- 1. E nzyme- L inkedI mmuno s orbentA ssay
2. Introduction
3. What is ELISA?
- Biochemical technique used mainly in immunology.
- first and most basic test to determine if an individual is
positive for a selected pathogen, such as HIV.
- 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of
96 wells, each of which are about 1 cm high and 0.7 cm in
diameter.
4. Types of ELISA
-
- Postive or Negative results
-
- optical density or fluorescent units of the sample is
interpolated into a standard curve, which is typically a serial
dilution of the target.
5. APPLICATIONS OF ELISA
- Serum Antibody Concentrations
- Detecting potential food allergens
- (milk, peanuts, walnuts, almonds and eggs)
- Disease outbreaks- tracking the spread
- e.g. HIV, bird flu, common, colds, cholera,
- e.g. pregnancy hormones, drug allergen,
- Detection of antibodies in blood sample for
- e.g. Lyme Disease, trichinosis, HIV, bird flu
6. Basic principles of ELISA
- the antibodies fixed to a solid surface, such as the inner
surface of a test tube;
- a preparation of the same antibodies coupled to an enzyme. This
is one (e.g.,-galactosidase ) that produces a colored product from
a colorless substrate.
7. Objectives 8.
- To be able to determine if a serum sample is positive or
negativewhich may help in the diagnosis and treatment of a
particular diseaselike HIV and leptospirosis and alike.
- To be able to apply the proper methods in achieving an
accurateresults.
- To be able to know the different reagents, their properties
andfunctions.
9. Methodology
- ELISA for Tracking Disease Outbreaks
10. Methods
- A yellow tube and plastic transfer was labeled.
- The bodily fluid was then transferred into the tube of another
group.The samples were then mixed and after which, the half of the
shared sample (750 l)was placed in the group s tube.
11. Methods
- The sharing protocol was repeated twice.
- The 12-well strip was then labeled.On the first 3 wells, it was
labeled as + while for the next 3 it was labeled as - .
- The remaining wells were labeled with two of the member s
initials.
12. Methods
- A fresh pipette tip was used to transfer 50 l of the positive
control into the + wells (the same was done for the - wells).
- 50l of the group s sample was transferred into the the
appropriately initialed three wells.
13. Methods
- There was a 5 minute waiting period so as to allow the proteins
in the samples to bind to the plastic wells.
- The microplate strip was then tapped onto the paper towels
provided.
- The well was then filled with wash buffer.
14. Methods
- A 50 l of primary antibody was then transferred into the 12
wells of the microplate strip.
- The fluids was then allowed to stand for another five minutes
so as to allow the antibody to bind.
- Washing of the wells was again performed.
15. Methods
- A 50 l of secondary antibody was then transferred into the 12
wells of the microplate strip.
- A 5 minute waiting period and washing of the microplate wells
was again performed (2x).
- A 50l of enzyme substrate was then transferred into the 12
wells of the microplate strip.
16. Methods
- The enzyme substrate was let to stand for five minutes.
- The results were then observed and recorded.
17. Results and discussion 18. Jumong and Hwang Jini Jang geum
&Lady Soshono 19. C4 1st Sharing 2nd Sharing 3rd Sharing
Results Lastikman Super Inggo Macoy Jang Geum Negative Super Inggo
Lastikman Jumong Baron Negative Zaido Kokey Ethel B. L. Soshono
Positive Kokey Zaido Hwang Jini Ruben Positive C5 1st Sharing 2nd
Sharing 3rd Sharing Results Macoy EthelB. Lastikman Hwang Jini
Positive Ethel B. Macoy Zaido Jumong Positive BaronRuben Jang geum
Super Inggo Negative Ruben Baron L. Soshono Kokey Positive C6 1st
Sharing 2nd Sharing 3rd Sharing Results Jumong Hwang Jini Super
Inggo Ethel B. Positive Hwang Jini Jumong Kokey Macoy Positive Jang
geum L. Soshono Baron Lastikman Negative L. Soshono Jang geum Ruben
Zaido Positive 20. Carriers