Presentation or ppt on immuno techniques By Keerti Gupta

1. TOPIC
IMMUNO TECHNIQUES

2. IMMUNO TECHNIQUES
IMMUNO TECHNIQUES ARE METHODS TO
STUDY IMMUNE SYSTEM AND TO
PRODUCTION AND USE OF ANTIBODIES
TO DETECT SPECIFIC PROTEINS IN
BIOLOGICAL SAMPLES.

3. USES OF IMMUNO
TECHNIQUES
IMMUNO TECHNIQUES ARE USED FOR
INDUCING,MEASURING AND CHARACTERIZING
IMMUNE RESPONSES. THESE CAN ALTER IMMUNE
SYSTEM THROUGH CELLULAR,MOLECULAR AND
GENETIC MANIPULATION.

4. PRODUCTION OF ANTIBODIES
• ANTIBODIES ARE PRODUCED FROM CELLS OR ANIMALS IN
SUFFICIENT QUANTITY TO BE USED AS EXPERIMENTAL REAGENT.
MONOCLONAL ANTIBODIES ARE PRODUCED BY INJECTING MICE
AND POLYCLONAL ANTIBODIES ARE PRODUCED BY INJECTING
RABBITS OR GOATS WITH LIVE OR INACTIVATED MATERIAL
AND COLLECT THE BLOOD OF THOSE ANIMALS THAT HAVE
HIGH TITERS TO THE TARGET AGENT.

5. ANTIBODY ISOLATION AND PURIFICATION
ANTIBODY ISOLATION AND PURIFICATION IS TO
KEEP SPECIFIC ANTIBODIES AND REMOVAL OF
NON-SPECIFIC ANTIBODIES.
PURE ISOLATED ANTIBODIES HAVE GREATER
ACTIVITY AND ARE LESS LIKELY TO CROSS REACT
WITH NON TARGET MOLECULES.

6. ANTIGEN ANTIBODY INTERACTIONS
ANTIBODIES ARE PRODUCED BY THE IMMUNE SYSTEM OF
VERTEBRATES AND ARE ESSENTIAL FOR PREVENTION AND
RESOLUTION OF INFECTION.
AN INDIVIDUAL ANTIBODY CAN REACT WITH ONLY ONE
ANTIGEN.
ANTIBODIES ARE GLYCOPROTEINS THAT BIND SPECIFICALLY TO
ANTIGEN.
THE INTERACTION BETWEEN AN ANTIGEN AND AN ANTIBODY
DEPENDS ON NON-COVALENT BONDS.
LOW AFFINITY ANTIBODY BIND ANTIGEN WEAKLY AND
TEND TO DISSOCIATE WHEREAS HIGH AFFINITY ANTIBODY
BIND ANTIGEN TIGHTLY AND REMAIN BOUND LONGER.

7. PRECIPITATION REACTION
IT OCCURS IN FLUIDS.
REACTION OF SOLUBLE ANTIGEN AND ANTIBODY IN AQUEOUS
SOLUTION FORM A LATTICE OR INSOLUBLE Ag-Ab COMPLEX
(WITHIN 1 MINUTE) THAT DEVELOPS INTO A VISIBLE PRECIPITATE
(IN 1-2 DAYS).
Ab THAT AGGREGRATE SOLUBLE Ag ARE CALLED PRECIPITINS.
FORMATION OF INSOLUBLE Ab-Ag COMPLEX OCCUR IN A
CONCENTRATION RANGE KNOWN AS ZONE OF EQUIVALENCE.

8. IMMUNODIFFUSION TEST
IT OCCURS IN GEL.
WHEN Ag AND Ab DIFFUSE TOWARD ONE ANOTHER IN AGAR
OR WHEN Ab IS INCORPORATED INTO THE AGAR AND Ag
DIFFUSES INTO THE Ab CONTAINING MATRIX ,A VISIBLE LINE
OF PRECIPITATION IS FORMED.
IT IS OF 2 TYPES.
(1) RADIAL IMMUNODIFFUSION(MANIMI METHOD)
(2) DOUBLE IMMUNO DIFFUSION(OUCHTERLONY METHOD)
BOTH ARE CARRIED OUT IN SEMISOLID MEDIUM SUCH AS
AGAR.

9. RADIAL IMMUNODIFFUSION
Ag SAMPLE IS PLACED IN A WELL AND ALLOWED TO DIFFUSE
WITH AGAR.
AGAR CONTAINS A SUITABLE DILUTION OF ANTISERUM.
AS THE Ag DIFFUSES INTO AGAR ,THE REGION OF EQUIVALENCE
IS ESTABLISHED AND A RING OF PRECIPITATION IS FORMED
AROUND THE WELL.

10. DOUBLE IMMUNODIFFUSION
IN THIS METHOD,BOTH Ag AND Ab DIFFUSE RADIALLY FROM WELLS
TOWARD EACH OTHER ESTABLISHING A CONCENTRATION GRADIENT.
AS EQUIVALENCE IS REACHED, A VISIBLE LINE OF PRECIPITATION IS
FORMED.

11. AGGLUTINATION REACTION
INTERACTION BETWEEN Ab AND PARTICULATE Ag RESULTS IN VISIBLE
CLUMPING IS CALLED AGGLUTINATION AND Ab IS CALLED
AGGLUTININS.
PROZONE EFFECT
EXCESS Ab INHIBITS AGGLUTINATION REACTION IS CALLED PROZONE
EFFECT.
CAUSES
AT HIGH Ab CONCENTRATION,THE NO. OF Ab BINDING SITES GREATLY
EXCEED THE NO. OF EPITOPE.SO,Ab VALENCIES WILL NOT BE FULFILLED
AND THERERFORE CAN NOT CROSS LINK TO Ag.
IT IS OF 3 TYPES.
(1)DIRECT AGGLUTINATION TEST
(2)INDIRECT OR PASSIVE AGGLUTINATION TEST
(3)HEMAGGULUTINATION TEST

12. DIRECT AGGLUTINATION
WHEN THE ANTIGEN IS AN INTEGRAL PART OF THE SURFACE OF A CELL
IT IS CALLED DIRECT AGGLUTINATION.
INDIRECT AGGLUTINATION
WHEN COATED CELLS ARE USED IT IS CALLED INDIRECT
OR PASSIVE AGGLUTINATON.
HEMAGGLUTINATION
CLUMING OF RBC IS CALLED HEMAGGLUTINATIN.

13. IMMUNOASSAYS
IMMUNOASSAYS ARE BIOANALYTICAL METHODS THAT
IDENTIFY AND QUANTIFY ANALYTES IN BIOLOGICAL SAMPLES.
IMMUNOASSAYS ARE BASED ON THE SPECIFIC Ab-Ag REACTION.
IN COMPETITIVE IMMUNOASSAYS LABELLED AND
UNLABELLED ANTIGENS ARE EXPOSED TO THE ANTIBODY.
IN NON COMPETITIVE IMMUNO ASSAYS THE LABELLED
ANTIBODY DETECT THE BOUND ANTIGENS.
IT IS OF 2 TYPES.

14. RADIOIMMUNOASSAYS(RIA)
RIA IS A TECHNIQUE FOR DETERMINING THE CONCENTRATION OF A
PARTICULAR ANTIGEN IN A SAMPLE, BASED ON COMPETITIVE BINDING
BETWEEN NON LABELLED AND LABELLED ANTIGEN FOR ITS SPECIFIC
ANTIBODY.
BASON YELOW DEVELOPED THE RIA METHOD.
IT IS MOST SENSITIVE TECHNIQUE FOR DETECTING Ag OR Ab.

15. ENZYME LINKED
IMMUNOSORBENT ASSAY(ELISA)
ELISA IS A SENSITIVE METHOD TO QUANTIFY OR TEST THE PRESENCE
OF ANTIGEN.ENZYME LINKED Ab ARE USED TO BIND AN ANALYTE
ON A SOLID SUBSTRATE AND CONVERT A COLOURLESS MOLECULE TO
A COLOURED PRODUCT FOR DETECTION.

16. IMMUNOFLUROSCENCE
Ab COULD BE LABELLED WITH MOLECULES THAT HAVE THE PROPERTY OF
FLUROSCENCE. FLUROSCENT MOLECULEABSORB LIGHT OF ONE WAVELENGTH AND
EMIT LIGHT OF ANOTHER WAVELENGTH.THE MOST COMMONLY USED FLUROSCENT
DYES ARE FLUROESCEIN AND RHODAMINE
IMMUNOBLOTTING
IMMUNOBLOTTING IS AN IN VITRO TECHNIQUE FOR DETECTING SPECIFIC PROTEINS
USING ANTIBODIES.PROTEIN MIXTURES ARE TRRASFERRED TO A FILTER MEMBRANE
EITHER DIRECTLY OR AFTER SEPARATION BY ELECTROPHORESIS,THEN PROBED WITH
ANTIBODIES THAT ARE EITHER LABELLED BY FLUROPHORE OR AN ENZYME OR
BOUND BY A LABELLED SECONDARY ANTIBODY FOR DETECTION.
IT IS ALSO CALLED WESTERN BLOTTING.

17. IMMUNOELECTROPHORESIS
COMBINES ELECTROPHORESIS AND DOUBLE IMMUNODIFFUSION.
• THE Ag MIXTURE IS FIRST ELECTROPHORESISED TO SEPARATE ITS
COMPONENT BY CHARGE.
• TROUGHS ARE THEN CUT INTO AGAR GEL PARALLEL TO THE
DIRECTION OF ELECTRIC FIELD.
• ANTISERUM IS ADDED
• Ag AND Ab THEN DIFFUSE TOWARDS EACH OTHER & PRODUCE LINES
OF PRECIPITATION.
• IT IS USED TO DETECT THE PRESENCE OF PROTEINS IN SERUM.

18. IMMUNOHISTOCHEMISTRY
• IT IS A TECHNIQUE FOR DETECTING MOLECULES OF INTEREST
WITHIN TISSUES USING ANTIBODIES PROBES OR ENZYMES
CONJUGATED TO PRIMARY ANTIBODIES THAT BIND THE
TARGET MOLECULE(SECONDARY ANTIBODIES).