the formation of immunocomplex from specific antibody and antigen
Vytášek 2008

2. The antibody-antigen complex
The binding of antibody to antigen is noncovalent and reversible and it is entirely dependent on noncovalent interactions
The many weak noncovalent interactions include hydrogen bonds, van der Waals forces, coulombic interactions and hydrophobic bonds
Strength of interaction is described by affinity constant
KA = [Ab-Ag] / [Ab].[Ag]
Affinity constant, e.g. strength of bond, is dependent on pH, ionic strength, the presence of detergents or chaotropic agents

3. Basic immunochemical methods
Immunoassays
Immunoblotting (Western blotting)
Immunoprecipitation
Immunoaffinity chromatography (specific kind of affinity chromatography)
Immunosensors

4. Immunoassays
The most frequently used immunochemical method for quantification of various substances.
Principle of method is in forming immunocomplex from free antigen and specific antibody with addition the small amount of labeled component (Ab or Ag) and subsequent separation of imunocomplex from its free components.
The level of labeling of immunocomplex is proportional the analytical amount of original free component

5. Types of immunoassays According the label
radioisotope - RIA radioimmunoassay
enzyme - EIA enzymimmunoassay
fluorescent dye (e.g. fluorescein)
Direct labelling
Indirect labeling (nonlabeled primary specific Ab is marked by addition labeled secondary Ab against Ig of promary Ig)
According separation of immunocomplex
Homogenous
Heterogenous

6. Antigen attached to solid phase
This basic arrangement is used for detection a quantification of specific antibodies (e.g. antiviral)

7. Antibody attached to solid phase

8. Sandwich EIA (two antibody assay)

9. Utilizing of immunoassay with attached antigen for estimation of free antigen – competetive EIA
Washing and incubation with secondary antibody labeled with enzyme or radioisotope

10. Estimation of antigen by competetive EIA
Concentration of antigen

11. Immunoblotting (Western blotting)
combines the resolution of gel electrophoresis with specifity of detection by antibodies
semiquantitative method
able to analyse antigens which are soluble even only in solvents with detergent (eg. SDS) or chaotropic compounds (urea, guanidin hydrochloride)

12. Immunoblotting procedure steps
Resolution of proteins by gel electrophoresis
Transfer of separated polypeptides to a membrane support (eg. nitrocellulose)
Blocking remaining nonspecific binding sites on membrane (usually by nonfat milk)
Incubation with primary specific antibody
Detection

13. Immunoblots of tryptic digests of COMP
0 mmol/l Ca2+
0°C, 30 min
12.5 mmol/l Ca2+ 37°C 6hod
12.5 mmol/l Ca2+ 0°C, 30 min

14. Densitometric evaluation of imunoblots of COMP from synovial fluid

15. Forming of the immunoprecipitate
(necessity of polyclonal antibodies and antigen with several different epitopes).

16. Heidelberger precipitation curve

17. Immunoprecipitation
Immunoprecipitation is the oldest imunological technique
Immunoprecipitation is used for detection in diffusion immunological techniques
Immunoprecipitation and subsequent separation of precipitated antigenic polypeptides is modern immunochemical technique for study of antigenic determinants occurred in various proteins under various conditions (e.g. fosfotyrosine during signalling or nitrotyrosine during tissue damage)

18. Immunosensors
principle is similar to heterogenous EIA but the determination of the amount of immunocomplex is done without labeled component by measurement of electric or optical changes on the surface of the sensor
QCM (quartz crystall microbalance) - decrease of frequence of quartz crystal oscilator is proportional to the bound mass to crystall
SPR (surface plasmon resonance) - optical phenomenon ,observed on surfaces of thin layer or noblr metal or on nanoparticles, is proportional to amount of absorbed mass on surface/nanoparticles