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

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

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

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

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

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

Antibody attached to solid phase

Sandwich EIA (two antibody assay)

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

Estimation of antigen by competetive EIA Concentration of antigen

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)

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

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

Densitometric evaluation of imunoblots of COMP from synovial fluid

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

Heidelberger precipitation curve

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)

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