Ms. Chnar Hussam Dr. Tola FARAJ__________________________________________________

Immunology_ 2nd Stage/2nd Semester

https://tiu.edu.iq/

2020 - 2021

TIU - Faculty of Science

Medical Analysis Department

ELISALab.9

Lab outlines:

5 minutes all settle in the lab. with

attendance

5 minutes Quiz

15-20 minutes pre-lab (10 ppt slides

including the Introduction and detail

of the practical part)

20 minutes practical part

Leave the lab 5 minutes earlier

Introduction to ELISA

What is an ELISA test?

• An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions. Antibodies are proteins that your body produces in response to harmful substances called antigens.

The principle

In ELISA, various antigen-antibody combinations are used, always including an enzyme-labeled antigen or antibody, and enzyme activity is measured calorimetrically.The enzyme activity is measured using a substrate that changes color when modified by the enzyme. Light absorption of the product formed after substrate addition is measured and converted to numeric values.

ELISA kitSample Diluent: A solution used to dilute the sample to the concentration proper for the ELISA test.

Conjugate: It contains the secondary antibody, which will bind to the antibody-antigen complex. And it is linked with an enzyme to react with substrate.

Substrate: Substrate react with enzyme to produce a color change, which is mean to measure the amount of the antibody or antigen. TMB (3,3’,5,5’-Tetramethylbenzidine) is a often used substrate in the ELISA Kit.

Stop Solution: Stop the reaction, it usually is the H2SO4 and HCI mixed solution.

Wash Buffer(s): When users remove the non-bound antibodies or antigens, they choose the proper concentrate wash buffer.

ELISA reader An ELISA reader measures and quantitates the color differences in the 12 wells of the plate. ELISA readers or micro plate readers do spectrophotometry; they emit light at one wave length, and measure the amount of light absorbed and reflected by an object such as a protein.

ELISA washer Microplate washers are laboratory instruments designed to control the procedure of washing experimental samples arranged in plate-based formats.

Microplate or micro-well is a solid phase most commonly has 96-well, and each well is noncovalently coated with antigen or antibody.

Incubator & shakers

• In certain ELISA systems, the plates are rotated during incubation for better antigen antibody reaction. The effect of rotating plates is to mix the reactants completely during the incubation step.

Microplate or micro-wells coverduring incubation wells should cover using cover plate.to prevent contamination

Multi-channel micropipitte it used to hold measured sample value

Micropipette tips

Direct ELISA

Indirect ELISA

Sandwich ELISA

Thanks