Hook Effect

The “Hook Effect” is characterized by the production

of artefactually low results from samples that have

extraordinarily high concentrations of antigen (analyte), far

exceeding the concentration of the upper standard in the

assay concerned.

The hook effect is most commonly found in single-step

immunometric assays, a popular format, chosen for its

specificity and speed, particularly with high-throughput

immunoassay analyzers. The assays most affected are

those that have analyte concentration that may range

over several orders of magnitude. For example, alpha

fetoprotein (AFP), CA-125, hCG, PSA, TSH, prolactin and

ferritin are most affected by Hook effect.

Reduction of Hook Effect

The incidence of Hook effect can be reduced (but not

eliminated) by careful assay design—incorporating a wash

step prior to addition of the second antibody, thereby

avoiding simultaneous saturation of both antibodies.

584 Concise Book of Medical Laboratory Technology: Methods and Interpretations Despite attempts to eliminate or reduce the Hook

effect by careful assay design, the only reliable method of

routinely eliminating the effect is to test the samples that

are likely to be affected by Hook effect in undiluted and

also at a suitable dilution. Such samples should be diluted

using either the assay diluent or serum from a normal

subject until a stable quantitative response is achieved.

Edge Effect

Sometimes with ELISA performed in a microwell plate

unexpectedly higher (or lower) optical densities (OD)

are measured in the peripheral wells than in the central

wells. This phenomenon is called “edge effect”. The

most probable causes of this effect are illumination or

temperature differences between the peripheral and the

central wells.

Light may cause edge effect if the substrate is

photosensitive (i.e. converted by light exposure) like the

H2O2/OPD substrate in the peroxidase system. Thus, if

strong light is coming from one side (e.g. sunlight from

a window) during the substrate reaction, the peripheral

wells closest to the light source may give elevated OD

values. Temperature difference, however, is the most

common cause of edge effect.

Incubation at 37°C instead of room temperature is often

used for shortening incubation time, which is not correct.

Also, a common mistake is to use reactant liquids straight

from a refrigerator and then incubate in a 37°C incubator

(or at room temperature). Temperature changes of these

magnitudes may, especially with short incubation times,

destroy the assay homogeneity in microwell plates. The

peripheral wells will normally be heated up first because of

their position closest to the lower edge of the plate, which

is in direct contact with the warm incubator shelf, which

may result in higher OD values in these wells, other things

being equal. The edge effect may be more pronounced if

plates are stacked during incubation, especially in plates

in the middle of the stack because their central wells are

shielded from the warmer surroundings by the plates

above and beneath.

To avoid the above-mentioned problems, the following

precautions should be taken:

¾ Incubations should take place in subdued light or in

the dark (if protocol requires)

¾ Reactant liquids (and plates) should be adjusted to the

temperature intended for incubation

¾ Plates should be sealed with adhesive tape or placed

in a 100% relative humidity environment during

incubation.

Assay Specificity

It is one of the most important requirements of

immunoassays. Interference occurs in all situations in

which the antibody is not absolutely specific for the analyte.

Consequently, assessment of specificity is a vital step in the

optimization of every new immunoassay. Poor specificity

results in interference from compounds of similar molecular

structure or which carry similar immunoreactive epitopes.

In determining the overall specificity of an assay, a major

factor is the crossreactivity of the antibody.

Some of the major specificity problem areas are related

to measurement of steroids and structurally related

compounds. All commonly used testosterone assays, cross

react in varying degrees with 5 α-dihydrotestosterone, and

all cortisol assays cross react with prednisolone.

Assessment of the specificity of immunometric assays

is complex and quite different from that used for singlesite assays. In most assays, two different antibodies are

employed, each having unique specificity for a different

epitope on the antigen. It is usual practice to employ at

least one monoclonal antibody, which can be selected by

epitope mapping to react only with predetermined sites on

the antigen molecule. Use of two monoclonal antibodies

can introduce extreme specificity.

Assay Sensitivity

The ability of a kit to detect very low concentrations of an

analyte (in quantitative ELISA) is mainly understood by

the sensitivity of the kit. Many manufacturers mention the

sensitivity and specificity after the result interpretation.

This is overlooked commonly. One should observe this

carefully. Higher sensitivity is a desirable property in

any kit. Some doubts have been expressed regarding the

value of ultrasensitive assays, which detect very minute

amounts of analyte, which may be below the clinically or

diagnostically significant values.

Most diagnostic kits are not exhausted overnight.

Repeated usage and storage exposes the kit to multiple

thermal shocks. This affects the performance of the kit

over a period of time due to lowering of sensitivity. This

shift in sensitivity affects the ultrasensitive kits lesser than

those with less sensitivity.

A good example of ultrasensitive kit is “Third Generation

TSH kits” which are very useful in the diagnosis of

hypothyroidism.

As compared to low sensitive kits, ultrasensitive kits are

more robust, more accurate that improve the reliability

of results and provide confidence to the clinicians on the

laboratory results.

Serology/Immunology 585

CHEMILUMINESCENCE: THE TECHNOLOGY

Introduction

“Chemiluminescence” is defined as the production of

electromagnetic (ultraviolet, visible or near-infrared)

radiation as a result of a chemical reaction. One of the

reaction products is in an excited state and emits light on

returning to its ground state.

The generation of signal and its estimation varies from

technology to technology. In RIA (radioimmunoassay)

the radioactive signal is measured in gamma counter. In

ELISA, the enzyme and substrate react to produce color,

which is measured using an ELISA reader. Fluorescence

immunoassays involve a similar principle where enzyme

and substrate react to produce a fluorophor, which is

measured fluorometrically. In case of chemiluminescence

immunoassays, the light is produced which is measured.

Measurement of light from a chemical reaction is

highly useful because the concentration of unknown

can be inferred from the rate at which light is emitted.

The rate of light output is directly related to the amount

of light emitted. This type of luminescence is frequently

compared with fluorescence, which also involves emission

of light as a result of relaxation of excited states. Since,

chemiluminescence does not involve initial absorption

of light, measurement of chemiluminescence emission

are made against a lower background noise that is not

possible with conventional fluorescence, thus potentially

allowing greater sensitivities of detection in chemiluminescent technology. This lack of inherent background

and the ability to easily measure very low and very high

light intensities with simple instrumentation provide a

large potential dynamic range of measurement. Linear

measurement over a dynamic range of 106

 or 107

 using

purified compounds and standards has become possible

with developments in the technology.

Light, as we see it, consists of billions of tiny packets

of energy called photons, which are measured in the

detection process. There are different factors that affect

the emission and measurement of light.

¾ The efficiency of light emission from a chemiluminescent molecule is expressed as the chemiluminescence

quantum yield, ÖCL, which describes the number of

moles of photons emitted per mole of reactant

¾ The signal

¾ The quantity of signal required to produce the emission

¾ The duration of emission

¾ Instrumentation employed for the quantification of

emission.

Components of Chemiluminescent System

The Signal

The signal (or substrate) used for generation of light

should have optimum stability. There are many signal

reagent available—luminol, 1,2 Dioxetanes, Acridinium

ester, ruthenium salts, etc. Luminol is preferred of all these

because of its stability and its advantage of being enhanced

by iodophenol and phenothiazine.