This lecture is presented by our volunteer M. Fida ur Rehman, he is from Pakistan, and he is covering ELISA topic. For video: https://www.youtube.com/watch?v=ZEEgRHUIbJ0

1. ELISA
Presented by
M.Fida Ur Rehman

2. ELISA
 Introduction
 Principle
 Steps
 Types
 Applications

3. ELISA (Enzyme Linked ImmunoSorbent Assay)
 It is sensitive plate base assay
 First introduce in 1971
 Used to detect and quantify substances such as antigens,
antibodies, proteins and glycoproteins in biological samples.

4. Principle
In the ELISA, an antigen or antibody is immobilized to the solid surface where
it form antigen antibody complex. The detection of antigen or antibody is
determined by the activity of conjugate enzyme via incubation with substrate
to produce a measureable product.

5. Steps
 Antigen or Antibody coating
 Blocking
 Incubation
 Developing solution

6. Antigen or Antibody coating
 Protein, peptide, crude protein
 Its choice depends on nature
 Bind due to hydrophobic interaction protein and plastic

7. Blocking
 Block surface to prevent from non specific binding
 Blocking buffers are used
 PBS Phosphate buffered saline
 TBS Tris-buffered saline
 Blocking solution contain protein attach to the surface of wells

8. Incubation
 Incubate with Primary and secondary antibody having enzyme
conjugate
 HRP Horse radish Peroxidase
 Alkaline Phosphate AP

9. Developing Solution
 Contains sbstrate/Chromogen
 TMB Tetra methyl benzidin

10. Types
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA

15. Application
Diagnosis of Hepatitis

16. ELISA for Hepatitis
 Hepatitis E (HEV)
 Hepatitis A (HAV)

17. Hepatitis E
This ELISA is of indirect type and detects IgG antibodies to hepatitis
E. The micro plates are coated with HEV recombinant antigens
specific to IgG HEV antibodies. The serum/plasma sample is first
diluted in a diluent, after which a specific amount of it is pipetted
into the wells, if the antibodies are present they are captured by the
antigens. After incubation, the plates are washed to remove all
components except the bounded antibodies.

18. .
The antigen antibody complex is detected by a anti
hIgG antibody conjugated with a enzyme named horseradish
peroxidase (the secondary antibody). After second incubation and
washing, a chromogen is added as substrate upon which the enzyme
reacts to convert it into a colored product. Color intensity is directly
proportional to the amount of IgG HEV antibodies in the sample.

19. Hepatitis A
HAV-IgM ELISA is a sandwich ELISA antibody capture ELISA that
detects IgM antibodies to hepatitis A. The micro plates are coated
with antibodies directed to IgM proteins. The serum/plasma
sample is first diluted in a diluent, after which a specific amount of
it is pipetted into the wells, if anti HAV IgM antibodies are present
in the sample, they are captured by the pre-coated antibodies.
After 1st incubation, the plates are washed to remove all
components (including the IgG antibodies if present) except the
bound antibodies.

20. .
After second incubation and washing, a chromogen
substrate is added upon which the enzyme reacts. Color intensity is
directly proportional to the amount of IgM HAV antibodies in the
sample.

21. References
1. Gauthier DK, et al. Am J Infect Control. 1989. PMID: 2672903
Review.See all similar articles
2. Aydin S. A short history, principles, and types of ELISA, and our
laboratory experience with peptide/protein analyses using ELISA.
Peptides. 2015 Oct;72:4-15. – PubMed
3. Engvall E. The ELISA, enzyme-linked immunosorbent assay. Clin
Chem. 2010 Feb;56(2):319-20. - PubMedShah K, Maghsoudlou P.

22. .
4. Enzyme-linked immunosorbent assay (ELISA): the basics. Br J Hosp
Med (Lond) 2016 Jul;77(7):C98-101. – PubMed
5. Konstantinou GN. Enzyme-Linked Immunosorbent Assay (ELISA).
Methods Mol Biol. 2017;1592:79-94. – PubMed
6. Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, Kuchel
GA. ELISA and multiplex technologies for cytokine measurement in
inflammation and aging research. J Gerontol A Biol Sci Med Sci. 2008
Aug;63(8):879-84. - PMC -

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