BB2903 ELISA Lab 6 C 2017

Objectives

• Gain a understanding of how antigens, antibodies, and secondary antibodies

interact

• Perform an ELISA (Enzyme Linked Immuno-Sorbent Assay)

Introduction

Vaccinations of people started in the late 18th Century with Edward Jenner who
discovered that inoculation of humans with cowpox or vaccinia induced protection
against human smallpox, an often fatal disease. Jenner called his process vaccination, a
term still used to describe the inoculation of healthy individuals with weakened or
attenuated pathogens to induce immunity. When Jenner introduced vaccination he knew
nothing of pathogens or immunology. It was not until late in the 19th century that Robert
Koch proved that infectious disease were caused by pathogenic microorganisms, each
one responsible for a particular disease. More vaccines against things like cholera and
rabies were developed.
These practical triumphs led to a search for the mechanism of protection. In 1890
Emil van Behring and Shibasaburo Kitasato discovered that the serum of vaccinated
individuals contained substances-which they called antibodies- that specifically bound to
the relevant pathogen. The idea that these antibodies might have a crucial role in
immunity was reinforced by Jules Bordet’s discovery in 1899 of complement, a
component of serum that acts in conjunction with antibodies to destroy pathogenic
bacteria. * taken from ImmunoBiology by Janeway and Travers, Garland Publishing
1994
It was soon clear that specific antibodies could be produced against a vast range
of antigens, which are defined as any substance that can induce the production of
antibodies. Antibody molecules as a class are now known as immunoglobulins,
commonly abbreviated Ig. Antibodies are produced by lymphocytes known as B-
lymphocytes. A given lymphocyte will only produce antibodies against a given antigen.
There are millions of different B-lymphocytes that will make antibodies directed against
millions of different antigens.
Since antibodies are so specific for their given substrate, investigators have used
this property in a variety of detection, purification, and medical procedures.
One such procedure is an ELISA (enzyme linked immuno–sorbent assay). ELISAs are
used in medicine, research and industry to detect proteins or other antibodies.

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Generally an ELISA is used to measure how much primary antibody to a specific antigen
is present in a sample (often a persons serum). Most commonly ELISAs are performed by
incubating serum or dilutions of serum on a card or in a microtiterplate that is coated with
the antigen of interest. If primary antibody is present in the serum it will bind to the
antigen that is stuck to the plate. Then to detect the presence of the primary antibody, we
add a second antibody that has a marker, often an enzyme, conjugated (bound to) to its
non-antigen binding domain. Often horseradish peroxidase (HRP) is used. This
enzyme will oxidize a substrate that contains stabilized hydrogen peroxide with an
associated chromogen causing a change in its absorbance to a visible absorbance range
that can be monitored. The color reaction is linear to the enzyme concentration which is
linear to the concentration of the second antibody which reacts with its antigen in a linear
fashion. Therefore we can determine how much primary antibody was present in the
sample.
You can also vary the components of ELISAs and conjugate an enzyme to a
protein or peptide other than an antibody. You can conduct competitive and inhibitory
assays. In the figure below is depicted the normal steps of an ELISA.

A Bind sample to support

B Add primary antibody; wash

C Add secondary antibody-enzyme conjugate; wash

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D Add substrate

Figure 1 Generalized ELISA protocol for detecting a target antigen. Enzyme (E) is
conjugated to secondary antibody. (Figure from Howard Hughes Medical Institute
website)

The ELISAs that we will be doing are designed as follows: Two types of coated
plates will be used, first a plate coated with protein G and secondly a plated coated with
antibodies directed against rabbit immunoglobulins. The first plate will bind to any
immunoglobulins (protein G has the ability to specifically bind the Fc region of
immunoglubulin molecules) and the second plate will bind or capture only
immunoglobulins made in rabbits.

We have three types of immunoglobulins isolated from serum of humans, goats,

and rabbits. Our secondary antibody is Goat Anti-Rabbit antibody with conjugated HRP.
It will detect any antibodies made by rabbits. The substrate for the HRP is 3,3`,5,5`
tetramethylbenzidine (TMB) and stabilized H2O2. TMB has an absorption maximum at
285 before oxidation. On oxidation, the maximal are 370 and 652 nm. The blue color
can be measured at 652 nm; however, addition of acid will create a yellow color, which
can be measured at 450 nm.

We will use both types of plates and then test the specificity of the secondary
antibody. We will determine if antibodies made in human or goat can be detected by the
anti-rabbit antibody.

Procedure
Each group will take 1 strip of G-coated wells and 1 strip of anti-rabbit coated plate.
Each strip contains 8 wells. Make sure you put label the strips so you will not confuse
the two.

You will be using all 8 wells on the strip. Follow the directions as to the time of additions
and number of washes etc. Wash all wells the same. When emptying tip the whole strip
upside down quickly so that fluid from one well will not enter another well. Remember
that there are a lot of incubation steps and you can be working on making things you need
for next week’s lab during that time and when you are finished with the ELISA. You can

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view https://video.wpi.edu/Watch/ELISA or http://youtu.be/2O4GF0N2jY0 to see a

demonstration of the procedure.

Directions

1. Obtain one goat anti rabbit 8 well strip and one 8 well strip coated with protein G.
You will find that they are slightly different sizes because the come from different
manufacturers so you will need two different plastic racks.

2. Pipette 200 ul of PBS (phosphate buffered saline) wash solution into each well
you will be using in both of the strips. Empty by tapping on a paper towel.
Repeat 2 more times.

3. Dilute the primary antibodies human, goat, and rabbit (TAs have done this for
you) in a proteinaceous blocking solution so that you have 1 ug/ml. Add 100 ul of
these diluted antibodies to the appropriate wells on both the goat anti rabbit and
protein G plates/strips. Human to well 3, goat to well 5, rabbit to wells 2,4,6.
Add 100 ul of PBS + BSA to any wells that don’t get antibody (1,7, & 8) Be sure
that the wells you are using never go dry at anytime during the process. They
should have at least PBS in them at all times.

4. Incubate 15 minutes at 37° C.

5. Empty your wells by inverting on a paper towel and wash the wells you are using
three times with 200 ul of the PBS+ Tween wash solution. Be sure to empty the
wells before continuing to step 7.

6. Add 100 ul of secondary antibody, diluted Goat Anti-Rabbit IgG conjugated with
HRP to the appropriate wells (1,3,4,5,6.). The TAs have diluted this 1: 100,000 in
the proteinaceous blocking solution. Add PBS+ BSA to wells 2, 7, & 8.

7. Incubate for 15 minutes at 37° C.

8. Empty and wash each well three times with 200 ul of the PBS+Tween wash
solution.

9. Add 150 ul of TMB-ELISA to the appropriate wells. (1,2,3,4,5, & 7). Add PBS
to wells 6 &8. Incubate for 15 min. at 37° C or until there is good color
formation. This may take less than 15 minutes. Keep checking

10. Stop the reaction by adding 100 ul of dilute sulfuric acid.

11. Determine the amount of color formation in each well qualitatively by observing
it visually and take a photo.

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12. Make a quantitative measurement of the color density in the wells using the
ELISA plate reader. See Appendix A below for instructions on using the plate
reader. If you aren’t sure how to operate the instrument or read the print out see
your TA or instructor for help.

List of key reagents that will have gone into each well.(not necessarily everything)

1. No primary antibody control. Add only secondary antibody and TMP

2. No secondary antibody control. Add only rabbit antibody and TMP
3. Human antibody. Add human antibody, secondary antibody and TMP
4. Rabbit antibody. Add rabbit antibody, secondary antibody and TMP
5. Goat antibody. Add goat antibody, secondary antibody and TMP
6. No TMP. Add only rabbit antibody and secondary antibody.
7. TMP only.
8. Nothing added

1 2 3 4 5 6 7 8

Appendix A. Using the Biotek Plate Reader

Logon to laptop with username: UVPuser password: DNAstring!
Power on the EL800 Biotek Reader, if not already on, and let the instrument go through
a self-test. The instrument will go into a ready state. On the laptop open the Gen5.1.11
application and the following screen appears:

Open the Protocol identified as BB2903.prt

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Under the File heading click on the top of the menu for New Experiment. Click OK.

This screen appears

Go to the top of the screen menu and select “Read” under the heading of “Plate”.

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Click on READ. Give a unique name for your data file that will be generated such as
shown in here and save it in the desktop folder for BB2903 for your lab section.

Save. Load plate onto carrier of reader, if not already done so. Press OK when ready.

This pop up window appears:

Data Appears:

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Click on the Excel icon above column 5 of the 96-well grid. An Excel file appears with
your data.

Save this in the desktop folder for BB2903 data in the subfolder for your lab section. E-
mail the Excel data to yourself or use a flash drive. Exit the Gen5 1.11 program.