Direct ELISA

DNase Activity

(Courtesy: Lilac Medicare)

Mfd: Orgentec

DNase Activity is a solid phase enzyme immunoassay

(ELISA) for the quantitative screening of DNase in human

serum or EDTA-plasma. The assay is intended for in vitro

diagnostic use only.

Principle of the Test

Specific DNase substrate is bound to microwells.

Any present DNase activity reacts with the specific

immobilized DNase substrate for 60 minutes at 37 °C.

Washing of the microwells removes nonreactive serum

and plasma components. Horseradish peroxidase (HRP)

conjugated anti-DNase substrate immunologically

detects the remaining DNase substrate immobilized

on the microplate. Washing of the microwells removes

unbound conjugate. An enzyme substrate in the

presence of bound conjugate hydrolyzes to form a blue

color. The addition of an acid stops the reaction forming

a yellow end-product. The intensity of this yellow color

is measured photometrically at 450 nm. The amount

of color is inversely proportional to the DNase activity.

Pathologic samples exhibit a higher activity reduction

(%AR).

604 Concise Book of Medical Laboratory Technology: Methods and Interpretations CLIA—Immunoenzymometric/Sandwich

(Streptavidin-Biotin) ELISA

Thyrotropin (TSH)

Intended Use: The quantitative determination of

thyrotropin concentration in human serum by a microplate

(CIA) chemiluminescence immunoassay.

Courtesy : Lilac Medicare.

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. The structure of human TSH is similar

to that of the pituitary and placental gonadotropins,

consisting of an 89 amino acid a subunit which is similar

or identical between these hormones and a 115 amino acid

β-subunit, which apparently confers hormonal specificity.

The production of the 2 subunits is separately regulated

with apparent excess production of the a subunit. The TSH

molecule has a linear structure consisting of the protein

core with carbohydrate side chains; the latter accounts for

16% of the molecular weight.

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

hy-pothyroidism) 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.

In this method, TSH calibrator, patient specimen

or control is first added to a streptavidin coated well.

Biotinylated monoclonal and enzyme labeled antibodies

(Abs) 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 light.

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 stearic

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

Serology/Immunology 605

BtnAb(m) = Biotinylated Monoclonal Ab (excess quantity)

AgTSH = Native Ag (variable quantity)

ENZAb(p) = Enzyme labeled polyclonal Ab (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 immobilized 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.

Materials Provided for 96-well Microplate

A. Thyrotropin calibrators — 1.0 mL/vial: Seven (7) vials

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