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ENZYME LINKED IMMUNO SORBENT 
ASSAY(ELISA)& 
RADIO IMMUNOASSAY (RIA) 
P.SAHITHI REDDY 
M.PHARMACY 
H.T.NO:256213886024 
Un...
Contents 
ELISA 
OVERVIEW PRINCIPLE PROTOCOL DIFFERENT TYPES MATERIAL REQUIRED MERITS&DEMERITS APPLICATIONS
OVERVIEW: 
Enzyme Linked Immuno sorbent 
Assay (ELISA) 
Term Was Coined By Engvall and 
Pearlmann in 1971 
is a quantitati...
• Antigen of interest is absorbed on to 
surface sorbent 
• Antigen is recognised only by the specific 
antibody immuno 
•...
PRINCIPLE: 
 ELISA is based on specific interaction between Ag and 
their corresponding Ab. One of the immuno reactant is...
MATERIALS REQUIRED 
• Test sample 
• Antibody /Antigen 
• Polystyrene microtiter plates 
• Blocking buffer 
• Washing buff...
Enzymes Used in ELISA 
 Horseradish peroxidase(very 
commonly used )  Alkaline phosphatase  Beta –galactosidase  Lacto...
The enzyme substrate should be colourless 
After degradation by the enzyme it should be strongly coloured 
fluorescent
TYPES: 
a)Indirect ELISA 
b)Sandwich ELISA 
c) Competitive ELISA
Sandwich ELISA direct method 
 2 Antibodies Required 
 Must Recognize Different Epitopes 
 1st Antibody Is Referred To ...
The ELISA plate is coated with Antibody to detect 
specific antigen
Protocol for sandwich 
ELISA 
Prepare a surface to which a known quantity of capture 
antibody is bound Block any non sp...
Wash the plate, so that the unbound antibody-enzyme 
conjugates are removed Apply a chemical which is converted by the e...
Indirect ELISA: 
• The protein antigen to be tested for is added to each 
well of ELISA plate, where it is given time to a...
Conti…. 
• Afterwards, a secondary antibody is added, which will bind to 
the antibody bound to the test antigen in the we...
COMPETIVE ELISA 
• The labelled antigen competes for primary antibody 
binding sites with the sample antigen (unlabelled)....
RESULTS 
 After reading the results the standard curve is drawn were the 
concentration is plotted on the X-axis and the ...
Advantages 
• Reagents are relatively cheap& have a long shelf life 
• Easy to perform and quick procedures 
• No radiatio...
Limitations 
 To carry out the test antibody must be 
available  Concentration may be unclear  Possibility of False pos...
Applications 
The various sections devoted to the uses are 
 Detection of mycobacterium antibodies in tuberculosis  Dete...
CONTENTS 
RADIO IMMUNOASSAY (RIA) 
OVERVIEW PRINCIPLE MATERIAL REQUIRED PROTOCOL INSTRUMENTAION MERITS&DEMERITS APP...
Overview: 
 Radio immuno assay was developed by  Berson&yalow(1956) for the quantitative 
measurement of insulin in huma...
An immunoassay is a test that uses antibody and antigen 
complex as means as a mean of generating a measureable 
result 
A...
Principle 
RIA is a competitive binding assay. 
The antibody & labelled antigen are always present as 
limiting factors an...
Unbound Ag* and Ag washed 
Radioactivity of bound residue measured 
Ligand conc is inversely related to radioactivity 
[Ag...
Materials required 
a) Preparation &characterisation of Antigen [Ligand to be 
analysed ] 
b) Radiolabelling of the Antige...
PREPARATION AND RADIOLABELLING 
OF THE ANTIGEN 
Antigens prepared by 
 synthesis of the molecule  isolation from natural...
PREAPRATION OF SPECIFIC 
ANTIBODY 
Antigen injected intradermal into rabbit 
antibody production Antibodies recovered fr...
ASSAY Procedure 
Add known amounts of the test sample+ labelled 
antigen into the microtitre wells 
 incubate – allow the...
Conti… 
Intensity of radioactivity is inversely correlated 
with the conc of the antigens in the test sample
INSTRUMENTATION: 
CENTRIFUGE: 
 swing bucket rotor : 1200-2500 rpm  Fixed angle head rotor :3500-4000 rpm 
RADIOACTIVE c...
The results obtained by plotting a graph 
antibody bound to labelled antigen Unlabelled antigen
Merits and demerits 
Merits : 
highly specific : immune reactions are specific 
high sensitivity : immune reaction are sen...
APPLICTIONS : 
Used in the estimation of pharmaceutical 
drugs like Morphine Clonazepam Barbiturates Neobentine Flura...
Limitations : 
 The limitations of the RIA are  Its expensive  Being hazardous Handling the radioactive material  The ...
References : 
A textbook of pharmaceutical analysis 
Biotechnology and its applications in pharmacy
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Elisa & ria

  1. 1. ENZYME LINKED IMMUNO SORBENT ASSAY(ELISA)& RADIO IMMUNOASSAY (RIA) P.SAHITHI REDDY M.PHARMACY H.T.NO:256213886024 Under the guidance of Mr Utham prasad
  2. 2. Contents ELISA OVERVIEW PRINCIPLE PROTOCOL DIFFERENT TYPES MATERIAL REQUIRED MERITS&DEMERITS APPLICATIONS
  3. 3. OVERVIEW: Enzyme Linked Immuno sorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 is a quantitative technique used in immunology to detect the presence of an antibody or an antigen in sample
  4. 4. • Antigen of interest is absorbed on to surface sorbent • Antigen is recognised only by the specific antibody immuno • This antibody is recognised by second antibody (immuno) which has enzyme attached (enzyme linked) • Substrate reacts with enzyme to produce product usually depicted by coloured
  5. 5. PRINCIPLE:  ELISA is based on specific interaction between Ag and their corresponding Ab. One of the immuno reactant is coated to a solid phase support such as polycarbonate, polyvinyl polyacrylamide tubes or microwells.  To the respective antibody or antigen is added  To antigen-antibody complex so formed an enzyme linked antibody is added. A colourless substance is added to well. This substance is a substrate for the enzyme which catalyses the conversion of colourless substance to coloured product.
  6. 6. MATERIALS REQUIRED • Test sample • Antibody /Antigen • Polystyrene microtiter plates • Blocking buffer • Washing buffer • Substrate • Enzyme
  7. 7. Enzymes Used in ELISA  Horseradish peroxidase(very commonly used )  Alkaline phosphatase  Beta –galactosidase  Lacto peroxidase  Tetra Methyl Benzidine (substrate)
  8. 8. The enzyme substrate should be colourless After degradation by the enzyme it should be strongly coloured fluorescent
  9. 9. TYPES: a)Indirect ELISA b)Sandwich ELISA c) Competitive ELISA
  10. 10. Sandwich ELISA direct method  2 Antibodies Required  Must Recognize Different Epitopes  1st Antibody Is Referred To As Capture Ab  2nd Antibody Detection Ab  2nd Antibody Is Biotinylated  Enzymes Commonly Used: HRP (Horse Radish Peroxidase) And AKP (Alkaline Phosphatase)  Substrate is TMB (Chromogen)
  11. 11. The ELISA plate is coated with Antibody to detect specific antigen
  12. 12. Protocol for sandwich ELISA Prepare a surface to which a known quantity of capture antibody is bound Block any non specific biding sites on the surface Apply the antigen – containing sample to the plate Wash the plate ,so that unbound antigen is removed Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen
  13. 13. Wash the plate, so that the unbound antibody-enzyme conjugates are removed Apply a chemical which is converted by the enzyme into a coloured product Measure the absorbency of the plate wells to determine the presence and quantity of antigen
  14. 14. Indirect ELISA: • The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to plastic through charge interactions • A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen • Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.
  15. 15. Conti…. • Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it • A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. • The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength •
  16. 16. COMPETIVE ELISA • The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabelled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal).
  17. 17. RESULTS  After reading the results the standard curve is drawn were the concentration is plotted on the X-axis and the absorbance on the Y-axis.
  18. 18. Advantages • Reagents are relatively cheap& have a long shelf life • Easy to perform and quick procedures • No radiation hazards during handling • Has wide usage to variety of infections
  19. 19. Limitations  To carry out the test antibody must be available  Concentration may be unclear  Possibility of False positive  Possibility of False negative
  20. 20. Applications The various sections devoted to the uses are  Detection of mycobacterium antibodies in tuberculosis  Detection of rotavirus in faeces  Detection of hepatitis B markers in serum  Detection of HIV antibodies in blood sample  Hormones  Proteins  Drug markers  Serum proteins  Vaccine quality control  Tumour markers
  21. 21. CONTENTS RADIO IMMUNOASSAY (RIA) OVERVIEW PRINCIPLE MATERIAL REQUIRED PROTOCOL INSTRUMENTAION MERITS&DEMERITS APPLICATIONS
  22. 22. Overview:  Radio immuno assay was developed by  Berson&yalow(1956) for the quantitative measurement of insulin in human plasma  RIA principles have found wide application in the field of drug analysis kinetic studies, immuno diagnosis  RIA is specific, sensitive &rapid
  23. 23. An immunoassay is a test that uses antibody and antigen complex as means as a mean of generating a measureable result An antibody :antigen complex is known as a immune complex. Immunoassays are different from other types of laboratory tests, such as colorimetric tests, because they use antibody: antigen complexes to generate a signal that can be measured . In contrast, most routine clinical chemistry tests utilize chemical reaction between the reagent and sample to generate a test result
  24. 24. Principle RIA is a competitive binding assay. The antibody & labelled antigen are always present as limiting factors and the concentrations of unlabelled antigens(sample) under examination is increased continually Uses an immune reaction (Antigen –antibody reaction ) to estimate ligand
  25. 25. Unbound Ag* and Ag washed Radioactivity of bound residue measured Ligand conc is inversely related to radioactivity [Ag: ligand to be measured ;Ag* radiolabelled ligand ]
  26. 26. Materials required a) Preparation &characterisation of Antigen [Ligand to be analysed ] b) Radiolabelling of the Antigen c) Preparation of the specific antibody d) Development of Assay system
  27. 27. PREPARATION AND RADIOLABELLING OF THE ANTIGEN Antigens prepared by  synthesis of the molecule  isolation from natural sources Radiolabelling [Tagging procedure ]  3H,14C,125I are used as radioactive tags  Antigens are tagged to 3H14c125I  tagging should NOT affect antigenic specificity and activity
  28. 28. PREAPRATION OF SPECIFIC ANTIBODY Antigen injected intradermal into rabbit antibody production Antibodies recovered from the serum Some ligand are not antigenic hormones ,steroids drugs HAPTENS E.g.: castrin morphine
  29. 29. ASSAY Procedure Add known amounts of the test sample+ labelled antigen into the microtitre wells  incubate – allow the reaction to reach completion Decant and wash the contents of the well-removes all unbound antigens Radioactivity remaining in the mocrotitre wells measured by a counter [GM counter, scintillation counter]
  30. 30. Conti… Intensity of radioactivity is inversely correlated with the conc of the antigens in the test sample
  31. 31. INSTRUMENTATION: CENTRIFUGE:  swing bucket rotor : 1200-2500 rpm  Fixed angle head rotor :3500-4000 rpm RADIOACTIVE counter  gamma counter : which is used for a gamma energy emitting isotopes. E.g. 125I. Scintillation counter : it is used for beta energy emitting isotopes.eg. Tritium 3H and 14C isotopes
  32. 32. The results obtained by plotting a graph antibody bound to labelled antigen Unlabelled antigen
  33. 33. Merits and demerits Merits : highly specific : immune reactions are specific high sensitivity : immune reaction are sensitive Demerits: radiation hazards :uses radiolabelled reagents Requires specially trained personnel Labs require special license to handle radioactive material Requires special arrangements for :requisition, storage of radioactive material radioactive waste disposal.
  34. 34. APPLICTIONS : Used in the estimation of pharmaceutical drugs like Morphine Clonazepam Barbiturates Neobentine Flurazepam And others Insulin Gastrin Glucogon Growth hormones
  35. 35. Limitations :  The limitations of the RIA are  Its expensive  Being hazardous Handling the radioactive material  The radio isotopes used 125I or 131I emit gamma radiation these requires a special counting machine
  36. 36. References : A textbook of pharmaceutical analysis Biotechnology and its applications in pharmacy

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