Implementation Details

a) ESTRADIOL/ESTROGEN (E2) ASSAY (Tietz, 1995) Principle

The E2 EIA is based on the Principle of competitive binding between E2 in the test specimen and E2-HRP conjugate for a constant amount of rabbit Estradiol. In the incubation, goat anti-rabbit IgG-coated wells were incubated with 25 μl E2 blank, controls, samples, 100 μl Estradiol-HRP Conjugate Reagent and 50 μl rabbit anti-Estradiol reagent at room temperature (18-25ºC) for 90 minutes. During the incubation, a fixed amount of HRP-labeled E2 competes with the endogenous E2 in the sample, or quality control serum for a fixed number of binding sites of the specific E2 antibody. Thus, the amount of E2 peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of E2 in the specimen increases. Unbound E2 peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 20 minutes, resulting in the development of blue color. The color development is stopped with the addition of 1N HCl, and the concentration is measured spectrophotometrically at 450 nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled E2 in the sample.

Procedure

The desired number of coated wells was placed in the holder. Twenty five (25) μl of reference reagents (blank), specimens and controls were dispensed into appropriate wells. Then 100 μl of estradiol-HRP conjugate reagent was added into each well. This was followed by 50 μl of rabbit anti-estradiol (E2) reagent to each well. The mixture was thoroughly mixed for 30 seconds and incubated for 90 minutes at 25ºC. The microwells were rinsed and flicked 5 times with distilled water. Then, 100 μl of TMB reagent were dispensed into each well and gently mixed for 10 seconds. The mixture was incubated for 20 minutes at temperature 25ºC. The reaction stopped by adding 100 µl of Stop Solution to each well. The final mixture was gently mixed for another 30 seconds. The concentrations were measured at 450 nm within 15 minutes.

41 b) ESTRONE (E3) ASSAY (Folan, 1988)

Principle

The Principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in blank, control and human samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microwell plate. The washing and decanting procedures remove unbound materials.

After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The intensity of the colour formed is inversely proportional to the concentration of estrone in the sample.

Procedure

The required number of microwell strips was placed in the holder. Then 50 μl of each blanks (control 1 and 2), sample control and specimen sample were Pipetted into the corresponding labelled wells. Another 100 μl of the conjugate working solution were pipetted into each well.

And incubated on a plate shaker of 200 rpm for 1 hour at 25ºC. The plate wells were washed 3 times with wash buffer, 300 μl/well for each wash and the plate firmly tapped against absorbent paper to ensure that it is dry. Then 150 μL of TMB substrate were pipetted into each well. And incubated on a plate shaker for 15 minutes at 25ºC temperature. Finally, 50 μl of stopping solution were dispenced into each well. The concentrations were read at 450 nm within 20 minutes.

c) ESTRIOL (E1) ASSAY (Speroff, 1983) Principle

The estriol ELISA kit is a competitive immunoassay for the quantitative determination of free estriol in biological fluids.The kit for the quantitative measurement of estriol uses a polyclonal antibody to estriol to bind, in a competitive manner, estriol in the sample which has estriol covalently attached to it. After a simultaneous incubation at room temperature the excess reagents are washed away and substrate is added.

After a short incubation time the enzyme reaction is stopped and the yellow color generated is read on a microplate reader at 405nm. The intensity of the bound yellow color is inversely proportional to the concentration of estriol in samples.

Procedure

The required number of microwell strips was placed in the holder. Then 100 μl of each blank (Reference Reagent), control and specimen sample were pipetted into the corresponding labelled wells. Then 50μl of blue conjugate was pipetted into each well, except the blank wells. Another 50 μL of yellow Antibody was pipetted into each well, except the Blank wells. And the resultant mixture was incubated on a plate shaker for 2 hours at 500 rpm at 25ºC. The contents of the wells were emptied and washed by adding 400 μL of wash solution to every well. The washing process was repeated 2 more times for a total of 3 washes. After the final wash, the wells were aspirated, and firmly tapped on a lint paper towel to remove any remaining wash buffer. Five (5) μL of the blue conjugate were added to the wells. Then, 200 μl of the pNpp Substrate solution was added to every well and incubate for 1 hour at 25ºC. Finally, 50μl of stop solution was added to every well.

The plate reader was blanked against the blank wells, and concentration was read at 405nm.

d) PROLACTIN ASSAY (Tietz, 1995) Principle

The prolactin quantitative test is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay system utilizes a mouse monoclonal anti-prolactin antibody for solid phase (microtiter wells) immobilization and another mouse monoclonal anti-prolactin antibody in the

42 antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the antibodies, resulting in the prolactin molecules being sandwiched between the solid phase and enzyme-linked antibodies. After 45-minute 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 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of stop solution, and the color is changed to yellow and measured spectrophotometrically at 450 nm. The concentration of prolactin is directly proportional to the color intensity of the test sample.

Procedure

The desired number of coated wells was placed in the holder. Then, 50 μl of blank (Reference Reagent), specimens, and controls were dispensed into appropriate wells. And 100 μl of enzyme conjugate reagent was added into each well. The mixture was gently mixed for 10 seconds. And incubated for 45 minutes at 25ºC. The incubation mixture was removed by flicking plate contents.

The flicking and rinsing of the microtiter wells was done 5 times with distilled water. The wells were striked sharply onto absorbent paper towels to remove residual water droplets. Then, 100 μl TMB reagent was dispensed into each well, and was gently mixed for 10 seconds. And incubated in the dark for 20 minutes at 25ºC. The reaction was stopped by addition of 100μl of stop solution to each well. The final mixture was gently mixed for another 30 seconds. The concentration was measured at 450nm within 15 minutes.

e) PROGESTERONE ASSAY (Kim, 1974) Principle

This kit is based on the Principle of competitive binding between progesterone in the test specimen and progesterone-HRP conjugate for a constant amount of rabbit anti- progesterone.

In the incubation, goat anti-rabbit IgG-coated wells are incubated with 25 μl progesterone blank, controls, samples, 100 μl progesterone-HRP conjugate reagent and 50 μl rabbit anti-progesterone reagent at room temperature (18-25°C) for 90 minutes.

During the incubation, a fixed amount of HRP-labeled progesterone competes with the endogenous progesterone in the sample, or quality control serum for a fixed number of binding sites of the specific progesterone antibody. Thus, the amount of progesterone peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of progesterone in the specimen increases. Unbound progesterone peroxidase conjugate is then removed and the wells washed.

Next, a solution of TMB reagent is then added and incubated at room temperature for 20 minutes, resulting in the development of blue color.

The color development is stopped with the addition of stop solution, and the concentration is measured spectrophotometrically at 450 nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled progesterone in the sample.

Procedure

The desired number of coated wells was placed in the holder. Twenty five (25)μl of blank (control 1 and 2), specimens, and controls were pipetted into appropriate wells. Then, 100μl of working progesterone-HRP conjugate reagent was dispensed into each well. And 50μl of rabbit anti-progesterone reagent as added to each well, and the mixture was thoroughly mixed for 30 seconds.

Then the plate and its contents were incubated for 90 minutes at 25°C. This was followed by rinsing and flicking the microtiter wells 5 times with distilled water. Then 100μl of TMB reagent was dispensed into each well and gently mixed for 10 seconds. The mixture was incubated at

43 25°C for 20 minutes. The reaction was stopped by adding 100μl of stop solution to each well, and gently mixed for another 30 seconds. The concentrations were measured at 450nm within 15 minutes.

f) TESTOSTERONE ASSAY (Hall, 1988) Principle

The Principle of the following enzyme immunoassay test follows the typical competitive binding schematic.

Competition occurs between an unlabeled antigen and an enzyme-labeled antigen (conjugate) for a limited number of antibody binding sites on the microwell plate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stop solution. The concentration is measured on a microtiter plate reader. The intensity of the color formed is inversely proportional to the concentration of testosterone in the sample.

Procedure

The required number of microwell strips was placed in the holder and 50μl of each calibrator, control and specimen sample were pipetted into correspondingly labeled wells. Then, 100μl of the conjugate working solution was pipetted into each well. And the mixture was incubated on a plate shaker at 200 rpm for 1 hour at 25°C. The wells were washed 3 times with 300 μl of diluted wash buffer per well, and tapped firmly against absorbent paper to ensure that it is dry. Another 150μl of TMB substrate was pipetted into each well. And incubated on a plate shaker for 15 minutes at 25°C. The reaction was stopped by pipetting 50μl of stop solution into each well. The concentration was measured at 450nm within 20 minutes.

g) LEUTEINIZING HORMONE (LH) ASSAY (Tietz, 1995) Principle

The Principle of the following enzyme immunoassay test follows a typical two-step capture or

‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for LH is immobilized onto the microwell plate and another monoclonal antibody specific for a different region of LH is conjugated to horse radish peroxidase (HRP). LH from the sample and blank are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second washing step, the enzyme substrate is added.

The enzymatic reaction is terminated by addition of the stopping solution.

The concentration is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of LH in the sample.

Procedure

The required number of microwells strips was placed in the holder and 25 μl of each calibrator, control and specimen sample were dispenced into correspondingly labelled wells. Then, 100 μl of assay buffer was pipetted into each well and incubated for 30 minutes at 25°C. The wells were washed 3 times with 300 μl of diluted wash buffer per well and tapped firmly against absorbent paper to ensure that it is dry. Another 100 μl of the conjugate working solution was pipetted into each well and incubated for 30 minutes at 25°C. The plate wells were washed again in the same manner as stated earlier. Then, 100 μl of TMB substrate was dispenced into each well, and incubated for 20 minutes at 25°C. Finally, 50 μl of stop solution was pipetted into each well. The concentration was measured at 450 nm within 20 minutes after addition of the stop solution.

44 h) FOLLICLE STIMULLATING HORMONE (FSH) ASSAY (Gore-Langton and Armstrong, 1988)

Principle

The Principle of the following enzyme immunoassay test follows a typical two-step capture or

„sandwich‟ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for FSH is immobilized onto the microwell plate and another monoclonal antibody specific for a different region of FSH is conjugated to horse radish peroxidase (HRP). FSH from the sample and blank are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stop solution. The concentration is measured on a microtiter plate reader. The intensity of the color formed by the enzymatic reaction is directly proportional to the concentration of FSH in the sample.

Procedure

The required number of microwell strips was placed in the holder and 25 μl of each calibrator, control and specimen sample were pipetted into correspondingly labeled wells. Then, 100 μl of assay buffer was added into each well, and incubated for 30 minutes at 25°C. The wells were washed 3 times with 300 μl of diluted wash buffer per well and tapped firmly against absorbent paper to ensure that it is dry. Again, 100 μl of the conjugate working solution was added into each well, and incubated on for 30 minutes at 25°C. The washing of the wells was repeated again in the same manner. Another 100 μl of TMB substrate was pipetted into each well and incubated on a plate shaker for 20 minutes at 25°C. Then, 50 μl of stop solution was added into each well to stop the reaction. The concentrations were read at 450 nm within 20 minutes after addition of the stop solution.

i) ANDROSTENEDIONE ASSAY (Kim, 1974) Principle

Androstenedione in vitro competitive ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of androstenedione in serum and plasma. A 96-well plate has been precoated with anti-androstenedione IgG.

Samples and the androstenedione-HRP conjugate are added to the wells, where androstenedione in the sample competes with the added androstenedione-HRP for antibody binding. After incubation, the wells are washed to remove unbound material and TMB substrate is then added which is catalyzed by HRP to produce blue coloration. The reaction is terminated by addition of stop solution which stops the color development and produces a color change from blue to yellow.The intensity of signal is inversely proportional to the amount of androstenedione in the sample and the intensity is measured at 450 nm.

Procedure

The required number of microwell strips was placed in the holder. Then, 25 μl of blank (Reagent), control and sample were added into their respective wells. Also, 200 μl androstenedione-HRP conjugate was added to each well except for the blank well. The wells were covered with the foil and incubate for 1 hour at 37°C. When incubation has been completed, the foil was removed and the content of the wells were aspirated and washed three times with 300 μl washing Solution.

Another 100 μl TMB substrate solution was added into all wells, and incubated for 15 minutes at 25°C in the dark. The reaction was stopped by the addition of 100μl stop solution into the wells and the microplate was shaked gently. The concentration was measure at 450 nm against blank within 5 minutes.

45 3.2.4.2. THYROID HORMONE ASSAY

a) THYROID STIMULATING HORMONE (TSH) ASSAY (Witherspoon and Shuler,1984)

Principle

The TSH ELISA test is based on the principle of a solid phase enzyme linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the intact TSH molecule. Mouse monoclonal anti-TSH antibody is used for solid phase immobilization. A goat anti-TSH antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the TSH molecules being sandwiched between the solid phase and enzyme-linked antibodies. After 60-minute incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB reagent is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of stop solution, changing the color to yellow. The concentration of TSH is directly proportional to the color intensity of the test sample. Concentration is measured spectrophotometrically at 450 nm.

Procedure

The desired number of coated wells were placed in the holder. Then, 100 μl of blank, specimens, and controls were dispensed into appropriate wells. After which 100 μl of enzyme conjugate reagent was added into each well. The mixtures were thoroughly mixed for 30 seconds. And incubated at 25°C for 60 minutes. The incubation mixture was removed by flicking plate contents. The microtiter wells were rinsed and flicked 5 times with distilled water and sharply striked onto absorbent paper towels to remove all residual water droplets. Another 100 μl of TMB reagent was dispensed into each well. The mixture was gently mixed for 10 seconds and incubated at 25°C for 20 minutes. The reaction was stopped by adding 100 μl of stop solution to each well and mixed for 30 seconds. The concentrations were read at 450 nm within 15 minutes.

b) THYROXINE (T4) ASSAY (Abuid et al., 1974) Principle

To measure T4 by competitive immunoassay techniques, a sample of serum or plasma containing the T4 to be quantified is mixed with labeled T4 and T4 antibody. In this T4 EIA, antibody to T4 is coated on a solid phase (microtiter well). A measured amount of patient serum and a constant amount of T4 labeled with horseradish peroxidase are added. During incubation, T4 in the patient sample and enzyme-labeled T4 compete for the limited binding sites on the T4 antibody. After 60-minute incubation at room temperature, the solid phase is washed with water to remove unbound-labeled T4. A solution of tetramethylbenzidine (TMB) is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of 1N HCI, and the resulting yellow color is measured spectrophotometrically at 450 nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of T4 in the sample.

Procedure

The desired numbers of coated wells were placed in the holder and 25μL of blank, specimens, and controls was pipetted into appropriate wells. Then 100 μL of working conjugate reagent was added into each well and was thoroughly mixed for 30 seconds. The mixture was incubated at 25°C for 60 minutes. The incubation mixture was removed by flicking plate contents into a waste container. The microtiter wells rinsed and flicked 5 times with distilled water and the wells were

46 striked sharply onto absorbent paper towels to remove all residual water droplets. Then 100μL of TMB reagent was dispenced into each well and was mixed gently for 5 seconds. Again, the mixture was incubated at 25°C, in the dark, for 20 minutes. Stop solution (100μL) was added to each well to stop the reaction. The content of the wells were gently mixed for 30 seconds. The concentration was measured at 450nm within 15 minutes.

c) TRIIODOTHYROXINE(T3) ASSAY (Abuid et al., 1974) Principle

Competitive Enzyme Immunoassay – Analog Method for Free T3

The essential reagents required for a solid phase enzyme immunoassay include immobilized T3 antibody, enzyme-T3 conjugate and native free T3 antigen. The enzyme-T3 conjugate should have no measurable binding to serum proteins especially TBG and albumin. The method achieves this goal.

Upon mixing immobilized antibody, enzyme-T3 conjugate and a serum containing the native free T3 antigen, a competition reaction results between the native free T3 and the enzyme-T3 conjugate for a limited number of insolubulized binding sites. The interaction is illustrated by the followed equation:

Ab c.w. = Monospecific Immobilized Antibody (Constant Quantity) Ag = Native Free Antigen (Variable Quantity)

EnzAg = Enzyme-antigen Conjugate (Constant Quantity) AgAb c.w. = Antigen-Antibody Complex

EnzAg Ab c.w. = Enzyme-antigen Conjugate -Antibody Complex Ka = Rate Constant of Association

k-a = Rate Constant of Disassociation K = ka / k-a = Equilibrium Constant

After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme concentration in the antibody-bound fraction is inversely proportional to the native free antigen concentration.

Procedure

The desired numbers of coated wells were placed in the holder. Then, 50 μl of the appropriate serum reference, control and specimen was pipetted into the assigned well. Again. 100μl of fT3-enzyme reagent solution was added to all wells. The microplate was swirled gently for 30 seconds to mix; it was then covered and incubated 60 minutes at 25°C. The content of the microplate was discarded by aspiration. Then 300μl of wash buffer was added to each wells and aspirated. The wash process was repeated two additional times for a total of three washes. Also, 100μl of working substrate solution was added to all wells and incubated for 15 minutes at 25°C. Finally, 50 μl of stop solution was added to each well and mixed for 20 seconds. The concentrations in each well were measured at 450nm.

47 3.2.4.3. INTERLUKINS (IL) ASSAY (Chan and Perlstein, 1987).

We assayed for interleukins 1- 33 except IL- 19, 20, 24, 28, and 29 because of the inability to get the test kits.

Principle

IL in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of IL in serum, plasma, buffered solutions and cell culture medium.

A monoclonal antibody specific for the particular IL has been coated onto the wells of the microtiter strips provided. Samples, including blank of known IL concentrations, control specimens or unknowns were pipetted into these wells. During the first incubation, the blank or samples and a biotinylated monoclonal antibody specific for the particular IL are simultaneously incubated. After washing, the enzyme Streptavidin- HRP, that binds the biotinylated antibody is added, incubated and washed. A TMB substrate solution is added which acts on the bound enzyme to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of IL present in the samples.

Procedure

The number of microwell strips required was placed in the holder. The microwell strips was washed twice with exactly 400 μl Wash Buffer prior to the analysis. The wells were thoroughly aspirated of its contents between washes. The Wash Buffer was allowed to sit in the wells for 15 seconds before aspiration. After the last wash step, wells were emptied and tapped on absorbent paper towel to remove excess Wash Buffer. The microwell strips were used immediately after washing. Exactly 100 μl of Sample Diluent was added to all blank wells. Another, 100 μl of prepared Reference Reagent was pipetted into the well. The contents of wells were mixed by repeated aspiration and ejection, and 100 μl of the mixture were transfered to another wells B1 and B2, respectively. Another, 100 μl of sample diluent was added to the blank wells. Exactly, 50 μl of sample diluent was added to the sample wells. Also, 50 μl of each sample was added to the sample wells. Then 50 μl of biotin-conjugate was added to all wells. Covered with an adhesive film and incubated at 25°C in the dark over night. After the incubation, adhesive film was removed and the wells emptied. Then the microwell strips was washed 6 times according to step two. Then 100 μl of diluted streptavidin-HRP to all wells. Covered with an adhesive film and incubated at 25°C for 1 hour on a microplate shaker set at 400 rpm in the dark. After the incubation, the adhesive film was removed and the wells emptied. The microwell strips was washed 6 times according to step two.

At the amplification stage, 100 μl of amplification solution I was added to all wells. Covered with an adhesive film and incubated at 25°C for 15 minutes on a microplate shaker set at 400 rpm in the dark. After the incubation, the adhesive film was removed and the wells emptied. The microwell strips was washed 6 times according to step two. Another, 100 μl of amplification solution II was added to all wells. Covered with an adhesive film and incubated at 25°C for 30 minutes, on a microplate shaker set at 400 rpm in the dark. After the incubation, the adhesive film was removed and the wells emptied. The microwell strips was washed 6 times according to step two. Again, another 100 μl of TMB substrate solution was pipetted to all wells and incubated at 25°C for 20 min. The enzyme reaction was stopped by pipetting 100 μl of stop solution into each well. The plate reader was blanked using the blank wells and concentration of both the samples and the control was determined at 450 nm as the primary wave length.