ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay. In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen. Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.

1. Enzyme-Linked Immunosorbent Assay
(ELISA)
By: Bhagyashree V B
23/Sep/2016

2. Why known as ......?
Enzyme Linked Immunosorbent Assay
2
1. ‘sorbent’ -Antigen of interest is absorbed on to plastic surface
2. ‘immuno’-Antigen is recognised by specific antibody
3. ‘enzyme-linked’-This antibody is recognised by second antibody which has
enzyme attached
4. Substrate reacts with enzyme to produce product, usually coloured.
5. The sample being analyzed.

3. Why ELISA ?
SENSITIVITY
QUANTITATIVE
REPRODUCIBLE
KIT FORMAT
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/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

4. APPLICATIONS
Serum Antibody Concentrations
Screening donated blood for evidence of viral contamination by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies) and Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C
Toxoplasma gondii
Detecting illicit drugs, allergens in food and house dust

5. ANTIGEN (Ag)
Any molecule that induces production of antibodies when introduced in the body of an animal
is called antigen.
OR
any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc.
 Protein molecule
 Carbohydrate molecule.
 Microorganisms
 Allergens.
 Viruses Etc.
5
SYMBOL FOR ANTIGEN

6. ANTIBODY ( Ab)
Proteins produced by the immune system which help defend against antigens
6
SYMBOL FOR ANTIBODY
Y

7. MATERIALS NEEDED FOR ELISA
 ELISA Plate
 Positive control
 Negative control
 Dilution Buffer
 Conjugate
 TMB Substrate
 Stop Solution

8. TYPES OF ELISA
Indirect Elisa
Direct Elisa
Sandwich Elisa
Competetive Elisa
Non -Competetive Elisa
Basic Assay Format : Solid phase = 96 / 384-well microplate

9. TYPES OF ELISA

10. Analyte = antibody Analyte = antigen
1. Coat solid phase with Analyte, Incubate overnight at 4℃, wash
STEPS INVOLVED
The most widely used coating buffers are: 50 mM Carbonate pH 9.6, 10 mM Tris pH 8.5, 10 mM PBS pH
7.2
Plate: polystyrene or derivatives
of polystyrene obtained by
chemical modification

11. 2. Block free binding sites: Incubate for 45-60 mins, Wash
Analyte = antibody Analyte = antigen
STEPS INVOLVED

12. 3. Add sample: Incubate for 1.5 to 2 hrs , Wash
Analyte = antibody Analyte = antigen
STEPS INVOLVED

13. 4. Add conjugate enzyme. Incubate for 45 mins, Wash.
STEPS INVOLVED
Analyte = antibody Analyte = antigen
E E E E

14. Add substrate and Incubate for 5-10 mins
Colorless
Stop Soln
STEPS INVOLVED
OD
CONCENTRATION
Measure Color Change

15. ENZYME SUBSTRATE
Initially the substrate should be colorless
After degradation by the enzyme it should be strongly colored or fluorescent.
15
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline Phosphatase
p-NPP (405 nm )
p-NPP 1 M NaOH
Horse radish Peroxidase
H2O2 (450 nm ) Tetramethylbenzidine
Phosphate
1 M H2SO4
Horse radish Peroxidase H2O2 (492 nm ) O – Phenylenediamine 1 M HCl

16. The ELISA assay yields three different types of data output:
 Quantitative: ELISA data can be interpreted in comparison to a standard curve in
order to precisely calculate the concentrations of antigen in various samples.
 Qualitative: ELISAs can also be used to achieve a yes or no answer
indicating whether a particular antigen is present in a sample, as compared to a blank
well containing no antigen or an unrelated control antigen.
 Semi-quantitative: ELISAs can be used to compare the relative levels of antigen in
assay samples, since the intensity of signal will vary directly with antigen
concentration.
ELISA RESULTS

17. ELISA RESULTS
The status of a sample are evaluated by the sample to positive ratio (S/P ratio):
Sample Mean - Negative Control Mean
Positive Control Mean - Negative Control Mean
(Mean Of Optical Absorbance)

18. ELISA RESULTS
POSITIVEor5.1
510.0053.0563.0
767.0053.0820.0



Example:
Sample mean= 0.820
Negative control mean=0.053
Positive control mean=0.563
Negative control = 0.150 or less
The difference between the positive and negative control means must be greater than 0.075
Values are relatively quantitative: a higher value indicates more antibody.

19. LABELED STREPTAVIDIN BIOTIN (LSAB) METHOD
The avidin-biotin complex is the strongest known non-covalent interaction
Streptavidin and avidin both possess four binding site for biotin
Tetravalent binding increase detection sensitivity.

20. ELISA STANDARDIZATION ASSAY
Antigen for coating : Protein/Peptide of interest (1mg/ml stock)
Primary Ab : Positive control (1mg/ml stock)
Secondary: IgG (h+l) -conjugated Enzyme ( 1:10,000 dilution)
Ag coated
/Dilution
of Ab
1:1000
(1µg/well)
1:2000(
500ng/wel
l
1:4000(250
ng/well)
1:8000(125
ng/well)
1:16000 1:32000 1:64000 1:128000 1:256000 1:512000 1:1024000 Blank
1ng /well 0.24085 0.1799 0.1497 0.14215 0.1394 0.0925 0.11495 0.0944 0.0853 0.0842 0.08595 0.05085
10ng/well 1.33505 1.2711 1.27795 1.1464 1.25685 0.981 0.68005 0.6831 0.5938 0.43305 0.29405 0.0486
50ng/well 2.71245 2.5166 2.1181 1.82695 1.6244 1.67685 1.52295 1.482 1.44625 1.35915 1.1782 0.05135
100ng/well 3.126 3.07745 3.06225 2.85965 2.9255 2.74745 2.42355 2.2168 2.11485 1.67645 1.31345 0.0645

21. ELISA STANDARDIZATION ASSAY
0
0.5
1
1.5
2
2.5
3
3.5
1ng /well 10ng/well
50ng/well 100ng/well
Dilution of positive control Ab

22. PRECAUTIONS
 Negative control with strong signal
The excessive background signal can be caused by inadequate rinsing of plates, reagents not sufficiently diluted,
inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative
control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.
 Positive control with no signal
 Microwell plates not coated properly.
 Reagents applied in wrong order or step omitted.
 Secondary antibody not matched to the species of primary antibody.
 Enzyme conjugate defective or inhibited by contaminant.
 ELISA with weak signal
 Wash buffer not adequately drained after every wash step.
 Inadequate incubation times.
 Enzyme conjugate defective or inhibited by contaminant.
 Substrate defective or contaminated.
 Microwell plates poorly coated.
 Loss of capture antibody during blocking/washing.