IMMUNOLOGY LABORATORY BY: VELASCO

IMMUNOLOGY
LABORATORY
(BCM 3201L)

Prepared by:
Rainier Velasco

ACTIVITY # ENZYME-LINKED IMMUNOSORBENT ASSAY

(ELISA)
I. Objectives
At the end of this exercise, the student should be able:
a. To familiarize oneself to the different types of ELISA
b. To identify the steps involved in the conduct of ELISA
c. To be able to know how to troubleshoot false positive results with ELISA
II. Introduction
Enzyme-Linked Immunosorbent Assay is one the most common immunoassay
type and used to detect the presence and measure the amount/concentration of specific
analytes such as antibodies (BIO-RAD, 2021). Developed in the early 1970s, this
immunoassay utilize antibodies or antigens covalently bound to enzymes which are
selected on the basis of their ability to catalyze the conversion of a substrate into a
colored, fluorescent, or chemiluminescent product. ELISA involves specific and non-
specific interactions via serial binding to a solid surface usually on a polystyrene
mutiwell plate hence its quantitative nature.

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There are four types of ELISA: Direct ELISA, Indirect ELISA, Sandwich ELISA
and Competitive/Inhibition ELISA, where each of these types has an advantage to one
another. Their differences will be explored in the succeeding sections.

III. Learning Platform

https://www.biointeractive.org/classroom-resources/immunology-virtual-lab
Microsoft Teams
Laboratory Manual
IV. Reagents

Coating
a. Sodium bicarbonate (NaHCO3)
b. Sodium carbonate (Na2CO2)
Blocking
c. Phosphate Buffer Saline (PBS)

Washing
d. Tween 20

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Detection

e. Conjugated Antibodies (HRP)

f. p-NitroPhenyl Phosphate (pNPP)

CHECKPOINT!
Identify the functions of each reagent indicated above. Why are the reagents segregated
into coating, blocking, washing, and detection?

V. Procedure:
Direct ELISA (adapted from BIO-RAD Protocol with modifications)
a. Coat the microtiter plate wells with 100uL of the coating antigen at 1 ug/mL in coating
buffer then incubate overnight at 4°C. Wash the plate after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60 minutes at 37 °C.
Wash
4 times with wash buffer
c. Add 100 uL of biotin-conjugated detection antibody, diluted in wash buffer, to each well.
Incubate for another hour then wash 3 times using the wash buffer.
d. Add 100 uL of enzyme-conjugated streptavidin, diluted in wash buffer, to each well.
Incubate for another hour then wash 3 times using the wash buffer.
e. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate for 30 minutes in the
dark.
f. Read absorbance values at 405nm

pNPP
Conjugated
Antibody
Antigen

Indirect ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of the coating antigen at 1 ug/mL in coating
buffer then incubate overnight at 4°C. Wash the plate after incubation.
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b. Add 150 uL of the blocking solution in each well, then incubate for 60 minutes at 37 °C.
Wash
4 times with wash buffer
c. Add 100 uL of unconjugated detection antibody, diluted in wash buffer, to each well.
Incubate for another hour then wash 3 times using the wash buffer.
d. Add 100 uL of enzyme-conjugated secondary antibody, diluted in wash buffer, to each
well. Incubate for another hour then wash 3 times using the wash buffer.
e. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate for 30 minutes in the
dark.
f. Read absorbance values at 405nm.

Unlabeled
Antibody pNPP
Unlabeled
Antibody
Antigen

Sandwich ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of the coating antibody at 1 ug/mL in coating
buffer then incubate overnight at 4°C. Wash the plate after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60 minutes at 37°C.
Wash 4 times with wash buffer
c. Dilute antigen and standards in wash buffer and dispense 100 uL to the microtiter wells in
triplicate. Incubate for 90 minutes at 37°C then perform washing.
d. Add 100 uL of enzyme-conjugated secondary antibody, diluted in wash buffer, to each
well. Incubate for another hour then wash 3 times using the wash buffer.
e. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate for 30 minutes in the
dark.
f. Read absorbance values at 405nm.

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Conjugated pNPP
Specific Antibody
Coating
Antigen
antibody

Inhibition ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of an antigen solution at 1 ug/mL in coating
buffer then incubate overnight at 4°C. Wash the plate after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60 minutes at 37°C.
Wash

4 times with wash buffer

c. Prepare antigen-antibody mixture by adding 50uL of antigen to 50uL of antibody for each
well in the assay and is incubated for 1 hour.
d. After incubation, 100 uL of the mixture is added onto each well. Incubate for 1 hour then
wash 3 times.
e. Add 100 uL of enzyme-conjugated secondary antibody, diluted in wash buffer, to each
well. Incubate for another hour then wash 3 times using the wash buffer.
f. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate for 30 minutes in the
dark.
g. Read the absorbance at 405 nm.

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LABORATORY WORKSHEET #
ENZYME-LINKED IMMUNOSORBENT ASSAY
Name: _______________________________________ Instructor:
____________________________
Year and Section: ____________________________ Score:
_________________________________

Guide Questions:
1. Differentiate the various types of ELISA in terms of their procedure and applications.

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2. Identify the specific functions/ concepts behind the steps in coating, blocking, washing and
detection. Why are the said stages performed in a stepwise manner? What will happen if the steps
are mixed up?

3. Why do we need to set a blank for each ELISA plate? Identify its specific function in a plate.

4. Troubleshooting:

Problem Proposed Solution

No enzyme signal detected (low absorption)
Blank sample exhibited coloration
Strong enzyme signal detected (high
absorption)
Slow color development

Conclusion:

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