4. A decrease in thyrotropin values has been reported

with the administration of propranolol, methimazol,

dopamine and d-thyroxine.

5. Genetic variations or degradation of intact TSH into

subunits may affect the biding characteristics of the

antibodies and influence the final result. Such samples

normally exhibit different results among various

assay systems due to the reactivity of the antibodies

involved. The interpretation of FT4 is complicated by

a variety of drugs that can affect the binding of T4 to

the thyroid hormone carrier proteins or interfere in its

metabolism to T3.

“Not intended for newborn screening.”

Expected Ranges of Values

A study of euthyroid adult population was undertaken to

determine expected values for the TSH ELISA Microplate

Test System. The number and determined range are given

in Table 22.1. A nonparametric method (95% Percentile

Estimate) was used.

It is important to keep in mind that establishment of

a range of values which can be expected to be found by

a given method for a population of “normal”-persons is

dependent upon a multiplicity of factors: The specificity of

the method, the population tested and the precision of the

method in the hands of the analyst. For these reasons each

laboratory should depend upon the range of expected

values established by the Manufacturer only until an inhouse range can be determined by the analysts using the

method with a population indigenous to the area in which

the laboratory is located.

Immunoenzymometric/Sandwich Sequential

(Streptavidin-Biotin) ELISA

Prolacting Hormone (PRL) Sequential Method

(Courtesy: Lilac Medicare)

Intended Use: The quantitative determination or

prolactin hormone concentration in human serum by a

microplate sequential immunoenzymetric assay.

TABLE 22.1: Expected values for the TSH ELISA test system (in µIU/mL)

Number 139

Low normal range 0.39

High normal range 6.16

70% Confidence intervals for 2.5 Percentile

Low range 0.28–0.53

High range 5.60–6.82

Principle

Immunoenzymometric Sequential Assay (Type 4)

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

antibodies (enzyme 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 antiprolactin antibody.

Serology/Immunology 603

Upon mixing monoclonal biotinylated antibody, and

a serum containing the native antigen, reaction results

between the native antigen and the antibody, forming an

antibody-antigen complex. The interaction is illustrated

by the following equation;

ka

Ag(prl) + BtnAb(m) Ag(prl)- BtnAb(m)

 k-a

BtnAb(m) = Biotinylated monoclonal antibody

(excess quantity)

Ag1(prl) = Native antigen (variable quantity)

Ag1(prl)- Btn Ab(m) = Antigen–antibody complex (variable

quantity)

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:

Ag(prl)- BtnAb(m) + Streptavidincw ⇒ Immobilized

complex (IC)

Streptavidincw = Streptavidinimmobilized on well

Immobilized complex (IC) = Ag-Ab bound to the well

After a suitable incubation period, the antibodyantigen bound fraction is separated from unbound

antigen by decantation of aspiration. Another antibody

(directed at a different epitope) labeled with an enzyme

is added. Another interaction occurs to form an enzyme

labeled antibody-antigen-biotinylated-antibody complex

on the surface of the wells. Excess enzyme is washed

off via a wash step. A suitable substrate is added to

produce color measurable with the use of a microplate

spectrophotometer. The enzyme activity on the well is

directly proportional to the native antigen concentration.

By utilizing several different serum references of known

antigen concentration, a dose response curve can be

generated from which the antigen concentration of an

unknown can be ascertained.

 kb

(IC) + EnzAb(x-prl) EnzAb(x-prl)- IC

 k-b

EnzAb(x-prt) = Enzyme labeled antibody (excess quantity)

EnzAb(x-prt)- IC = Antigen-antibodies complex

kb = Rate constant of association

k-b = Rate constant of dissociation

“Mu Capture” Immunocapture ELISA

HAV-IgM

Courtesy: Lilac Medicare

Mfd: Equipar

Enzyme “Capture” Immunoassay for the qualitative

determination of IgM class antibodies to Hepatitis A virus

in human serum and plasma.

For in vitro diagnostic use only.

Principle of the Assay

Microplates are coated with a monoclonal anti-IgM

antibody that in the first step captures specifically this class

of antibodies. After washing out all the other components

of the sample, bound anti-HAV specific IgM are detected

by the addition of a preformed immunocomplex, made of

HAV antigens and a virus specific antibody, labeled with

peroxidase (HRP). The captured enzyme, acting on the

substrate/chromogen mixture, generates an optical signal

that is proportional to the presence of anti-HAV IgM in the

sample.