serum references of known antigen concentration, a dose

response curve can be generated from which the antigen

concentration of an unknown can be ascertained.

Immunoenzymometric/Sandwitch

(Streptavidin-Biotin) ELISA

Thyrotropin (TSH)

(Courtesy: Lilac Medicare)

Intended use: The quantitative determination of

thyrotropin concentration in human serum by a

microplate immunoenzymometric assay.

mfd: Monobind Inc.

Summary and Explanation of the Test

Measurement of the serum concentration of thyrotropin

(TSH), a glycoprotein with a molecular weight of 28,000

daltons and secreted from the anterior pituitary, is

generally regarded as the most sensitive indicator available

for the diagnosis of primary and secondary (pituitary)

hypothyroidism. Increase in serum concentrations of

TSH, which is primarily responsible for the synthesis and

release of thyroid hormones, is an early and sensitive

indicator of decrease thyroid reserve and in conjunction

with decreased thyroxine (T4) concentrations is diagnostic

of primary hypothyroidism. The expected increase in

TSH concentrations demonstrates the classical negative

feedback system between the pituitary and thyroid glands.

That is, primary thyroid gland failure reduces secretion of

the thyroid hormones, which in turn stimulates the release

of TSH from the pituitary.

Additionally, TSH measurements are equally useful

in differentiating secondary and tertiary (hypothalamic)

hypothyroidism from the primary thyroid disease. TSH

release from the pituitary is regulated by thyrotropin

releasing factor (TRH), which is secreted by the hypothalamus, and by direct action of T4 and triiodothyronine (T3),

the thyroid hormones, at the pituitary. Increase levels

of T3 and T4 reduces the response of the pituitary to the

stimulatory effects of TRH. In secondary and tertiary

hypothyroidism, concentrations of T4 are usually low

and TSH levels are generally low or normal. Either pituitary TSH deficiency (secondary hypothyroidism) or

insufficiency of stimulation of the pituitary by TRH (tertiary

hypothyroidism) causes this. The TRH stimulation test

differentiates these conditions. In secondary hypothyroidism, TSH response to TRH is blunted while a normal or

delayed response is obtained in tertiary hypothyroidism.

Further, the advent of immunoenzymometric assays

has provided the laboratory with sufficient sensitivity

to enable the differentiating of hyperthyroidism from

euthyroid population and extending the usefulness of

TSH measurements. This method is a second-generation

assay, which provides the means for discrimination in the

hyperthyroid-euthyroid range. The functional sensitivity

(< 20% between assay CV) of the one-hour procedure

is 0.195 µIU/mL while the two-hour procedure has a

functional sensitivity of 0.095 µIU/mL.

In this method, TSH calibrator, patient specimen

or control is first added to a streptavidin coated well.

Biotinylated monoclonal and enzyme labeled antibodies

are added and the reactants mixed. Reaction between the

various TSH antibodies and native TSH forms a sandwich

complex that binds with the streptavidin coated to the

well.

After the completion of the required incubation period,

the antibody bound enzyme thyrotropin conjugate

is separated from the unbound enzyme thyrotropin

conjugate by aspiration or decantation. The activity of the

enzyme present on the surface of the well is quantitated by

reaction with a suitable substrate to produce color.

Serology/Immunology 599

The employment of several serum references of known

thyrotropin levels permits construction of a dose response

curve of activity and concentration. From comparison to

the dose response curve, an unknown specimen’s activity

can be correlated with thyrotropin concentration.

Principle

Immunoenzymometric Assay

The essential reagents required for an immunoenzymometric assay include high affinity and specificity

antibodies (enzyme conjugated and immobilized), with

different and distinct epitope recognition, in excess, and

native antigen. In this procedure, the immobilization takes

place during the assay at the surface of a microplate well

through the interaction of streptavidin coated on the well

and exogenously added biotinylated monoclonal anti-TSH

antibody.

Upon mixing monoclonal biotinylated antibody, the

enzyme-labeled antibody and a serum containing the

native antigen, reaction results between the native antigen

and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. The interaction

is illustrated by the following equation:

 ka

EnzAb(p) + AgTSH + BtnAb(m) EnzAb(p)-AgTSH-BtnAb(m)

 k-a

BtnAb(m) = Biotinylated monoclonal antibody

 (excess quantity)

AgTSH = Native antigen (variable quantity) EnzAb(p) = Enzyme-polyclonal antibody

(excess quantity) EnzAb(p) - AgTSH-BtnAb(m)= Antigen-Antibodies

 Sandwich complex

ka = Rate constant of association

k-a = Rate constant of dissociation

Simultaneously, the complex is deposited to the well

through the high affinity reaction of streptavidin and

biotinylated antibody. This interaction is illustrated below:

EnzAb(p)-AgTSH-BtnAb(m) + StreptavidinC.W.

⇒ immobilized complex

StreptavidinC.W. = Streptavidin immobolized on well

Immobilized complex = Sandwich complex bound

to the solid surface

After equilibrium is attained, the antibody-bound

fraction is separated from unbound antigen by decantation

or aspiration. The enzyme activity in the antibody-bound

fraction is directly proportional to the native antigen

concentration. By utilizing several different serum

references of known antigen values, a dose response curve

can be generated from which the antigen concentration of

an unknown can be ascertained.

Reagents

Materials Provided

A. Thyrotropin calibrators—1 mL/vial: Seven (7) vials

of references for TSH Antigen at levels of 0(A), 0.5(B),

2.5(C), 5.0(D), 10(E), 20(F) and 40(G) µIU/mL. Store

at 2–8°C. A preservative has been added.

 Note: The calibrators, human serum based, were

calibrated using a reference preparation, which was

assayed against the WHO 2nd IRP 80/558.

B. TSH enzyme reagent—13 mL/vial: One (1) vial

containing enzyme labeled affinity purified polyclonal

goat antibody, biotinylated monoclonal mouse IgG in

buffer, dye, and preservative. Store at 2–8°C.

C. Streptavidin coated microplate—96 wells: One 96-

well microplate coated with streptavidin and packaged

in an aluminum bag with a drying agent. Store at 2–8°C.

D. Wash solution concentrate—20 mL: One (1)

vial containing a surfactant in buffered saline. A

preservative has been added. Store at 2-30°C.

E. Substrate A—7 mL/vial: SA One (1) bottle containing

tetramethylbenzidine (TMB) in buffer. Store at 2–8°C.

F. Substrate B—7 mL/vial: One (1) bottle containing

hydrogen peroxide (H2O2) in buffer. Store at 2–8°C.

G. Stop solution—8 mL/vial: One (1) bottle containing

a strong acid (1N HCl). Store at 2–30°C.

Note 1: Do not use reagents beyond the kit expiration date.

Note 2: Opened reagents are stable for sixty (60) days when

stored at 2–8°.

Note 3: Above reagents are for a single 96-well microplate.

For In Vitro Diagnostic Use

Not for Internal or External Use in Humans or Animals

Precautions

All products that contain human serum have been

found to be nonreactive for Hepatitis B surface antigen,

HIV 1 and 2 and HCV antibodies by FDA required tests.

Since no known test can offer complete assurance that

infectious agents are absent, all human serum products

should be handled as potentially hazardous and capable

of transmitting disease. Good laboratory procedures for

handling blood products can be found in the Center for

Disease Control/National Institute of Health, “Biosafety

in Microbiological and Biomedical Laboratories,” 2nd

edition, 1988, HHS.

600 Concise Book of Medical Laboratory Technology: Methods and Interpretations Specimen Collection and Preparation

The specimens shall be blood serum in type and the

usual precautions in the collection of venipuncture

samples should be observed. For accurate comparison

to established normal values, a fasting morning serum

sample should be obtained. The blood should be collected

in a plain redtop venipuncture tube without additives

or gel barrier. Allow the blood to clot. Centrifuge the

specimen to separate the serum from the cells.

Samples may be refrigerated at 2–8°C for a maximum

period of five (5) days. If the specimen(s) cannot be

assayed within this time, the sample(s) may be stored at

temperatures of –20°C for up to 30 days. Avoid repetitive

freezing and thawing. When assayed in duplicate, 0.100mL

of the specimen is required.

Required but not Provided

1. Pipette(s) capable of delivering 50 µL and 100 µL

volumes with a precision of better than 1.5%.

2. Dispenser(s) for repetitive deliveries of 0.100 mL and

0.300 mL volumes with a precision of better than

1.5%.

3. Microplate washer or a squeeze bottle (optional).

4. Microplate Reader with 450 nm and 620 nm

wavelength absorbance capability (The 620 nm filter

is optional).

5. Adjustable volume (200–1000 µL) dispenser.

6. Container(s) for mixing of reagents (see below).

7. Absorbent Paper for blotting the microplate wells.

8. Plastic wrap or microplate cover for incubation

steps.

9. Vacuum aspirator (optional) for wash steps.

10. Timer.

11. Storage container for storage of wash buffer.

12. Distilled or deionized water.

13. Quality control materials.

Reagent Preparation

1. Wash Buffer

 Dilute contents of Wash Concentrate to 1000 mL

with distilled or deionized water in a suitable storage

container. Store at room temperature 20–27°C for up

to 60 days.

2. Working Substrate Solution

 Pour the contents of the vial labeled Solution ‘A’

into the vial labeled Solution ‘B’. Mix and store at

2–8°C. Use within 60 days. Or for longer periods of

usage determine the amount of reagent needed and

prepare by mixing equal portions of Substrate A and

Substrate B in a suitable container. For example, add

1 mL of A and 1 mL of B per two (2) eight well strips (A

slight excess of solution is made. Discard the unused

portion).