Immunochemical methods are based on the formation of immunocomplex from specific antibody and...

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

Transcript of Immunochemical methods are based on the formation of immunocomplex from specific antibody and...

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

Immunochemical methods

are based on the formation of immunocomplex from specific

antibody and antigen

Vytášek 2008

Page 2: 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

Page 3: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Basic immunochemical methods

Immunoassays

Immunoblotting (Western blotting)

Immunoprecipitation

Immunoaffinity chromatography (specific kind of affinity chromatography)

Immunosensors

Page 4: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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

Page 5: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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

Page 6: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Antigen attached to solid phase

This basic arrangement is used for detection a quantification of specific antibodies (e.g. antiviral)

Page 7: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Antibody attached to solid phase

Page 8: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Sandwich EIA (two antibody assay)

Page 9: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Washing and incubation with secondary antibody labeled with enzyme or radioisotope

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

Page 10: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Concentration of antigen

Estimation of antigen by competetive EIA

Page 11: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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)

Page 12: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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

Page 13: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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

Page 14: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Densitometric evaluation of imunoblots of COMP from synovial fluid

Page 15: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Forming of the immunoprecipitate

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

Page 16: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

Heidelberger precipitation curve

Page 17: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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)

Page 18: Immunochemical methods are based on the formation of immunocomplex from specific antibody and antigen Vytášek 2008.

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