Immunochemical methods

are based on the formation of immunocomplex from specific

antibody and antigen

Vytášek 2008

The antibody-antigen complexThe 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

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 label1) radioisotope - RIA radioimmunoassay2) enzyme - EIA enzymimmunoassay3) fluorescent 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 immunocomplex1) Homogenous2) 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)

Washing and incubation with secondary antibody labeled with enzyme or radioisotope

Utilizing of immunoassay with attached antigen for estimation of free antigen – competetive EIA

Concentration of antigen

Estimation of antigen by competetive EIA

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 min12.5 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