with antibody, while labeled and unlabeled antigens (from

the patient sample) compete for the antibody.

In a competitive ELISA, the amount of color developed

is inversely proportional to the amount of Ag-specific

patient Ig present. Careful standardization is required to

interpret the results.

Analytes tested by competitive ELISA

¾ Thyroid hormones T3, T4, FT3, FT4

¾ Steroid hormones:

FIG. 22.10: Indirect ELISA FIG. 22.12: Antibody capture

FIG. 22.11: Antigen capture

574 Concise Book of Medical Laboratory Technology: Methods and Interpretations • Androgens, testosterone, androstenedione, DHEA-S

• Estrogens: Estradiol (E2), estriol (E3)

• Progesterones: Progesterone, 17-OH progesterone.

• Cortisol

• Hepatitis Markers: HAV, HBc Antibody.

Streptavidin-Biotin ELISA

This is also a type of sandwich ELISA. In this, the solid

phase is coated with streptavidin instead of antigen or

antibody.

Avidin, found in hen egg white, is a fascinating protein

because of its high binding affinity for the vitamin Biotin

(vitamin H). In fact, avidin and a related protein, streptavidin

(found in the bacteria Streptomyces avidinii), exhibit the

highest known affinity in nature between a protein and

a ligand (Ka.1015 M/L). The rate constant for the avidin/

biotin association reaction is also fast (K=7 × 107

 M-1s-1).

Because of its extraordinary binding capacity, biotin is

used to develop modern ultrasensitive quantitative enzyme

immunoassays. The tetrameric avidin/streptavidin system

(cross-linking with biotinylated molecules) has been used

for developing third generation ultrasensitive quantitative

endocrine and other related immunoassays.

The active form of avidin is a tetramer composed of

four glycosylated subunit (mol. wt = 62,400). The avidin

tetramer has the capacity to bind up to four biotin

molecules through noncovalent linkages. Each avidin

monomer consists of 128 residues arranged in an orthogonal eight-stranded “B barrel”. Biotin binds with the

barrel towards one end only.

Avidin-biotin relation of Ag-Ab binding

Every antibody has two antigen binding sites (Fab), the

structure and shape of the particular antigen-binding site of

an antibody (also termed as paratope) and its corresponding

antigen (more specifically epitope) is such that they tightly

fit (high attraction force and minimum repulsive force

between participating molecules) to each other. Similarly,

biotin gets tightly fit into the avidin molecule because of the

following structural characteristics of avidin:

1. Strong hydrogen bonding between the monomers

of avidin makes streptavidin an extremely stable

molecule.

2. Properly placed hydrophobic and hydrophilic residues

that create a tight fit for the biotin molecule.

3. Limited access to other parts of the protein molecule

for non-specific binding.

The avidin-biotin system is well suited for use as a

bridging or sandwich system in association with antigenantibody reactions. The biotin molecule can be easily

coupled to either antigens or antibodies, and avidin can be

conjugated to enzymes and other immunological markers.

Advantages of Streptavidin

Streptavidin is used in preference to avidin because of the

following reasons:

¾ It has a neutral isoelectric point and it does not contain

carbohydrates

¾ Streptavidin is more inert in assay systems

¾ It reportedly exhibits less non-specific binding than

avidin; and hence, offer, greater specificity.

Streptavidin-biotin based IEMA systems are a better

choice for Indian laboratories because of the following:

Stability: The binding of avidin and biotin is not disturbed

by extremes of salt, pH or temperature.

Specificity and sensitivity: Avidin has a very high binding

affinity for biotin and so the system avidin-biotin is highly

specific; moreover, the rate constant for the avidin-biotin

association is also fast; and as a result, assay protocols

become rapid and simple.

Speed of the reaction: The solid phase is coated with avidin

and the capture antibody is biotinylated, this minimizes

the need for the other coating methods and facilitates

the use of antibodies with high affinities, reducing overall

assay incubation time.

Temperature stability and other problems: Non-bound

avidin is very thermostable for the folded-unfolded

transition, Tm=85 degree Celsius (pH 7–9). When biotin

is bound, the protein acquires greater thermostability

(Tm=132 degree celsius).

Thus, the avidin-biotin is more resistant to high

temperature. This greater thermostability of avidin-biotin

system overcomes/reduces the problems faced during

transportation, storage use and handlin

Serology/Immunology 575

Significance of Coating Streptavidin as Solid Phase

Streptavidin possesses greater electrostatic attraction for

the microwell/plastic tubes.

Streptavidin-biotin based IEMA systems use a

biotinylated antibody (biotin-labeled 1st antibody/

capture). This is because biotin can be attached to the

Fc portion of an antibody in relatively high proportion

without loss of immunoreactivity.

The binding ratio of avidin to biotin is 1: 4. One

molecule of streptavidin, which is a tetramer can bind

with four molecules of biotin/biotinylated 1st antibody.

In a traditional enzyme immunoassay, a limited space is

normally available for coating the capture/1st antibody

in the bottom of the microwell/plastic tube. Ideally, if one

can increase the number of capture/1st antibodies coated

on the microwell. The assay sensitivity goes up because

more number of antigen-binding sites becomes available

in case of low concentration of analytes (antigens) present

in the sample.

Streptavidin biotin based systems coat streptavidin on

the microwell/plastic tubes instead of directly coating the

capture/1st antibody. Capitalizing the tetrameric valency

of streptavidin binds with four molecules of biotinylated

capture/1st antibody thus providing an excess of binding

sites to the system, which ensures four-fold higher

sensitivity of the IEMA system (Fig. 22.14).

Analytes tested by Streptavidin-biotin ELISA

Pituitary hormones: TSH, FSH, LH, PRL

Tumor markers: PSA, CEA, AFP, CA 125, CA 15.3, CA 242,

hCG

Antibody estimation: Anti-thyroglobulin, anti-thyroid

peroxidase (TPO), anti-H. pylori.

Immunocapture ELISA

This is also a type of sandwich ELISA and is commonly

known as µ-capture/IgM-capture ELISA. It is mainly

used for the identification of IgM class of antibodies. In

this, there is an “immunocomplex” (antigen complexed

with conjugate) is used. The microwell is coated with

anti-human IgM, which is IgG in nature, which is specific

against the µ-chain of IgM class antibody (from the patient

sample). After binding the conjugate, it is added followed

by substrate (Fig. 22.15).

Analytes tested by immunocapture ELISA

Infectious serology IgM panels:

TORCH infections: Toxo, Rubella, CMV, HSV

Hepatitis markers: HAV IgM, HBc IgM, HDV IgM.

Interference Corrected ELISA

It is also one type of sandwich ELISA and is used for

infectious diseases immunoassays. This ELISA is best

FIG. 22.14: Streptavidin-biotin ELISA

FIG. 22.15: Immunocapture ELISA

576 Concise Book of Medical Laboratory Technology: Methods and Interpretations for overcoming all the false positives and false negatives,

which affect the correct result. For TORCH (Toxo, Rubella,

CMV, HSV) IgM assays, many factors can give false

positives and false negatives. This leads to wrong reporting

and wrong diagnosis. “Interference correction” is a

principle by which one can remove all these factors. A few

manufacturers have this feature. For TORCH assays, it is

always suggested to use interference corrected ELISA kits.

Based on separation steps, ELISA can be classified as:

Homogeneous ELISA

Do not require separation of free and bound label. Bound

label selectively separates or label is inactive when bound.

Latex Particle Agglutination Immunoassay (LPAIA)

A large number of latex agglutination immunoassays have

been adopted from clinical chemistry. These assays are

based on the visualization of antigen-antibody complexes by

the attachment of latex particles or gold colloids. Entities of

this type with dimensions in the nanometer or micrometer

range can be quantified by turbidimetry, nephelometry,