Lysozyme: Strongly associates with proteins having low

isoelectric points (pI). Immunoglobulins have a pI of

around 5 and lysozyme may form a bridge between the

solid–phase IgG and the signal antibody.

Endogenous hormone-binding proteins: These are

present in varying concentrations in all serum and plasma

samples and may markedly influence assay performance.

For example:

SHBG (sex hormone binding globulin) interferes in

immunoassay of testosterone and estradiol.

TBG, (thyroxine binding globulin) and NEFA (non

esterified fatty acid) interfere with the estimation of free

T4.

Abnormal forms of endogenous binding proteins: These

are present in the plasma of some patients. They are

present in familial dysalbuminemic hyperthyroxinemia

(FDH) in which albumin molecules bind to thyroxine (T4).

Individuals with FDH can be diagnosed as thyrotoxic, in

spite of being normal.

Heterophilic antibodies: They may arise as a consequence

of intimate contact, either intentional or unintentional,

with animals. The most familiar effect of heterophilic

antibodies is observed in two-site sandwich reagent-excess

assays, in which a ‘bridge’ is formed between the two

antibodies forming the sandwich. Assays that are affected

by heterophilic antibodies include for CEA, CA 125, hCG,

TSH, T3, T4, free T4, Prolactin, HBsAg and Digoxin.

Serology/Immunology 567

Mechanical Interference

Fibrinogen from incompletely clotted samples interferes

with sampling procedures on automated immunoassay

instruments and may produce spurious results.

Paraproteinemia causes interferences in many assays by

increasing the viscosity of the sample. They may also nonspecifically bind either analytes or reagents that may affect

the result.

Nonspecific Interference

Non-specific interference may arise from excessive

concentrations of other constituents of plasma. Free fatty

acids affect some assays for free T4 by displacement of T4

from endogenous binding proteins.

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, α Feto protein

(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.

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.

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 cross reactivity of the antibody.

Some 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 5α-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.

What is the difference between an antigen and

immunogen?

The word “antigen” is conventionally used to describe

as antibody generators, i.e. that can generate antibody

against itself. Also, anything that is foreign to the body is

also known as antigen. This definiton of foreignness has

become irrelevant with autoantigens. Antigen can be

defined as those that bind with the antibody. They need

not be foreign in nature. Some antigens also require a

carrier/helper to bind with the antibody.

Immunogens, as the name goes are those that can

elicit an immune response. It may be either T-cell or

B-cell response. All immunogens can be antigens. But all

antigens need not be immunogens.

1. What are the different types of epitopes?

There are two different types — sequential and conformational. Sequential epitopes are made of linear region

of peptides. Conformational epitopes are formed when

the protein chain is folded. Disulfide bonds are important

for maintaining the conformational integrity.

2. What is Hook effect?

Sometimes, the value of an analyte obtained by laboratory

testing will be very low in spite of suspicion that it will

be high. This false low values derived in spite of it being

very high is known as Hook effect. This is due to very

high concentration in the blood. The levels are so high

that they actually mask the binding sites available in the

immunoassay system, leading to very low values. (Imagine

one hundred persons fighting to sit in 5 chairs. Even

though there were hundred the actual number of people

who sat were only 5). This is observed in parameters like

PSA,hCG, CEA, etc. The solution for this is to dilute the

sample and run the assay.

568 Concise Book of Medical Laboratory Technology: Methods and Interpretations 3. What is the difference between chemiluminescence

and fluorescence? Which is better?

Fluorescence is a phenomenon where molecule absorsbs

light in one wavelength and emits in another wavelength.

In this, there is a source of excitation. Chemiluminescence

is the production of light by a chemical reaction. The main

difference is that there is no radiation is absorbed. The

energy required to emit light comes from the energetics

of chemical reaction. Definitely chemiluminescence is a

better technology for use in immunoassays.

4. What is meant by apoptosis?

In an organism such as a human, the number of new cells

created must be balanced by an equal number of cells

dying. Sometimes cell death occurs as a result of injury;

most often, however, it is a planned, natural process called

apoptosis. Apoptosis is sometimes called cellular suicide

because it is a cell’s own gene products that carry out its

death. While it kills a cell, apoptosis is beneficial to the host

as a whole - it is important, for example, in development,

in the immune response, etc.

5. How does secondary response differ from primary

response?

It differs mainly in three ways:

¾ It involves an amplified population of memory cells

¾ The response is more rapid

¾ Higher levels of antibodies are formed than primary

response.

6. What are primary and secondary lymphoid organs?

Primary lymphoid organs are the bone marrow and

thymus. These organs function as sites for B-cell and T-cell

maturation, respectively. Secondary lymphoid organs

are spleen, lymph nodes and various mucous associated

lymphoid tissues. All these trap antigens and provide sites

lymphocytes can interact with antigen.

7. What is the difference between active and passive

immunity?

Active immunity Passive immunity

Produced actively by the host Received passively by the host

Induced by infection Conferred by introduction of

readymade antibodies

Durable and effective protection Protection transient and less

effective

Immunity effective only Immunity effective immediately

after a lag time

Immunological memory present No immunological memory

Negative phase may occur No negative phase

Not applicable in immunodeficient host

Applicable in immunodeficient

hosts

8. What is the difference between analytical and

functional sensitivity?