Immunochemical methodsare based on the formation of immunocomplex from specific antibody and antigenVytek 2008

The antibody-antigen complexThe binding of antibody to antigen is noncovalent and reversible and it is entirely dependent on noncovalent interactionsThe many weak noncovalent interactions include hydrogen bonds, van der Waals forces, coulombic interactions and hydrophobic bondsStrength of interaction is described by affinity constantKA = [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 methodsImmunoassaysImmunoblotting (Western blotting)ImmunoprecipitationImmunoaffinity chromatography (specific kind of affinity chromatography)Immunosensors

ImmunoassaysThe 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 immunoassaysAccording the labelradioisotope - RIA radioimmunoassayenzyme - EIA enzymimmunoassayfluorescent dye (e.g. fluorescein)

Direct labellingIndirect labeling (nonlabeled primary specific Ab is marked by addition labeled secondary Ab against Ig of promary Ig)

According separation of immunocomplexHomogenousHeterogenous

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

Antibody attached to solid phase

Sandwich EIA (two antibody assay)

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

Concentration of antigenEstimation of antigen by competetive EIA

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

Immunoblotting procedure stepsResolution of proteins by gel electrophoresisTransfer of separated polypeptides to a membrane support (eg. nitrocellulose)Blocking remaining nonspecific binding sites on membrane (usually by nonfat milk)Incubation with primary specific antibodyDetection

Immunoblots of tryptic digests of COMP 0 mmol/l Ca2+0C, 30 min12.5 mmol/l Ca2+ 37C 6hod12.5 mmol/l Ca2+ 0C, 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

ImmunoprecipitationImmunoprecipitation is the oldest imunological technique Immunoprecipitation is used for detection in diffusion immunological techniquesImmunoprecipitation 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)

Immunosensorsprinciple 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 sensorQCM (quartz crystall microbalance) - decrease of frequence of quartz crystal oscilator is proportional to the bound mass to crystallSPR (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