The Effects Of Metformin And Diabinese On Female Sex Hormone Of Type 2 Diabetes Mellitus Patients
[UNIVERSITY OF ILORIN TEACHING HOSPITAL (UITH), ILORIN, KWARA STATE]

3.7.5    DERMINATION OF PROGESTERONE
Progesterone was determined in serum samples by using an ELISA kit (Progesterone ELISAkit, Product code: 4825-300, lake forest,USA ) according to the manufacturer’s instructions. The kit comprises a 96-well clear microtitreplate coated with anti-progesterone IgG, standard solutions containing 0(S0), 0.2 (S1), 1.0 (S2), 8.0 (S3), and 40.0 (S4) ng/ml of progesterone, progesterone-horseradish peroxidase (HRP) conjugate (tracer), a TMB-based chromogenic substrate for HRP, and a stop solution containing sulphuric acid to stop enzymatic activity.
3.7.6    PROGESTERONE ASSAY PRINCIPLE
The essential reagents required for a enzyme immunoassay include antibody, enzyme-antigen conjugate and native antigen. Upon mixing biotinylated antibody, enzyme-antigen conjugate and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme-antigen conjugate for a limited number of antibody binding sites. The interaction is illustrated by the followed equation:

AbBtn= Biotinylated Antibody (Constant Quantity)
Ag = Native Antigen (Variable Quantity)
EnzAg = Enzyme-antigen Conjugate (Constant Quantity)
AgAbBtn = Antigen-Antibody Complex
EnzAg AbBtn = Enzyme-antigen Conjugate -Antibody Complex
ka = Rate Constant of Association
k-a = Rate Constant of Disassociation
K = ka / k-a = Equilibrium Constant
A simultaneous reaction between the biotin attached to the antibody and the streptavidin immobilized on the microwell occurs. This effects the separation of the antibody bound fraction after decantation or aspiration.
AgAbBtn + EnzAgAbBtn + StreptavidinCW⇒  immobilized complex StreptavidinCW = Streptavidin immobilized on well.
3.7.7     PROGESTERONE ASSAY PROCEDURE
•    The micro plates wells were formatted in duplicate for each serum reference, control and patient specimen to be assayed.
•    25μl of the appropriate serum reference, control or specimen was pipette into the assigned well.
•    50µl of Progesterone Enzyme Reagent was added to all wells.
•    The micro plate was swirl gently for 20 seconds to mix and cover.
•    50 µl of progesterone Biotin Reagent was added to all wells
•    The microplate was swirl gently for 20 seconds.
•    It was incubated for 60 minutes at room temperature.
•    The content of the micro plate was discarded by decantation and the plate was tapped and blotted dry with absorbent paper.
•    350ul of wash buffer was added, decant (tap and blot). This was repeated three times.
•    100ul of the working substrate solution was added to all the wells.
•    It was incubated at room temperature for 20 minutes.
•    50ul of stop solution was added to each well and mixed gently for 20 seconds.
•    The absorbance of each well was read at 450nm in a micro plate reader.