AG - AB REACTION by akashyap

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									Precipitation reactions
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Precipitation is the result of the reaction between soluble antigen and antibody, precipitate settles down Requires at least two antigenic determinants per molecule and bivalent antibody Each antibody combines with two different antigen molecules Not widely used, mostly used for ag detection

Precipitation Reactions
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A lattice forms until the complex is large enough to become insoluble and precipitate Precipitin - an antibody involved in precipitation reactions Flocculation is the result when the precipitate floats. E.g, VDRL test and RPR test for syphilis,

Zone phenomenon/ lattice hypothesis
prozone

postzone

Types of precipitation
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Precipitation in liquid medium – capillary ppt (ring ppt), slide ppt and tube ppt Preicipitation in gel – test can be done as a single diffusion (only one reactants diffuse) or as double diffusion ( both reactants diffuse)

Fluid phase precipitation
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Passive diffusion of antigen and antibody into each other, forms precipitate Carried out in slide/ tube/capillary tube Antigen solution is layered over antibody in a capillary tube

Precipitation in fluid phase
RPR

E.g: capillary precipitation for streptococcal typing , Ascoli’s thermoprecipitation for anthrax diagnosis Eg: Slide ppt- VDRL test and RPR test for syphilis E.g: Tube ppt: Kahn flocculation test for syphilis

Precipitation in gel
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Single diffusion in one dimension Double diffusion in one dimension Single diffusion in two dimension – single radial immunodiffusion – Mancini’s technique Double diffusion in two dimensions – Ouchterlony procedure Immunoelectrophoresis Countercurrect immunoelectrophoresis Rocket immunoelectrophoresis

Single radial immunodiffusion (Mancini)
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Antiserum (antibody) is incorporated into the gel Wells are punched in the gel Antigen solution is placed in the well Precipitation occurs in a ring at the zone of equivalence Uses: estimation of Ig and complement components

Ab in gel
Ag

Ag

Ag

Ag

Diameter2

Ag Concentration

Radial immunodiffusion

Ouchterlony procedure
Wells are punched in the gel  One well is filled with the antigen and one well filled with antibody  Both antigen and antibody diffuse  Types of reactions a. Identity b. Non-identity c. Partial identity
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Test can be also used to detect different antigens

Immunoelectrophoresis
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Combines electrophoretic separation, diffusion and precipitation Makes use of principle that proteins ( ag) are negatively charged in an alkaline Ph and have different molecular wt Used to separate serum proteins and identify the various fractions

Immunoelectrophoresis
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Method
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Ags are separated by electrophoresis

– Ab is placed in trough cut in the agar

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Ag Ag
Ab

Ag Ab

• Interpretation
– Precipitin arc represent individual antigens

COUNTERCURRENT IMMUNOELECTROPHORESIS(CI E)
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Modification of Ouchterlony procedure Ag and Ab are driven towards each other in an electric field Ag are strongly negatively charged and Ab are weakly charged

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Parallel columns of wells punched in gel Antigen in one side and antibody in the other Electric current is passed through the gel At pH 8.6 the antigen migrates to the anode, and antibody to the cathode Used in Rapid detection diagnosis of meningococcal ag /cryptococcal Ag in CSF,HBsAg in blood, alpha foetoproteins

Countercurrent electrophoresis
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Method
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Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges

Ag Ab

+

• Qualitative
–Rapid

Rocket immunoelectrophoresis( Laurel technique)
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Antiserum is incorporated into the gel Antigen is placed in the well and electrophoreses done Precipitation occurs along lateral boundaries of migration pattern

contd

RADIOIMMUNOASSAY
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The technique was introduced as an assay to estimate concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.

RIA

The Technique
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A mixture is prepared of radioactive antigen (I 131 or I125) & antibodies against that antigen. Known amounts of unlabeled antigen are added to samples of the mixture. These compete for the binding sites of the antibodies. At increased concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured. From these data, a standard binding curve can be drawn

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Diagnosis of allergies Endocrinology – estimation of hormones Detection of drug molecules Detection of viral antigens – Hepatitis B ag in blood Very sensitive to detect the antigen

Immunofluorescence
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Principle: Fluorescent dyes like fluorescein isothiocyanate or rhodamine can be conjugated to the ab without altering the specificities. These labeled antibodies can be used to detect ag on microorganisms and tissues. Uses: -Detection of viral ag in tissues -Detection of antinuclear ab in autoimmune diseases

IMMUNOFLUORESCENCE
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Tests for Cell Associated Antigens Lattice formation not required

Immunofluorescence
• Direct
– Ab to tissue Ag is labeled with fluorochrome
Eg . • Detection of rabies antigen on cells •Disadvantage – separate fluorescent conjugates necessary

Fluorochrome Labeled Ab

Ag Tissue Section

Indirect Immunofluorescence
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Indirect
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To Known tissue Ag attached on slide unknown serum is added Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab.

Fluorochrome Labeled Anti-Ig Unlabeled Ab

Ag

• Eg .FTA –ABS for treponema pallidum

Tissue Section

• Qualitative to SemiQuantitative

Immunofluorescence

Immunofluorescence

Immunofluorescence -- Flow Cytometry & (FACS)
• In FAT-Quantitation is difficult • Cells in suspension are labeled with fluorescent tag •Direct or Indirect Fluorescence – Cells analyzed on a flow cytometer – using laser beam, light detector to count single intact cells in suspension
Flow Tip FL Detector

Light Scatter Detector
Laser

Immunofluorescence
• Flow Cytometry cont.
– Data displayed, based on color coded tag, exact no. can be counted
One Parameter Histogram
Green Fluorescence Intensity Red Fluorescence Intensity

Two Parameter Histogram

Unstained cells Number of Cells

FITC-labeled cells

Green Fluorescence Intensity

COMPLEMENT FIXATION TEST
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Lattice formation not required Complement , a protein component of normal serum is utilized during the ag-ab interaction. This phenomenon forms the basis of CFT Requires ag, ab, C and indicator system

CFT
• Methodology
- Ag is mixed with test serum to be assayed for Ab

-Standard amount of complement is added
– Erythrocytes coated with Abs is added (sheep RBC coated with rabbit ab – amboceptor) – Amount of erythrocyte lysis is determined

– Hemolysis indicates NEGATIVE TEST – No hemolysis is Positive test

CFT

Ag
Ag

No Ag
Patient’s serum

Ag

Enzyme Linked ImmunoSorbent Assay (ELISA)
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Ag or Ab bound to solid phase Patients serum containing either AG/ Ab Whether or not these ab bind to ag/ab can be determined by the addition of enzyme conjugated with antihuman immunoglobulin To detect this binding an substrate added, colored products are formed. Intensity of color is measured using spectrophotometer –ELISA reader

ELISA
Done on polyvinyl Microtitre plate Enzyme substrate color • Horse radish o-phenyl-diamine red/ • peroxidase dihydrochloride orange
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Alkaline Phosphatase

p-nitro phenyl phosphatase

yellow

TYPES OF ELISA
Detection of Ag : Sandwich ELISA Direct sandwich Indirect sandwich
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Detection of Ab : Indirect ELISA IgM capture ELISA Competitive ELISA
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ELISA
Sensitivity : High  Specificity : GOOD  Advantages: -- Rapid --Automation Possible --Many samples tested at same time  Disadvantages: False positive results
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Direct ELISA (sandwich ELISA)
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To detect ag in sample Plates coated with specific ab against the ag Ag will bind to coated ab on plate Reaction detected by adding conjugate (antibody conjugated with enzyme) Add Substrate to detect the reaction – colored compound will form Reading done with naked eye or ELISA reader

Ag coated

sera

Indirect ELISA ABs

INDIRECT ELISA
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6

ELISA Plates

Cassette ELISA
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Specific antigens of a virus like HIV or dengue are immobilized on nitrocellulose membrane in a cassette Test serum added ,antibody will bind if positive Wash the unbound Ab ,add conjugate & then the substrate If positive colored spots will appear along with control spot

EXAMPLES
ELISA for Ab: HIV Anti HBsAg after vaccination, HAV,HCV,HEV Rubella, CMV, IgM leptospira Toxoplasma  ELISA for Ag: Bacterial exotoxins, Rota virus in stool Hepatitis B surface ag, HBe Ag Chlamydial ag from cervix
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IMMUNOBLOTTING(WESTERN BLOTTING )

Uses
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Confirmatory test for detection of HIV antibodies Legionella antibodies


								
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