immuno assay by SYAMKRISHNAN M.PHARM

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Types of immuno assay,Different techniques,sensitivity for pharmacy and various other students

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PRESENTED BY: SYAMKRISHNAN T T M.PHARM PHARMACEUTICS 2011-13 GOVT.COLLEGE OF PHARMACEUTICAL SCIENCE,CALICUT MEDICAL COLLEGE

Immuno assay:

Immuno assay “A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample . ”

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4 Classification of immunoassays: Competitive & Non-competitive immunoassays. 2.Homogeneous & Heterogeneous immunoassays. 3.Limited reagent assays & Excess reagent assays.

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5 Competitive format Amount of antigen is indirectly related to the amount of label (signal) in competitive formats

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6 One step competitive immunoassay Two step competitive immunoassay

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7 Noncompetitive (Sandwich) Method Noncompetitive sandwich method of immunoassay

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8 Amount of antigen is directly related to the amount of label (signal) in competitive formats

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9 Homogeneous and Heterogeneous Immunoassay Methods Homogeneous and heterogeneous immunoassays

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10 Limited reagent assay: Many conventional RIAs follow limited reagent assay protocols. The following scheme depicts the AgAb reaction: Ag AgAb + Ab Ag* Ag* Ab With limited amount of Ab , the unlabeled antigen ( analyte ) competes with the labeled antigen Ag* for limited binding sites. Bound fraction [ AgAb ] is separated from free [ Ab ], and the signal [Ag* Ab ] complex i.e. the Ab fraction not occupied by the analyte is measured. The amount of analyte is inversely proportional to the bound [Ag* Ab ] complex in a hyperbolic function.

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11 Excess-Reagent assay This protocol is utilized by- 1. Immunoradiometric Assays (IRMA). 2. Two-site or sandwich Assays. Here the excess Ab is labeled.(In case of sandwich assay an excess amount of first Ab used to capture analyte from sample matrix, and a labeled second Ab provides the signal for quantitation .) IRMA : Ag + Ab * AgAb * Sandwich assay: Ag + Ab1 Ag-Ab1 + Ab2* Ab1-Ag-Ab2* Bound fraction is separated from free; the signal [ AgAb *] or [Ag-Ab1-Ab2*] complex is measured. The amount of analyte is proportional to the bound complex in a hyperbolic function.

Immuno diffusion techniques:

Immuno diffusion techniques 2 types 1,Radial immuno diffusion 2,Double immuno diffusion

Double immunodiffusion Pattern of precipitin line:

Double immunodiffusion Pattern of precipitin line Identity Non identity Partial identity

Immuno assays:

Immuno assays 1.Immunoelectrophoresis. 2.Radio immuno assay. 3.Enzyme linked Immuno sorbent assay(ELISA). 4.ELISPOT Assay. 5.Western Blotting. 6.Immunoprecipitation. 7.Immunofluorescence. 8.Flow cytometry and Fluorescence. 9.Immunoelectron Microscopy.

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Immunoelectrophoresis: Separation by electrophoresis with identification by double immunodiffusion . Technique is useful in determining whether a patient produces abnormally low amounts of one or more isotypes , characteristic of certain immunodeficiency disease. Or overproduce some serum protein such as albumin, immunoglobulin,or transferrin . Eg:distorted arc in myeloma

Rocket electrophoresis:

Rocket electrophoresis - vely charged antigen is electrophoresed in a gel containingAb . The ppt formed b/w Ag and Ab has the shape of a rocket,the height of which is propotional to the concentration of Ag in the well

Radioimmunoassay S.A.BERSON & ROSALYN YALOW:

Radioimmunoassay S.A.BERSON & ROSALYN YALOW PRINCIPLE: competitive binding of labelled Ag and Unlabelled Ag to a high affinity Ab. Step1:( Ab+labelled Ag known concn )+unlabelled Ag (unknown concn ). Labels used tritium,I 125 Test Ag= serum, body fluids, etc

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Step2. labelled Ag is added excess. The added unlabelled Ag will decrease the amount of labelled Ag bound on the Ab. If the concn of unlabelled Ag is increased that will dcrease the amount of bound labelled Ag.

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I 125 HBsAg bound to anti- HBsAg % Concn of unlabelled HBsAg

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To understand the amount of labeled Ag bound, The Ag-Ab complex is separated from free Ag. And the Radioactivity is measured from the ppt. After removing complex the free labeled Ag (supernatant) can be measured using radiation counter. Subtracting this value from total labeled Ag= Labeled Ag bound.

ELISA:

ELISA E- Ab + [s] = coloured product. E= are alkaline phosphatase , horseradish peroxidase , beta galactosidase.etc

types:

types Indirect ELISA Sandwich ELISA Competitive ELISA Chemiluminescence

Indirect ELISA:

Indirect ELISA

Sandwich ELISA:

Sandwich ELISA

Competitive ELISA:

Competitive ELISA Wash Wash Ag coated well Ad enzyme coated 2°Ab Add substrate measure color Ag- Ab ( incubate Ab with Ag to be measured ) Ag- Ab Ag E- Ab - Ab -Ag S E- Ab Ab -Ag

chemiluminescence:

chemiluminescence Highly sensitive alternative to Absorbance measurements in ELISA assays. Eg : Oxidation of compound Luminol by H2O2 and the enzyme horseradish peroxidase (HP) produce light. Ab -HRP+ Ag ==== Ab -HRP-Ag======light luminol+H2O2

ELISPOT ASSAY:

ELISPOT ASSAY Modification of ELISA assay Quantitative determination of the number of cells in a population that are producing Ab specific for a given Ag

Western Blotting:

Western Blotting Identification of a specific protein in a complex mixture of proteins can be accomplished by a technique known as Western blotting. proteins are separated in a SDS PAGE. protein bands are transferred to a nitrocellulose membrane with Radiolabeled or E .

Immunoprecipitation:

Immunoprecipitation Centrifuge or magnet Ab Ag- Ab Ag/ ce Ag- Ab - Ab - Ag Ag Ab Ag- Ab - Ab - Ag- Ab - Ab -

Immunofluorescence:

Immunofluorescence Ag- Ab -F + Fluorescence microscope equipped with u.v light source. Fluorescent dye or Fluorochrome used 1,Fluorescein 2,Rhodamine 3,Phcoerythrin

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inverted microscope

applications:

applications Identify sub populations of Lymphocytes, CD8+,CD4+ T cell Bacterial population,Ag-Ab complex in Autoimmune disease, Ags in tissue sections

Flow cytometry and Fluorescence:

Flow cytometry and Fluorescence Quantitative measurement. FACS: analysis and separation of cell stained with fluorescent Ab.

sorting:

sorting A-B+ cells A+B- cells A-B- cells A+B+ cells

Immunelectron microscopy:

Immunelectron microscopy Ferritin & colloidal gold used as a electron cocentrating agent. Different size of this can be used to identify different Ag. They appeared as dark spot when viewed under electron microscopy.

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Sensitivity of various immunoassays Assay (g antibody/ml) Precipitation reaction in fluids 20–200 Precipitation reactions in gels Mancini radial immunodiffusion 10–50 Ouchterlony double immunodiffusion 20–200 Immunoelectrophoresis 20–200 Rocket electrophoresis 2 Radioimmunoassay 0.0006–0.006 ELISA 0.0001–0.01 ELISA using chemiluminescence 0.0001–0.01† Immunofluorescence 1.0 Flow cytometry 0.06–0.006

REFERENCES:

REFERENCES Kuby’s Immunology.Page no.155-169 en.wikipedia.org/wiki/ Antibody en.wikipedia.org/wiki/ Immunoassay

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