MEASUREMENT OF HORMONE BY ELISA

INTRODUCTION

• ELISA= Enzyme-Linked Immunosorbent Assay

• Immuno - refers to an immune response that causes the body to generate
antibodies
• Assay - refers to check or test the amount of a substance in a given mixture.
• ELISA is a widely used method for measuring the concentration of particular
molecule such as antibody or antigen in a sample.
• An enzyme conjugated with an antibody reacts with a colourless substrate to
generate a coloured reaction product. Such a substrate is called a chromogenic.
STEPS

There are four main general step to completing ELISA immunoassay. The steps are:
• Coating(with either antigen or antibody)
• Blocking(typically with the addition of bovine serum albumin(BSA)
• Detection
• Final read

• A number of enzymes have been employed for ELISA, including alkaline

phosphate, horseradish peroxidase and beta- galactosidase.
• Detection is carried out by the addition of a substrate that can generate a colour.
TYPES
Direct Elisa
Different types of ELISA kits

• There are now two major suppliers in the field, Buhlmann Laboratories and IBL
International-Tecan. Some other are:
• Abcam
• Kamiya
• The cloud- clone corp
• Enzo life sciences, Inc
• Biomatik
• Novus biological, LLC
• The Elabscience
MEASUREMENTS OF HORMONE BY
ELISA
Follicle-stimulating hormone

• FSH is a glycoprotein secreted by the basophilic cells of the anterior

pituitary.
• Gonadotropins- releasing hormone(GnRH) produced in the
hypothalamus controls the release of FSH from the anterior pituitary.
• FSH consists of subunits designated as alpha and beta
• In the female, FSH stimulates the growth and maturation of ovarian
follicle by acting directly on the receptors located on the granulosa
cells.
Principle of the assay

• The assay system utilizes a monoclonal anti-α-FSH for solid phase(microtiter wells)
immobilization and monoclonal anti-β-FSH antibody-enzyme(horseradish peroxidase)
conjugate solution.
• The test sample is allowed to react simultaneously with the antibodies resulting in the
FSH molecules being sandwich between the solid phase and enzyme-linked antibodies.
• After a 45minute incubation at room temperature, the wells are washed with water to
remove unbound-labeled antibodies.
• A solution of TMB reagent is added and incubated at room temperature for 20min
Resulting in the development of a blue colour. The colour development is stopped with
the addition of stop solution, and the colour is changed to yellow and measured
spectrophometrically at 450nm
Material required

• Precision pipettes: 50μl, 100μl and 1.0ml

• Distilled water
• Disposable pipette tips
• Vortex mixture
• Absorbent paper or paper towel
• A microtiter plate reader at 450nm
Wavelength, with a bandwidth of 10nm
• Graph paper
Assay procedure

• Secure the desired number of coated wells in the holders.

• Dispense 50μl of standards, specimen and controls into appropriate wells.
• Dispense 100μl of enzyme conjugate reagent into each well.
• Thoroughly mix for 30seconds.it is very important to have a complete mixing in
this setup.
• Incubate at room temperature(18-25c) for 45 minutes.
• Remove the incubation mixture by flicking plate contents into waste container.
• Rinse and flick the microtiter wells 5times with distilled or dionized water.
• strike the wells sharply onto absorbent paper or towels to remove all residual
water droplets.
• Dispense 100μl of TMB reagent into each well. Gently mix for 10seconds.
• Incubate at room temperature in the dark for 20minutes
• Stop the reaction by adding 100μl of stop solution to each well.
• Gently mix for 30 seconds. It is important to make sure that all the blue colour
changes to yellow colour completely.
• Read the optical density at 450nm with the microtiter plate reader within
15minutes.
sensitivity

• Based on random selected outpatient clinical laboratory samples, the mean FSH
values in males(N=100) and females(N=150) are 11 and 12mlU/ml, respectively.
• The mean FSH values in postmenopausal(N=60) and pregnant female(N=60) are
94 and 1.0mlU/ml, respectively
• The minimum detectable concentration of FSH by this assay is estimated to be
2.5mlU/ml.