2. ENZYME LINKEDIMMUNOSORBENT ASSAY 3. INTRODUCTION TO ELISA
ELISA, or enzyme-linked immunosorbent assay,are quantitative
immunological procedures inwhich the Ag- Ab reaction is monitored
byenzyme measurements. The term ELISA was first used by Engvall
&Perlma in 1971. The ELISA test, or the enzyme
immunoassay(EIA), was the first screening test commonlyemployed for
HIV. It has a high sensitivity. 4. Enzyme LinkedImmunosorbant Assay
5. ELISA is the abbreviation of ENZYME-LINKEDIMMUNOSORBENT ASSAYIt
is useful & powerfulmethod in estimating ng/mL to pg/mL ordered
materialsin the solution . 6. Why known as ......? Enzyme Linked
Immunosorbent Assay1. Antigen of interest is absorbed on to plastic
surface (sorbent).2. Antigen is recognised by specific antibody
(immuno).3. This antibody is recognised by second antibody (immuno)
which has enzyme attached (enzyme- linked).4. Substrate reacts with
enzyme to produce product, usually coloured. 7. BASIC PRINCIPLE OF
ELISA Use an enzyme to detect the binding of antigen (Ag)antibody
(Ab). The enzyme converts a colorless substrate(chromogen) to a
colored product, indicating thepresence of Ag : Ab binding. An
ELISA can be used to detect either the presenceof Antigens or
antibodies in a sample depending howthe test is designed. ELISA was
dveloped in 1970 and became rapidlyaccepted 8. Secondary antibody
SubstrateColouredproductPrimaryantibody Different antigens in
sample 9. ELISA Qualitative/Quantitative Qualitative determines
antigen or antibody is present or absent Quantitative determines
the quantity of the antibody Titer The highest dilution of the
specimen usually serumwhich gives a positive reaction in the test
10. Basic Steps OfEnzyme-LinkedImmunosorbantAssay 11. ANTIGEN (Ag)
Any molecule that induces production of antibodieswhen introduced
in the body of an animal is calledantigen.OR any thing, foreign to
the immune system. e.g.bacteria, viruses, (or their parts), pollen,
etc. Protein moleculeSYMBOL FOR ANTIGEN Carbohydrate molecule.
Microorganisms Allergens. Viruses Etc. 12. ANTIBODY ( Ab) Antibody:
proteins produced by the immunesystem which help defend against
antigensSYMBOL FORANTIBODY Y 13. Antibodies (Immunoglobulins) 14.
Materials Needed Testing sample Antibody (1st, 2nd) / Antigen
Polystyrene microtiter plate Blocking buffer Washing buffer
Substrate Enzyme 15. Specimen Sample For ELISASERUM CSF
SPUTUMURINESEMENSUPERNATANT OF CULUTRESTOOL 16.
WorkstationInventoryLab Equipment and Supplies:Micro plate strips,
pipettes, pipette tips,transfer pipette, wash buffer, paper
towels,marking pen 17. Equipment for performing theELISA test
Pipettes IncubatorELISA reader 18. ELISA READER THERMOLAB
SYSTEM(USA) 19. PRINCIPLE OF INSTRUMENT 20. TYPES OF ELISA Solid
phase immunoassay Enzyme Label Competitive assay Non-competitive
assay 21. Competitive Elisa Used to determine small molecule
antigens.(T3,T4,progesterone etc.) antibody coated microwell serum
antigen and labelled antigen added together--competition.
antibody-antigen-enzyme complex bound is inversely related to
theconcentration of antigen present in the sample. The bound enzyme
conjugate reacts with the chromogenic substrate added toproduce a
color reaction (blue to yellow color). . Increased serum antigen
results in reduced binding of the antigen-enzymeconjugate with the
capture antibody producing less enzyme activity and color(yellow)
formationSubstrate product concentration is inverselyproportional
to the concentration of standard or testantigen added 22.
DIAGRAMMATIC 23. Noncompetitive Sandwich Assay Direct Assay Antigen
capture ELISA Antigen adsorbed directly detected by labeled enzyme
Antibody capture ELISA Antibody adsorbed directly by labeled
enzyme. Indirect Assay Antigen directly adsorbed onto the solid
phase is first incubated with patient serum, and then with a
labeled antibody specific for human immunoglobulin. Detection of
infectious agents(HIV,HBV,HCV) and auto antibodiesIn Indirect ELISA
color change is directly proportionalto the concentration of
specific antibodies in specimen 24. Sandwich Assay Antigens such as
tumor markers, hormones and serum proteins may bedetermined Antigen
in the sample binds with the capture antibody on the microwelland
becomes immobilized. The antibody of the enzyme conjugate binds
with the immobilizedantigen to form a sandwich of
antibody-antigen-antibody/enzyme boundto the microwell.Enzyme
reaction product is directly proportional toconcentration of
standard or analytical antigen 25. Diagrammatic 26. to detect Ab
(HIV, HCV) to detect Ag ( Tumor Markers, Hormones ) to detect Ag (
Free Testosterone)Comparison between Indirect Sandwich &
Competitive ELISA 27. Results 28. Importance of incubation step:-
During the test performance incubation time andmentioned
temperature is must required For theproper binding between antigen
and antibody andalso binding with conjugate and color developmentof
substrate. Importance of Washing :- For the removal of anyunbound
Antibody/Antigen proper washing andtaping is required other wise we
get the incorrectresult. So incubation & washing is much
important for goodresults. 29. Enzymes Used in Elisa Horseradish
peroxidase (most commonly used) Alkaline Phosphatase -galactosidase
Lactoperoxidase Tetra Methyl benzidine In case of peroxidase, the
substrate hydrogen peroxide is convertedinto water and o2 in the
presence of electron donors . (likediaminobenzidine or
4-chloronaphthol which themselves oxidized inthe reaction).
Oxidation of diaminobenzidine produces dark brown color while
thatof 4-chlorornaphthol yields purple color which is the basis of
ELISA 30. ENZYME SUBSTRATE Initially the substrate should be
colorless After degradation by the enzyme it should bestrongly
colored or fluorescent.ENZYME SUBSTRATE CHROMOGENSTOPPINGAlkaline
p-NPP p-NPP+ 1 M NaOHPhosphatasediethandamine+Mg Cl2Horse radish
H2O2Tetramethylbenzidi 1 M H2SO4Peroxidase ne + Phosphate Citrate
bufferHorse radish H2O2O 1 M HClPeroxidase Phenylenediamine + HCl
31. ELISA KIT FOR DETERMINATION OF IgA, IgG orIgM anti-Mycobacteria
antibodies in human serumANDA-TB DETECTION OF MYCOBACTERIAIn vitro
diagnostic test for the determination of IgA, IgG and IgM
antibodies against mycobacteria inhuman liquid (serum, CSF, pleural
fluid, sputum, saliva, etc...) 32. Components of Kit Pre-Coated,
Stabilized 96-well Microtiter Plate. Sample Diluent Standards and
controls Conjugated Detection Antibody 10X Wash Solution Substrate
Stop Solution 33. What Are TheReagents?Antigen: Elisa plate coated
with the A60And antigen-antibodies complexes.What Function Primary
antibody: Human Serum IgG, IgA, &IgMDo TheySecondary antibody:
Peroxidase-labelled anti-Perform?human IgA, IgG, or IgM antibodies
that bindto the antibobdy complexes .Enzyme substrate: 3,3,5,5
tetramethylbenzidine (TMB) a colorlesssolution that when oxidized
by HRP turnsblue.Stop Solution: Sulphuric Acid 0.5N (H2So4) 34.
Elisa PlateMicrotitrewellsGenerally 96wellsMarked on oneside
alphabeticallyNumerically onthe other sideComes with thekit 35.
Complete forms and specimenidentification Fill in the information
on tube (identifying information, date of collection, and other
information as required). Fill in the laboratory form that will
accompany specimens. For TB Gold used heparin Tube FFFI 36.
Collection and processing ofserum Collect blood in a tube that does
not contain any chemicals or anticoagulants. Collect 5mL of whole
blood (for very small children collect 1mL). Place tube upright for
30-60 minutes then when firm clot has formed, centrifuge tube for
20 minutes at 2500rpm. Remove serum with a pipette and place in a
plastic storage tube (2-3mL microtube or cryovial). If 5mL of blood
was collected it will result in about 2mL of serum. 37. TEST
PERFORMANCE Usinga cleanPipette , add 100 Lof diluted serumsample
(Dilute thesera to be tested1:100 in the samplediluents) to
eachwell. Incubate 1 hour at37C . 38. After incubation empty out
contents ofwells into waste container. Using pipette, fill wells
with washingbuffer then empty out. Tap wells upside down on paper
towel. Wash the wells 5 times. At the end of thewashing process,
the wells must beentirely dry after the last wash. 39. Distribute
100L ofanti-humanimmunoglobulin-PODconjugate in each well.Incubate
30 minutes at37C. 40. ELISA PLATE READYFOR READINGMeasures the
absorbanceat 450nm With the help ofELISA READER.Calculate the
absorbancefor each sample andreference.We used Ascent Softwarefor
Calculation of theresult 41. ELISA READER THERMOLAB SYSTEM(USA) 42.
RESULT DETERMINATION:-A) IgA and IgG Tests:- Plot the O.D. result
of each reference , except for the negative reference on
thevertical axis (Y-axis) in relation to the number of
corresponding units on thehorizontal axis (X-axis).Using the
absorbance value for each sample , determine the
correspondingconcentration of antibodies expressed in units/ml from
the reference curve.B) IgM Tests :- We can calculate the cut off
value A450nm sample / A450nm Positive limitreference the normalized
value of the positive reference is 1 . All samples whose value is
comprised between 0.8 and 1.0 are considereddubious and all samples
whose normalized value is above 1.0 are consideredpositive for IgM
antibodies. 43. What is Cut-off Value ?Cut-off: provided in the
kits by the manufacturer. The cut-off valuedefines a range in which
90% of the normal population is negative belowthe cut-off value and
10% of the normal population is positive above thecut-off value.
ELISA is semiquantitative method. The calculation is done
asfollows. The units of ELISA is OD ratio:Sample value= sample
OD/cut-off OD 44. RANGE OF IgM IgG & IgAIgM- < 0.8 Negative
, 1.0Borderline, > 1.0 PositiveIgG - < 125 Negative 125-225
Borderline,> 225 PositiveIgA - < 200 Negative, 201
300Borderline Negative, 301 350Borderline positive, > 350
Positive 45. Advantages of ELISA Reagents are relatively cheap
& have a longshelf life ELISA is highly specific and sensitive
No radiation hazards occur during labelling ordisposal of waste.
Easy to perform and quick procedures Equipment can be inexpensive
and widelyavailable. ELISA can be used to a variety of infections.
46. Disadvantages of ELISA Measurement of enzyme activity can be
more complexthan measurement of activity of some type
ofradioisotopes. Enzyme activity may be affected by
plasmaconstituents. Kits are commercially available, but not cheap
Very specific to a particular antigen. Wont recognizeany other
antigen False positives/negatives possible, especially
withmutated/altered antigen 47. LimitationsResults may not be
absoluteAntibody must be availableConcentration may be unclearFalse
positive possibleFalse negative possible 48. APPLICATIONS OF
ELISA1- Hormones 7- Vaccine Quality Control2- Proteins 8- FOR GMO
(Genetically modifiedorganism)3- Infectious Agent ( Viral,
Bacterial, 9- For Rapid TestParasitic, Fungal )4- Drug Markers 10-
IgG, IgM, IgA5- Tumor Markers11- In New Born Screening6- Serum
Proteins 12- In Clinical Research 49. Sensitivity of various
immunoassays 50. A New technique:- Reverse ELISA A new technique
uses a solid phase made up of an immunosorbent polystyrene rod with
4-12 protruding ogives. The entire device is immersed in a test
tube containing the collected sample and the following steps
(washing,incubation in conjugate and incubation in chromogenous)
are carried out by dipping the ogives in microwells of standard
microplates pre- filled with reagents. Advantage of this technique:
1- The ogives can each be sensitized to a different reagent,
allowing the simultaneous detection of different antibodies and
different antigens for multi-target assays. 2- The sample volume
can be increased to improve the test sensitivity in clinical, food
and environmental samples. 3- One ogive is left unsensitized to
measure the non-specific reactions of the sample. 4- The use of
laboratory supplies for dispensing sample aliquots, washing
solution and reagents in microwells is not required, facilitating
ready-to-use lab kits and on-site kits. 51. Questions &
Answers
