ELISA
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Transcript of ELISA
Total slides : 115 1April 7, 2023
ELISAImmunochemistryIsfahan University of Medical Science, School of Pharmacy
Department of Clinical Biochemistry
Created by:
A.N. Emami Razavi
Enzyme Linked Immunosurbent Assay
ELISAELISA
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ELISAImmunochemistry
Outlines
What is ELISA History Application Mechanism & Reagents Types of ELISA Methods Quality control HIV test
What is ELISA?
ELISAELISA
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ELISAImmunochemistry
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
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ELISAImmunochemistry
Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
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ELISAImmunochemistry
Enzyme-linked immunosorbent assay
Name suggests three componentsAntibody
Allows for specific detection of analyte of interest
Solid phase (sorbent)Allows one to wash away all the material that is not
specifically captured
Enzymatic amplificationAllows you to turn a little capture into a visible color
change that can be quantified using an absorbance plate reader
History
ELISAELISA
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ELISAImmunochemistry
Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.
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ELISAImmunochemistry
The American physicist Rosalyn S.Yalow (born 1921) made her most outstanding contribution to modern medicine in developing radioimmunoassay (RIA), for which she received a Nobel Prize in physiology/medicine (1977).
The American physicist Rosalyn S.Yalow (born 1921) made her most outstanding contribution to modern medicine in developing radioimmunoassay (RIA), for which she received a Nobel Prize in physiology/medicine (1977).
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ELISAImmunochemistry
Dr. Solomon A. Berson, M.D. '45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959.
Dr. Solomon A. Berson, M.D. '45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959.
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ELISAImmunochemistry
Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce.
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ELISAImmunochemistry
Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966
In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA
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ELISAImmunochemistry
Eva Engvall is one of the scientists who invented the ELISA test in 1971. She is shown at her home near Buellton, Calif. Engvall is currently a professor at the Burnham Institute in La Jolla, Calif.
Eva Engvall is one of the scientists who invented the ELISA test in 1971. She is shown at her home near Buellton, Calif. Engvall is currently a professor at the Burnham Institute in La Jolla, Calif.
Applications
ELISAELISA
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ELISAImmunochemistry
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.
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ELISAImmunochemistry
What is it used for?
Measure antibody levels (allergies, vaccines) Detect viruses (hepatitis, HIV, venereal
diseases) Detect hormonal changes (pregnancy) Detect circulatory inflammatory markers
(cytokines)
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ELISAImmunochemistry
Advantages of ELISA
Sensitive: nanogram levels or lower Reproducible Minimal reagents Qualitative & Quantitative
Qualitative Eg. HIV testing quantitative assays Eg. Drug Monitoring
Wells can be coated with Antigens OR Antibodies Suitable for automation high speed NO radiation hazards
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ELISAImmunochemistry
Sensitivity
Relative sensitivities of tests (approx)
Usual operating range [Ab] or [Ag]
precipitationimmunoelectrophoresisdouble/radial diffusion
10 g/ml - 1 mg/ml
immunofluorescence 0.1 - 10 g/ml
ELISA (colour) 0.1 - 10 ng/ml (chemiluminescence) 0.01 - 10 ng/ml
radioimmunoassay 0.01 - 10 ng/ml
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ELISAImmunochemistry
Enzymes with Chromogenic Substrates
High molar extinction coefficient (i.e., strong color change)
Strong binding between enzyme and substrate (low KM)
Linear relationship between color intensity and [enzyme]
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ELISAImmunochemistry
Limitations
Results may not be absolute
False positive possible
False negative possible Antibody must be available
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ELISAImmunochemistry
Materials needed
Testing sample Antibody (1st, 2nd) / Antigen Polystyrene microtiter plate Blocking buffer Washing buffer Substrate Enzyme
Mechanism & Reagents
ELISAELISA
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ELISAImmunochemistry
Mechanism
The basic mechanism involved in these test utilizes absorption of antigen to a solid surface which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labeled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction. Two distinct test mechanisms are “noncompetitive " and "competitive".
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ELISAImmunochemistry
Reagents Antigens may be produced using any of the standard
techniques. They may be purified by precipitation and ultra-centrifugation or by column chromatography before being absorbed onto the surface. An alternative method which allows the use of relatively impure antigens is to bind specific antibody to the surface and allow it to absorb the antigen from the preparation. Since the latter approach usually adds an extra stage to the test this is not the preferred approach for commercial test kits. In most cases the antigen is delivered pre-absorbed onto plates, though they need to be carefully dried and packed to maintain their stability at refrigerator temperature for a reasonable period.
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ELISAImmunochemistry
The labeled anti-globulin used in the standard test is included in the test kits as is the appropriate substrates and stop-solutions for use with it. All of these reagents may be purchased separately from a number of sources however their titration to a standard level of activity for a specific purpose is rarely worthwhile for a commercial laboratory carrying out routine serology.
Reagents
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ELISAImmunochemistry
Finally, and most importantly, standard negative sera and positive sera of known potency are required. These are even more important than those used in other serological tests since it is common practice to express the results of the test sera by comparing them with those of the control sera (serum-to-positive or serum-to-negative ratios). The objective of this is to remove some of the variation due to operator, environment and plate effects.
Reagents
Types of ELISA
ELISAELISA
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ELISAImmunochemistry
Noncompetitive binding assay or Sandwich method
Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled antibodies]
Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies]
Competitive binding assay Titrewells coated with antibodies ; Enzyme labelled
antigens
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ELISAImmunochemistry
Noncompetitive or Sandwich Assay Antigen measuring system
Titre wells coated with suitable antibody Add patient sample containing the antigen Incubate: till antigen antibody reaction is complete Wash remove unbound antigen Add Antibody labelled with Enzyme Incubate till antigen binds labelled antibody Wash remove unbound labelled antibody Add substrate ; incubate Enzyme + Substrate Product measure colour Colour proportional to antigen in patient sample
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ELISAImmunochemistry
Noncompetitive or Sandwich Assay Antibody measuring system
Titre wells coated with suitable antigen Add patient sample containing the antibody Incubate: till antigen antibody reaction is complete Wash remove unbound antibody Add Antiantibody labelled with Enzyme Incubate till labelled antiantibodies binds antigen-
antibody complex Wash remove unbound labelled antiantibody Add substrate ; incubate Enzyme + Substrate Product measure colour Colour proportional to antibody in patient sample
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ELISAImmunochemistry
Competitive binding assay
Titrewells coated with antibodies Known quantities of patient sample containing
antigen + antigen labelled with enzyme Incubate: till antigen antibody reaction is complete Wash remove unbound antigens Add substrate ; incubate Enzyme + Substrate Product measure colour Colour inversely related to antigen in patient sample
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ELISAImmunochemistry
Enzyme labels
Enzyme labels should have high specific reactivity Should be easily coupled to ligands & the labelled
complex must be stable The reactivity should be retained after linking of the
enzyme to the antigen/antibody The chosen enzymes should not be normally present
in the patient samples Examples of enzyme labels
Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase
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ELISAImmunochemistry
Enzyme-Mediated Detection
Enzyme Horseradish Peroxidase
Substrate Abbrev. Color
Diaminobenzidine DAB Brown
Enzyme Alkaline Phosphatase (AP)
Substrate Abbrev. ColorBromochloroindolylphosphateNitro Blue Tetrazolium
BCIP (AP substrate) NBT (Enhance color)
Purple
Methods
ELISAELISA
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ELISAImmunochemistry
BASIC FORMAT
Solid phase = 96 / 384-well microplate
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ELISAImmunochemistry
96 Well 350µL Polypropylene Plates
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ELISAImmunochemistry
96 Well 500µL Polypropylene Plates
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ELISAImmunochemistry
96 Well 600µL Polypropylene Plates
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ELISAImmunochemistry
96 Well 1mL Polypropylene Plates
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ELISAImmunochemistry
96 Well 2mL Polypropylene Plates
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ELISAImmunochemistry
384 Well Polystyrene Assay Plates
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ELISAImmunochemistry
384 Well Polypropylene Microtiter Plates
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ELISAImmunochemistry
Analyte = antibody Analyte = antigen
Incubate, wash
1. Coat solid phase withantigen when analysing antibody
antibody when analysing antigen
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ELISAImmunochemistry
2. Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen
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ELISAImmunochemistry
3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen
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ELISAImmunochemistry
4. Add conjugate. Incubate. Wash.
Analyte = antibody Analyte = antigen
E E E E
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ELISAImmunochemistry
5. Add substrate
6. Incubate, stop, measure colour change
Colourless
ENZYME
OD
CONCENTRATION
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ELISAImmunochemistry
COATING THE PLATE
Dilute the Ag in PBS containing 0.02% NaN3 (5-10 ug/ml) and despense 50 ul into each well of 96-well microtiter plate using a multipipette/dispenser. (Micro plates vary in their ability to bind Ag. So each resercher should test for the plates of choice for their specific Ag). Seal plates with adhesive plate sealer and incubate overnight at 4° C or 2 to 3 h at 37° C (coated plates can be prepared and stored in the refrigerator for several weeks).
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ELISAImmunochemistry
WASHING THE PLATE
Wash the plate three time with PBS using the Nunc-Immuno wash device (this device simultaneously delivers and aspirates fluid)
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ELISAImmunochemistry
BLOCKING THE PLATE
block any residual binding capacity by adding 50 ul of a blocking buffer (PBS containing 5% BSA, 0.02% NaN3) to each well. Incubate the plate for 1 h at room temperature.
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ELISAImmunochemistry
SAMPLE
Dilute in buffer-Tween 20 Include known positive and negative samples Standards……. recombinant protein
International standard antibodyDouble-dilute from 10 pg/ml - 10 ng/ml
100 l/well, duplicates 2 - 4 hours 20/37oC or overnight 4oC 3 - 6 washes with buffer-Tween 20
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ELISAImmunochemistry
CONJUGATE
For assays of (human) antibodies use anti-(human) Ig-enzyme
For assays of antigens use enzyme-conjugated antibody
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ELISAImmunochemistry
Conjugating antibodies to Alkaline Phosphatase
Centrifuge 5 mg of alkaline phosphatase suspension (supplied as a suspension in 65% (NH4)SO4) in a microfuge for 5 to 10 min. resuspend the enzyme pellet in a total volume of 1 mg of the antibody to be labeled.
Dialyzed overnight against 0.1 M sodium phosphate buffer, pH 6.8 to remove any contaminating free amino groups.
In a fume hood, slowly add 50 ug 1% glutaraldehyde solution while stirring for 5 min. Incubate at room temperatyre for 2 to 3 h, add 100 ul of 1 M ethanolamine, pH 7, and then incubate for additional 2 h at room temperature.
Dialyze overnight against PBS and remove any insoluble materials by centrifugation at 40,000 x g for 30 min.
Store the conjugate (supernatant) in the presence of 50% glycerole,1mM MgCl2 1mM ZnCl2, and 0.02% NaN3 at 4ºC
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ELISAImmunochemistry
AMPLIFICATION
E
Directly conjugated developingantibody may give weak signal
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ELISAImmunochemistry
amplify with
unlabelled (rabbit) anti-(human) Ig followed by
anti-(rabbit) Ig-enzyme
EE
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ELISAImmunochemistry
or
Biotin-labelled anti-Ig followed by
streptavidin-enzyme
E-S B S-E
ES
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ELISAImmunochemistry
SUBSTRATES
See Sigma catalogue for list of conjugates and substrates
Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP)
Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm Spectrophotometer
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ELISAImmunochemistry
Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm Methyl umbelliferone Spectrophotometer
365 nm 445 nm Fluorimeter
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ELISAImmunochemistry
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ELISAImmunochemistry
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ELISAImmunochemistry
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ELISAImmunochemistry
Micro-titre plate with a standard Elisa test seen from below. The first 2 wells at top right are the negative controls. The following 2 are positive controls. The remaining sera are field test sera. Usually at least 2 wells with a known laboratory positive control are included on each plate.
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ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
• screening hybridoma supernatants• detecting clinically important antibodies - autoantibodies - anti-pathogens - anti-allergens
1. Antigen
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ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
2. Sample (human) antibody
1. Antigen
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ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
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ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
Useful when pure antigen not available or antigen coats poorly
1. Specific antibody
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen eg tissue homogenate
1. Specific antibody
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
E E5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
6. Substrate
5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
eg. hormones drugs tumour antigens cytokines
1. Anti-analyte
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ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
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ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Anti-analyte-enzyme
E E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Or: anti-analyte-biotin followed by streptavidin-enzyme
E-S B S-E
ES
E S E-S B S-E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
1. Anti-analyte
2. Sample
4. Substrate
3. Or: anti-analyte-biotin followed by streptavidin-enzyme
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Analyte
(antigen-coated plate)
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ELISAImmunochemistry
2. Anti-analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
E E E
E EE
EE
E
E
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
3. Wash
2. Anti-analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
E E E
E
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
3. Wash
2. Anti-analyte-E + sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Anti-analyte
(antibody-coated plate)
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ELISAImmunochemistry
2. Analyte-E + sample
1. Anti-analyte
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
3. Wash
2. Analyte-E + sample
1. Anti-analyte
E E E E E E E
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
3. Wash
2. Analyte-E + sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
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ELISAImmunochemistry
CYTOKINES
12 1
2
HEALTH
CANCER VIRUSES
MYCOBACTERIAASTHMA, ALLERGY
LUPUS
RHEUMATOID ARTHRITISMULTIPLE SCLEROSIS DIABETES
IL2IL12IFNTNF
IL4 IL5 IL6 IL10
Type 1 Type 2
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ELISAImmunochemistry
Detection of cytokines by ELISA
Plasma or supernatant of cultured mononuclear cells
Coat plate with anti-CK (Pharmingen) 0.5 g/ml in bicarbonate buffer, 4o overnight
Wash x 2 with PBS-T
Block with PBS + 10% FCS, 2 hours RT
Wash x2 with PBS-T
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ELISAImmunochemistry
Add standards (recombinant CK 10 pg-10 ng /ml), controls and samples 4o overnight
Wash x3 with PBS-T
Add biotinylated anti-CK (Pharmingen) 0.5 g/ml
RT 60 minutes
Wash x6 with PBS-T
Add streptavidin-peroxidase
RT 30 min
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ELISAImmunochemistry
Wash x8 with PBS-T
Add OPD substrate
15 min RT, dark
Stop with N H2SO4
Read A490
2
1
0
[rCK]
HIV Test
ELISAELISA
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ELISAImmunochemistry
HIV
The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.
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ELISAImmunochemistry
HIV
An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.
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ELISAImmunochemistry
An ELISA plate
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ELISAImmunochemistry
The ELISA Method Partially purified, inactivated HIV antigens pre-
coated onto an ELISA plate Patient serum which contains antibodies. If the
patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.
Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.
Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.
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ELISAImmunochemistry
Negative ELISA Test
Positive ELISA Test
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ELISAImmunochemistry
ELISA data from three patients
Above is ELISA data from three patients. Numbers are expressed as optical density at 450 nm. The cutoff value indicating a positive result is 0.500. Optical densities of 0.300 to 0.499 are indeterminate and need to be retested. Values below 0.300 are considered to be negative. In most cases, a patient will be retested if the serum gives a positive result. If the ELISA retests are positive, the patient will then be retested by western blotting analysis.
Positive Control Negative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
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ELISAImmunochemistry
Sources of Error for HIV EIA Tests
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ELISAImmunochemistry
Sources of False Negative Results
Early Seroconverters
the window between infection and an antibody response to the virus
Operator Error Fail to add serum or reagent to the correct well Reagent diluted in wrong diluvent or in wrong dilution
Equipment Error
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ELISAImmunochemistry
Source of False Positive Results
MULTIPLE PREGNANCY
MULTIPLE TRANSFUSION
AUTO IMMUNE DISORDER
CHRONIC HEPATITIS,
CHRONIC ALCOHOLIC
HBV VACCINATION
ANTIBODY TO POLYSTERENE
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ELISAImmunochemistry
Trouble shooting EIA Kit integrity
Controls (kit and in-house)
Equipment
Cross contamination
Sample quality
Personnel training
Correct validation and interpretation of results
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ELISAImmunochemistry
Kit integrity
Was cold chain maintained during kit transport?
Has the kit expired?
Has the kit been stored properly in your lab?
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ELISAImmunochemistry
Controls
Kit Controls should be run on each EIA plate. Indicates whether the kit components are functioning. Used to validate EIA run, calculate cut off
In-house controls Kit controls are designed to be quite robust and do not reflect subtle
changes in testing. In-house controls should be calibrated to test as a low positive (above
cut-off, below maximum)
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ELISAImmunochemistry
Equipment : Pipettes
Single and Multi channel Pipettes should be calibrated on a monthly basis.
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ELISAImmunochemistry
Equipment : Microplate Washers
Daily Prime the washer with wash solution before running sample plates
Set the washer to wash the recommended number of times (with correct volume)
Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head
Listen for changes in the sound the washer makes, this can indicate a vacuum leak
At the end of the day prime the washer with DI water
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ELISAImmunochemistry
Equipment : Microplate Washers Weekly
If a washer is not used during the week rinse it out with DI water to reduce microbial growth.
Monthly Run a 10% solution of ethanol through the washer to disinfect.
This can also be done if the washer exhibits signs of contamination (high background).
Thoroughly rinse the washer after alcohol is used.
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ELISAImmunochemistry
Equipment : Micro-plate Reader Daily
Each time a reader is turned on it runs a self test, it will then report any errors.
Weekly Run a control plate weekly. Variations in positive or negative specimens
could be a sign of a bad diode or a spill on a diode.
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ELISAImmunochemistry
Cross contamination
Can be caused by:
Reusing pipette tips (contaminated with + plasma)
Splashes from one well to another during removal of plate covers
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ELISAImmunochemistry
Sample Quality
Properly collected (no haemolysis)
Transport conditions
Storage conditions
Number of freeze/thaw cycles
Age of sample
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ELISAImmunochemistry
Validation and Interpretation of Results
Positive and Negative controls must fall within a certain range.
Controls are used to calculate a cut-off.
Samples below cut-off are negative, those above are positive
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ELISAImmunochemistry
Test your self
1. What does ELISA measure?
2. What if serum were left out?
3. Omission of the wash step
4. Which patient is HIV positive?
Use these problems to test your understanding of this topic.
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ELISAImmunochemistry
Problem 1
What is the ELISA test intended to measure?Antibody to HIV only
Antigen to HIV only
Presence of free, circulating virus in the patient
Antibodies directed against HLA molecules
BB
AA
CC
DD
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ELISAImmunochemistry
Problem 2
What would happen if serum were omitted from the ELISA, but all other steps remained the same and were performed properly?
Anti-human Ig-conjugate would not bind and be washed away.
Anti-human Ig-conjugate would bind non-specifically to the ELISA plate.
The O.D. values would be nearly the same as the assay control.
Both A and C.
BB
AA
CC
DD
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ELISAImmunochemistry
Problem 3
What would happen if the anti-human Ig-conjugate were not washed free of the well before the substrate was added? The ELISA would not develop when the substrate was
added. The ELISA would develop normally.
All wells would show uniform over development due to unbound and excess anti-human Ig enzyme conjugate.
Both A and B.
BB
AA
CC
DD
Total slides : 115 115April 7, 2023
ELISAImmunochemistry
Problem 4
From the ELISA data, which patient is seropositive for HIV?
Patient A Patients A and B
Patient B Patient C
Positive Control Negative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
BB
AA CC
DD
Thank youThank you
Questions Questions
Total slides : 115 117April 7, 2023
ELISAImmunochemistry
Antibody to HIV only
Antibody from patient serum made in response to various HIV proteins binds to antigen.
AA
Correct!
Total slides : 115 118April 7, 2023
ELISAImmunochemistry
Antigen to HIV only
HIV antigens are already coated onto the plate. Antibody from the patient binds to the HIV antigens.
BB
Incorrect!
Total slides : 115 119April 7, 2023
ELISAImmunochemistry
Presence of free, circulating virus in the patient
Serum antibody is detected, not circulating virus in the serum.
CC
Incorrect!
Total slides : 115 120April 7, 2023
ELISAImmunochemistry
Antibodies directed against HLA molecules
Although there may be anti-HLA antibodies present in the serum, the test is not designed to measure them.
DD
Incorrect!
Total slides : 115 121April 7, 2023
ELISAImmunochemistry
Anti-human Ig-conjugate would not bind and be washed away.
but there is another correct answer
AA
Correct!
Total slides : 115 122April 7, 2023
ELISAImmunochemistry
Anti-human Ig-conjugate would bind non-specifically to the ELISA plate.
The anti-human Ig conjugate is specific for human Ig and has very little nonspecific binding activity.
BB
Incorrect!
Total slides : 115 123April 7, 2023
ELISAImmunochemistry
The O.D. values would be nearly the same as the assay control.
but there is another correct answer.
CC
Correct!
Total slides : 115 124April 7, 2023
ELISAImmunochemistry
Both A and C.
Since no serum was added, the anti-human Ig conjugate would not be able to bind to human Ig and the O.D. values would be the same as the assay control.
DD
Correct!
Total slides : 115 125April 7, 2023
ELISAImmunochemistry
The ELISA would not develop when the substrate was added.
The substrate would overdevelop because an excess of enzyme coupled to the anti-human Ig was present.
AA
Incorrect!
Total slides : 115 126April 7, 2023
ELISAImmunochemistry
The ELISA would develop normally.
The substrate would overdevelop because an excess of enzyme coupled to the anti-human Ig was present.
BB
Incorrect!
Total slides : 115 127April 7, 2023
ELISAImmunochemistry
All wells would show uniform over development due to unbound and excess anti-human Ig enzyme conjugate.
Since the enzyme which acts on the substrate is in excess, it would turn all the wells a uniform color whether they were truly positive or not.
CC
Correct!
Total slides : 115 128April 7, 2023
ELISAImmunochemistry
Both A and B.
The substrate would overdevelop because an excess of enzyme coupled to the anti-human Ig was present.
DD
Incorrect!
Total slides : 115 129April 7, 2023
ELISAImmunochemistry
Patient A
Patient A has an O.D. of 0.055 and would be considered negative.
AA
Incorrect!
Total slides : 115 130April 7, 2023
ELISAImmunochemistry
Patient B
Patient B would be considered indeterminate and would need to be retested.
BB
Incorrect!
Total slides : 115 131April 7, 2023
ELISAImmunochemistry
Patients B and C
Patient A has an O.D. of 0.055 and would be considered negative and patient B would be considered indeterminate and would need to be retested.
CC
Incorrect!
Total slides : 115 132April 7, 2023
ELISAImmunochemistry
Patient C
Serum from patient C showed an O.D. of 1.999 which is even higher than the positive control O.D. for the assay.
DD
Correct!
Total slides : 115 133April 7, 2023
ELISAImmunochemistry
Total slides : 115 134April 7, 2023
ELISAImmunochemistry
Total slides : 115 135April 7, 2023
ELISAImmunochemistry
Extinction coefficient
The extinction coefficient for a particular substance is a measure of how well it scatters and absorbs electromagnetic radiation (EM waves). If the EM wave can pass through very easily, the material has a low extinction coefficient. Conversely, if the radiation hardly penetrates the material, but rather quickly becomes "extinct" within it, the extinction coefficient is high.
A material can behave differently for different wavelengths of electromagnetic radiation. Glass is transparent to visible light, but many types of glass are opaque to ultra-violet wavelengths. In general, the extinction coefficient for any material is a function of the incident wavelength. The extinction coefficient is used widely in ultraviolet-visible spectroscopy.