The document discusses various proteomics techniques including ELISA and ELISpot assays. It provides an overview of proteomes and proteomics, different proteomics techniques such as molecular techniques, separation techniques, protein identification techniques, and protein structure techniques. It then describes ELISA in detail, covering the basic principles, types of ELISA (direct, indirect, sandwich, competitive), equipment used, and applications. Finally, it discusses ELISpot assays and provides examples to demonstrate how ELISA and ELISpot can be used to analyze specific proteins and immune responses.

1. Methods in Proteomics, ELISA
and ELISpot Assays
Suleiman, Shamsudeen Kyauta (B.Sc., M.Sc.(in view))

2. Proteomes and Proteomics
• Proteomes: These are the sets of all expressed proteins in a cell, tissue,
or an organism
• Proteomics: A science that focuses on the analysis and study of proteins
(their roles, structures, localization, interactions, and other factors).
• SO, there should be methods to understand these items!
6/3/2019 2Yoithapprabhunath et al., 2015

3. Proteomics techniques
• Molecular techniques
• Separation techniques
• Protein Identification techniques
• Protein Structure techniques
6/3/2019 3(Twyman, 2004; Aslam, et al., 2016)

4. Molecular techniques
• DNA Microarrays or Gene
• Differential Display
• Northern/Southern Blotting
• RNAi (small RNA interference)
• Serial Analysis of Gene Expression (SAGE)
• Yeast two-hybrid analysis
6/3/2019 4(Twyman, 2004; Aslam, et al., 2016)

5. Separation techniques
• 1D Slab Gel Electrophoresis
• 2D Gel Electrophoresis
• Capillary Electrophoresis
• Chromatography (HPLC)
• Protein microarray
6/3/2019 5(Twyman, 2004; Aslam, et al., 2016)

6. Protein Identification techniques
• Edman sequencing
• Microsequencing
• Mass spectroscopy
6/3/2019 6(Twyman, 2004; Aslam, et al., 2016)

7. Protein Structure techniques
• NMR(Nuclear magnetic resonance spectroscopy)
• X-ray crystallography
• Computational prediction
6/3/2019 7(Twyman, 2004; Aslam, et al., 2016)

8. CASE REPORTS
• Bose had unprotected sex with Kola. Few weeks later she developed the
following symptoms- GI disturbances, diarrhoea, night sweats, fever, red body
rashes, sore throat. She went to the hospital and was referred to the lab for
test. Conventional HIV test showed a negative result. The doctor was not
convinced so he ordered an ELISA test be done and the test was positive.
HOW?
• Basirat went to the hospital to see the doctor for her routine antenatal check.
A sample of her urine was taken and sent to the lab for hCG screening. What
ELISA method is used?
6/3/2019 8

9. CASE REPORTS
• Dr. Tokunbo Sadeeq and his Neuroscience team conducted an
experiment inducing neurotoxicity in an animal model focusing on the
Schwann cells in the cerebral cortex. A plant extract was used with
the intent of ameliorating the neurotoxic effect. The animals were
sacrificed and tissue homogeneity was done. Histology confirmed
neurodegeneration. How will they determine if actually the plant
extract has neuroregenerative propties?
6/3/2019 9

10. ELISA
• This is a common laboratory technique which is used to measure the
concentration of an analyte (usually antibodies or antigens) in solution.
• They are performed in a 96-well polystyrene plates which bind antibodies and
proteins
• The reactants are immobilized to the microplate surface, this makes it easy to
separate bound from non-bound material during assay
• The ability to wash away nonspecific bound materials makes ELISA a powerful
tool for measuring specific analytes within a crude preparation
6/3/2019 10Aydin, 2015

11. General Principles
Solid phase: a 96-well microtitre
plate made of polypropylene or
polysterene
Enzymes: Alkaline phosphatase
(AP) and horseradish peroxidase
(HRPO) are commonly used.
Substrates: PNPP for AP and
TMB or OPD hydrochloride
combined with H2O2 for HRPO.
Washing solutions: PBS
containing 0.05 % Tween 20
The reaction is usually completed
within 30-60mins. The reaction can
be stopped using NaOH, HCL or
H2SO4 depending on the kit used
Results : The colour produced is
measured using a
spectrophotometer.
Sensitivity and specificity: Present
ELISA formats have sensitivity in the
range of 98-100 per cent, and
specificity of 95-98 per cent
Aydin, 20156/3/2019 11

12. Types of ELISA
6/3/2019 12
Aydin, 2015

13. Direct ELISA
Developed simultaneously in
1971 by Engvall and
Perlmann and by Van
Weeman and Schuurs
The primary Ab conjugate
directly binds to the target Ag
that is immobilized to the plate
(solid phase)
The enzyme linked to the primary Ab
reacts with its substrate to produce a
visible signal that can be measured and
the Ag of interest is detected
6/3/2019 13Aydin, 2015
http://www.cusabio.com/c-20659.html

14. Indirect ELISA
Developed in
1978 by Lindstrom
and Wager
Uses both
primary and
secondary Ab
The secondary Ab and
not the primary Ab is
labelled with an
enzyme
The primary Ab binds to the Ag
immobilized to the plate and then
the enzyme-labellled secondary Ab
binds to the primary Ab
Finally, the enzyme-linked to the
secondary Ab reacts with its
substrate to produce a visible
signal that can be measured
(assayed)
6/3/2019 14
http://www.cusabio.com/c-20659.html
Aydin, 2015

15. Sandwich ELISA
Developed in
1977 by kato
and his co-
workers
In this, it is the Ab
that is immobilized
to the plate, this Ab
is called Capture Ab.
It also employs the use of
detection Ab which includes the
unlabeled primary detection Ab
and the enzyme-labelled
secondary detection Ab.
Firstly, the Ag of interest
binds to the capture Ab
immobilized to the plate
Secondly, the primary
detection Ab binds to
the Ag.
Thirdly, the secondary detection Ab binds
to the primary detection Ab and then the
enzyme reacts with its substrate to
produce a visible signal that can be
measured
6/3/2019 15
http://www.cusabio.com/c-20659.html
Aydin, 2015

16. Competitive ELISA
Developed in
1976 by Yorde
and his co-
workers
In this the inhibitor Ag
and the Ag of interest
compete for binding to
the primary Ab.
Firstly, the unlabeled primary Ab is incubated
with the sample containing the Ag of interest,
forming Ag-Ab complex. The Ab is excessive
compared with the Ag, so there are free Ab
left.
Secondly, the Ag-Ab mixture is added to the
plate coated with the inhibitor Ag that can also
bind to the primary Ab. The free Ab in the
mixture binds to the inhibitor Ag on the plate
while the Ag-Ab complexes in the mixture do not
bind and are therefore washed off.
Thirdly, the enzyme-labelled
secondary Ab is added to the
plate and binds to the
primary Ab bound to the
inhibitor Ag on the plate
Finally, a substrate is
added to react with the
enzyme and emit a visible
signal for detection
6/3/2019 16
Aydin, 2015

17. Equipment used in ELISA
1) Microwell Plate:
Flat bottom
polystyrene plate, contains 8 x 12 wells
holding 350 μL each.
2) Multipipette :
An 8-channel 100 μL pipette is a good help for even small-scale
work.6/3/2019 17

18. 3) Washing Device:
• manually operated washing devices.
• may be of use particularly when there is a risk
that the samples tested in ELISA contain
infectious material.
4) Microplate washer:
• These are very efficient with unusually low carry-over
contamination.6/3/2019 18

19. ELISpot
A sensitive method used to
quantify immunoglobulin-
secreting cells (cytokines) at
the single cell level
Developed in 1983
by Cecil Czerkinsky
Used to investigate specific
immune responses to
infections, cancer, allergies
and autoimmune diseasesIt has a detection level as low as one
cell in 100,000 this makes it one of
the most sensitive cellular assays
6/3/2019 19https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle

20. 6/3/2019 20
https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle/elispot-step-step

21. Answers to Case report 1
• The HIV genome has 2 major protein products in its envelope region (gp120 and
gp41). Beneath the env. Are 2 capsid protein (gag) protecting the nucleic acid
they are p17 and p24.
• It takes 4-6wks for anti-p24 and anti-gp 41 antibodies to become detectable after
infection this period is called serological window period
• During this period, p24 antigen is detectable by ELISA
6/3/2019 21(Rao., Kapila., and Gupta., 1997)

22. Answer to Case report 2
• Sandwich ELISA
• The specimen is sandwiched between the capture Ab and the primary Ab, secondary
Ab-Enzyme conjugate at different epitopes increasing specificity and sensitivity
• In the presence of hCG, a specific Ab-hCG-Ab enzyme complex will form on the surface
of the microtiter well.
• Washing is done in PBS, to remove unbound enzyme conjugate and incubated in TMB
reagent
• A blue colour develops in the well, this indicates hCG positive
• In less than 10mins elevated level of hCG as little as 20mIU/ml is detected
6/3/2019 22https://www.genwaybio.com/human-chorionic-gonadotropin-hcg-2668

23. Answer to Case report 3
• Indirect ELISA
• The well are precoated with Schwann cells Ag immobilized to the
plate
• The tissue homogenate is added to the well forming Ag-Ab complex
• The reaction is washed to remove unbound Ab
• A reagent containing enzyme-labelled sec. Ab is added (MBP) and
washed
• A chromogen (colour substrate) is added to make the reaction visible
using the spectrophotometer
• Presence of colour shows presence of Ab (MBP) for quantification
6/3/2019 23

24. References
• Aslam, B., Basit, M., Atif Nisar, M., Khurshid, M., Rasool, M. (2016). Proteomics: Technologies
and Their Applications. Journal of Chromatographic Science. 55. 10.1093/chromsci/bmw167.
• Aydin, Suleyman. (2015). A short history, principles, and types of ELISA, and our laboratory
experience with peptide/protein analyses using ELISA. Peptides. 72.
10.1016/j.peptides.2015.04.012.
• Mishra C. Introduction to Proteomics: Principles and Applications. John Wiley & Sons, 2011;
pp. 1-38.
• Rao, M., Kapila, K., and Gupta, M. (1997). ENZYME LINKED IMMUNOSORBENT ASSAYS
REVISITED. Medical journal, Armed Forces India, 53(1), 45–51.
• Twyman R. Principles of Proteomics. Garland Science, 2004; pp. 1-22
• Yoithapprabhunath, R., Nirmal, M., Santhadevy, A., Anusushanth, A., Charanya, D., Rojiluke,
and Yamunadevi, A. (2015). Role of proteomics in physiologic and pathologic conditions of
dentistry: Overview. Journal of pharmacy & bioallied sciences, 7(Suppl 2), S344–S349.
6/3/2019 24

25. References Cont’d
• http://www.cusabio.com/c-20659.html
• https://www.genwaybio.com/human-chorionic-gonadotropin-hcg-2668
• https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle
• https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-
principle/elispot-step-step
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26. 6/3/2019 26
THANK YOU