Immuno Assay i

ENZYME IMMUNO ASSAY

S Rubesh Kumar M.Pharm, (Ph.D),

Department of Pharmaceutical Analysis

J N T U A – O T R I

ANANTAPUR - 515001

Enzyme Immuno Assay 2012

S RUBESH KUMAR Dept. Pharma. Analysis JNTUA- OTRI Page 2

ENZYME IMMUNO ASSAY

EIA: Assay systems involving use of antigens, haptens, or antibodies labeled with

an enzyme have recently been applied to the measurement of substances in

biological fluids. These assay systems have been given various names: enzyme-,

enzymic-, enzymatic-, and enzymoimmunoassay (EIA),’ enzyme-linked immunoassay,

enzyme-labeled immunoassay, enzyme-coupled immunoassay, immunoenzymatic

assay, and enzymelinked immunosorbent assay (ELISA). In addition, there is a type

of EIA, Homogeneous EIA, in which, unlike the other types, separation of free and

bound labeled material is not required. Homogeneous EIA has also been called the

enzyme multiplied immunoassay technique (EMIT) and enzyme-inhibition

immunoassay.

Immunoassays combine the principles of chemistry and immunology enabling

scientific tests, e.g. enzyme immunoassays and immunoblotting for a specific and

sensitive detection of the analytes of interest. The basic principle of these assays

is the specificity of the antibody-antigen reaction. Though being very specific and

sensitive immunoassays are easy to perform which has contributed to the

widespread use and tremendous success. In immune blotting like the Western Blot

of electrophoretically separated proteins a single one can be identified by its

antibody.

RIAs (Radioimmunoassay) and enzyme immunoassays like ELISA (Enzyme-linked

immunosorbent assay), LIA (luminescent immunoassay), and FIA (fluorescent

immunoassay) are widely used in research, drug discovery and diagnostics for



highly specific and cost efficient detection of analytes not detectable with other

techniques.

The antibodies – either used as primary or secondary ones – are labelled with

radioisotopes (e.g. 125I), fluorescent dyes (e.g. FITC) or enzymes (e.g. HRP or AP)

which catalyse fluorogenic or luminogenic reactions.

Immunoassays involve tests using antibodies as reagents.

Enzyme immunoassays (EIA) make use of enzymes attached to one of the

reactants in an immunoassay to allow quantification through the development of

color after addition of a suitable substrate/chromogen.

As indicated above ELISAs involve the step-wise addition and reaction of reagents

to a solid phase bound substance, through incubation, and separation of bound and

free reagents using washing steps.

An enzymic reaction is utilized to yield color and is used to quantify the reaction,

through use of an enzyme labeled reactant.

A very brief definition of terms used in ELISA is needed.

Antibodies (also known as immunoglobulins abbreviated Ig) are gamma

globulin proteins that are found in blood and are used by the immune system

to identify and neutralize foreign objects, such as bacteria and viruses.

i) The Antibody: An immunoglobulin, a specialized immune protein, produced

because of the introduction of an antigen into the body, and which

possesses the remarkable ability to combine with the very antigen that

triggered its production (specific affinity)



ii) The antibody recognises and bind to the antigenic determinant region of

the antigen

Antigens A substance that when introduced into the body

stimulates the production of an antibody. An antigen is any substance that

causes the immune system to produce antibodies against it. The substance

may be from the environment or formed within the body. The immune system

will kill or neutralize any antigen that is recognized as a foreign and

potentially harmful invader. The term originally came from antibody

generator ]and was a molecule that binds specifically to an antibody, but the

term now also refers to any molecule or molecular fragment that can be

bound by a major histocompatibility complex (MHC) and presented to a T-

cell receptor. "Self" antigens are usually tolerated by the immune system;

whereas "Non-self" antigens can be identified as invaders and can be

attacked by the immune system.

Immunoassay A laboratory technique that makes use of the binding between

an antigen and its homologous antibody in order to identify and quantify the

specific antigen or antibody in a sample

Analyte The sample being analyzed and in immunoasssays the analyte is

either Antibody or Antigen







Types of Enzyme Immuno Assay

1. Competitive EIA for antigen: Labeled antigen competes with unlabeled antigen

for binding to a limited quantity of antibody. The antibody-bound antigen is

separated from the free antigen by the use of solid phase antibody or a second

antibody with specificity for the first. The enzyme activity in either the bound or

free fraction is determined and related to the concentration of the unlabeled

antigen. This procedure is analogous to the classical RIA method. In the sequential

saturation variant of the competitive assay, the addition of the labeled antigen is

delayed until the binding between the antibody and the unlabeled antigen is

complete. This method is analogous to the sequential RIA.

2. Immunoenzymometric: assay for antigen. Antigen reacts with excess labeled

antibody and, after incubation; excess solid-phase antigen is added. The solid-

phase antigen reacts with the free labeled antibody remaining and, after

separation of the solid-phase, the enzyme activity associated with soluble antigen

is measured and related to the concentration of antigen. This assay is analogous to

the immunoradiometric assay.

3. Sandwich EJA for antigen: This procedure requires the antigen to have at

least two binding sites. Antigen reacts with excess solid-phase antibody, and after

incubation followed by washing, the bound antigen is treated with excess labeled

antibody. After further washing the bound label is assayed, and this provides a

direct measure of the amount of antigen present. This assay is analogous to the

“two-site” immunoradiometric procedure. A variation of this method

(“double sandwich” EIA) involves a third antibody. This antibody carries the label

and reacts with unlabeled second antibody already bound to the antigen. As before,

the amount of antigen is found by measuring the amount of bound label.



4. EIA for antibody. Antibody binds to excess solid-phase antigen and, after

incubation followed by washing, labeled second antibody with specificity for the

first antibody is added. The bound label is assayed after further washing and it

provides a direct measurement of the amount of specific antibody present.. This

assay is analogous to the radioallergosorbent technique. The system may also be

used to assay antigens.

5. Homogeneous EIA for haptens. This type of EIA does not require separation

of free and bound label, because the assay depends on inhibition or activation of

the enzyme label by antibody binding. In the first of these assays the haptenic

group, a morphine derivative, was attached to lysozyme. When antibodies to

morphine were bound to this modified lysozyme the enzyme was inhibited, possibly

because the large substrate, the cell wall peptidoglycan of Micrococcus luteus, was

unable to gain access to the catalytic site. However, when free morphine was

present it competed with the enzyme-bound morphine for the antibody binding

sites. Thus fewer antibodies were bound to the enzyme and consequently inhibition

of its catalytic activity was decreased. Work has also been done on an assay in

which a morphine derivative was attached to malate dehydrogenase. In this case

the inhibition of the enzyme’s action on binding to antibody appears to be caused

by a conformational change induced in the enzyme rather than by steric hindrance.

In a similar system, attachment of a thyroxine derivative to malate dehydrogenase

caused inhibition of the enzyme but this inhibition was reversed when antibodies

against thyroxine were bound to the modified enzyme.3 In this assay the presence

of free thyroxine prevents binding of the antibody to the enzyme and thus the

enzymic activity decreases.



6. Other EIAs. Other types of EIA are possible and two additional EIAs for

antibodies have been briefly described . In the first (Type 6a), labeled and

unlabeled antibody compete for solid-phase antigen. In the second assay (Type 6b)

labeled antigen reacts with antibody; solid-phase antibody is then added and the

remaining labeled antigen reacts with it. The amount of antibody is determined in

both cases by measuring the enzyme activity in the free or bound fractions after

centrifugation.

ELISA technique

Is a biochemical technique used mainly in immunology to detect the presence of an

antibody or an antigen in a sample.

The technique is divided into

Direct ELISA Indirect ELISA

SandwichELISA Competitive ELISA



Direct method



Coating (Steps 2 and 3)

Antigen is diluted in a buffer and added to the solid phase, commonly high pH (9.6)

carbonate/bicarbonate buffer or neutral phosphate buffered saline. The key is

that the buffer contains no other proteins that might compete with the target

antigen for attachment to the plastic solid phase. Antigens are mainly protein in

nature, and attach passively to the plastic during a period of incubation. The

temperature and time of the incubation is not so critical, but standardization of

conditions is vital and the use of incubators at 370C is favoured, (since they are

widely available in laboratories).

Washing (Steps 4 and 7)

Washing is necessary to separate free from bound reagents. In Step 4, any

unadsorbed antigen is removed, in Step 7 it is necessary to remove free antibody

enzyme from that bound specifically to the antigen. Washing can be simply flooding

and emptying wells, using a neutral buffered solution (e.g., PBS).

Conjugate addition (Step 6)

Antibodies conjugated with an enzyme are added directed specifically against

antigenic sites on the solid-phase bound reagent. The conjugated antibodies are

diluted in a buffer containing some substances which inhibits passive adsorption of

protein, but which still allows immunological binding. Such substances are either

other proteins, which are added at high concentration to 'compete' for the solid

phase sites with the antibody protein; or detergents at low concentration and are



termed "blocking " agents, and the buffers they help formulate are called "blocking

buffers". On incubation, antibodies bind to the antigen.

A simple washing step is then used to get rid of unbound antibodies (Step 7). The

complex then on plate is antigen combined with specific enzyme labeled antibody.

Substrate/Chromphore Addition (Step 9)

This involves addition of a suitable substrate or substrate/chromogen combination

for the particular enzyme attached to the antibodies. The objective is to allow

development of a color reaction through enzymic catalysis. The reaction is allowed

to progress for a defined period after which the reaction is stopped by the

alteration in pH of the system, or addition of an inhibiting reactant. Finally, the

color is quantified by the use of a spectrophotometer reading at the appropriate

wavelength for the color produced.



Indirect ELISA



Steps 1-5 are similar to the Direct system where wells are coated with antigen.

Step 6 involves the addition of unlabeled antibodies, which are diluted in a buffer to

prevent non-specific attachment of proteins in antiserum to solid phase, (blocking buffer).

This is followed by incubation and washing away excess (unbound) antibodies, to achieve

specific binding

Step 7 involves washing away unbound antibodies leaving the complex in Step 8, consisting

of antigen and antibody, attached to solid phase.

Step 9 is the addition of the conjugate (enzyme-labeled), anti-species antibodies, diluted

in blocking buffer, again followed by incubation and washing to achieve binding of

conjugate. After washing (Step 10) the complex on the solid phase has increased with the

enzyme conjugate detecting the specific species of antibody detecting the antigen (Step

11)

Step 12 involves the addition of the substrate/chromophore and allowing the colour to

develop (Step 13). Stopping the color is Step 14 and reading is Step 15.

The Indirect system is similar to the Direct system, in that antigen is directly attached to

the solid phase and 'targeted' by added antibodies, (detecting antibodies). However, these

added antibodies are not labeled with enzyme but are themselves 'targeted' by antibodies

linked to enzyme. Such antibodies are produced against the immunoglobulins of the species

in which the detecting antibodies are produced and termed "anti-species" conjugates.

Thus, if the detecting antibodies are produced in rabbits, the enzyme labelled antibodies

would have to be anti-rabbit immunoglobulin in nature. This allows great flexibility in use of

anti-species conjugates in that different specificities of conjugate can be used to detect

particular immunoglobulins binding in the assay and there are literally thousands of

commercially available conjugates available. For example, the anti-species conjugate could

be anti-IgM, anti IgG1, IgG2 , etc.

The Indirect system offers the advantage that any number of antisera can be examined

for binding to a given antigen using a single anti-species conjugate. Such systems have



been heavily exploited in diagnostic applications particularly when examining (screening)

large numbers of samples.

The protein antigen to be tested for is added to each well of ELISA plate,

where it is given time to adhere to the plastic through charge interactions

A solution of non-reacting protein is added to block any plastic surface in

the well that remains uncoated by the protein antigen

Then the serum is added, which contains a mixture of the serum antibodies,

of unknown concentration, some of which may bind specifically to the test

antigen that is coating the well.

Afterwards, a secondary antibody is added, which will bind to the antibody

bound to the test antigen in the well. This secondary antibody often has an

enzyme attached to it

A substrate for this enzyme is then added. Often, this substrate changes

colour upon reaction with the enzyme. The colour change shows that

secondary antibody has bound to primary antibody, which strongly implies

that the donor has had an immune reaction to the test antigen.

The higher the concentration of the primary antibody that was present in

the serum, the stronger the colour change. Often a spectrometer is used to

give quantitative values for colour strength





Sandwich ELISA

The term sandwich comes from the fact that antigens are sandwiched between

detecting antibody both on the solid phase and acting as an enzyme labeled

conjugate. The antibody attaching to solid phase is sometimes called-Capture

antibody, the detecting antibody-Detector.

The sandwich systems vary depending on the source of the antibodies used. To

simplify terminology, the following definitions will be used.

Sandwich Direct

A. The detecting antibody is labelled (conjugate)

B. The capture and detecting antibodies can be from the same sample

C. The detecting antibody can be from different species

Sandwich Indirect

A. The detecting antibody is NOT labelled

B. The detecting antibody is not prepared in same species as capture antibody

C. The detecting antibody is detected using an anti-species conjugate

There are complications using the same specificities outlined after the overviews.

Sandwich Direct Sandwich Indirect







This involves the passive attachment of antibodies to the solid phase. These

antibodies then bind antigen(s). The antigen(s) are diluted in a blocking buffer to

avoid non-specific attachment to the solid phase. Here the components of the

blocking buffer should not contain any antigens which might bind to the capture

antibodies.

After incubation and washing an antibody-antigen complex is attached to the solid

phase.

The captured antigen (sometimes referred to as "trapped"), is then detected by

addition and incubation of enzyme labelled specific antibodies in blocking buffer

Thus, this is a direct conjugate binding with the antigenic targets on the captured

antigen.

This second antibody can be the same as that used for capture, or be different in

terms of specific animal source or species in which it was produced.

After incubation and washing,the bound enzyme is developed by the addition of

substrate/chromogen and then stopped and read using a spectrophotometer.

• Since a single enzyme-conjugated antibody is used, the system is limited to the

specificities and properties inherent in that particular antibody 'set'. This limits

the versatility of the test, e.g., each antibody preparation used must be labelled

(for different antigens), in the same way as the Direct ELISA was limited to single

antibody preparations.



• The system also is limited in that antigens have to have at least two antigenic

sites, since both the capture and the detecting antibodies need to bind. This can

limit the assay to relatively large antigenic complexes.

• The capture antibody (on the solid phase) and the detecting antibody, can be

against different epitopes on an antigen complex. This can be helpful in orienting

the antigenic molecules so that there is an increased chance that the detecting

antibodies will bind. It can also be an advantage when investigating small

differences between antigenic preparations by use of different detecting

antibodies and a common capture antibody.

• More versatile and hence appropriate systems are dealt with below (Sandwich

ELISA-Indirect), for this purpose. The use of exactly the same antibodies for

capture and detection (e.g., use of monoclonal antibodies) can lead to problems

whereby there is a severe limitation of available binding sites for the detector.







This has similar steps to the Sandwich Direct for coating with capture antibody

and addition of antigens. Thus, antibodies are passively attached to the solid phase

and antigen(s) are captured.

However, the detection of the antigen involves the addition of unlabelled

antibodies.

After incubation and washing, the detecting antibodies are themselves 'detected'

by addition and incubation with an anti-species enzyme conjugate.

The bound conjugate is then processed as described in the other systems.

The advantage of this assay is that any number of samplescontaining antibodies can

be added to the captured Ag, provided that the species in which it was produced is

not the same as the capture Ab. More specifically, that the enzyme conjugated

anti-species antibody, does not react with the antibodies used to capture the

antigen.

It is possible to use the same species of antibody, if immunochemical techniques

are used to select and produce particular forms of antibodies and with attention to

the specificity of the enzyme conjugate used.

Thus, as an example, the capture antibody could be processed to a bivalent

molecule without the Fc portion (a so called FAb2 fraction). The detecting

antibodies could be untreated. The enzyme conjugate could then be an anti-species

anti-Fc portion of the immunoglobulin molecule. Thus, the conjugate would only

react with antibodies containing Fc (and not therefore the capture molecules).



The need to devise such assays depends on the reagents available. It may be that a

monoclonal antibody is available that confers a desired specificity as compared to

polyclonal sera or that one wishes to screen a large number of monoclonal

antibodies against an antigen which must be captured (it may be at low

concentration or in a mixture of other antigens). In this case use of polyclonal sera

is unsuccessful, therefore the preparation of FAb2 fragments for the capture

antibody is worthwhile, and in facts relatively easy to use kits are available for this

purpose. The use of a commercially available anti-mouse Fc completes the

requirements.



Competitive ELISA

The labelled antigen competes for primary antibody binding sites with the

sample antigen (unlabeled). The more antigen in the sample, the less labelled

antigen is retained in the well and the weaker the signal).



Immunometric Assays

Numerous schemes have been developed that use antibodies to capture and

measure analytes. The details of some can be complex, but most are designed

around two basic strategies. Perhaps the easiest to understand is the

immunometric assay (Figure 3):

• Antibodies immobilized onto a plastic surface (most often a 96-well microtiter

plate) are used to capture the target antigen present in the sample.

• A second antibody linked to an enzyme (the conjugate) is then added. It binds to

a different location on the target antigen.

• Plate wells are washed to remove unbound components.

• Substrate is added. Bound enzyme present reacts with the substrate, yielding

color.

• The enzymatic reaction is stopped in order to establish a consistent time period

for all wells. After stopping, the color is measured.



Immunometric assays are also commonly used to measure antibodies as the analyte

(Figure 5). In this case a capture antigen is fixed to the plastic surface, and the target

antibody binds to it. An antibody-enzyme conjugate that binds to the target antibody is

then added, the plate is washed, and the conjugate reacts with the substrate to produce

the color.

Because the analyte in an immunometric assay is surrounded on two sides, the

procedure is often referred to as a sandwich assay. The acronym ELISA (Enzyme

Linked Immuno Sorbent Assay) is also often associated with sandwich assays, but

some authorities prefer to use the term in a more general sense for all sorts of



microtiter plate immunoassays that involve enzymatic labels. The acronym IEMA is

used more formally to refer to the Immunoenzymometric assay.

Advantages of EIA

Specific and sensitive assays of wide applicability

Equipment required is relatively cheap and is widely

available

Reagents are relatively cheap and have a long shelf-life

Manipulations are simple

Assays may be very rapid

A separation step may not be required

The variety of labels available may allow multiple,

simultaneous assays to be performed

Potential for automation

No radiation hazards





An example of an ELISA experiment

The sample is added to plate in duplicate or triplicate and then the mean

result is calculated

The quality control sample which is provided with the kit is treated as the

test samples

After reading the results the standard curve is drawn were the

concentration is blotted on the X-axis and the absorbance on the Y-axis

The standards concentrations is specified on the x-axis and the reading of

each standard is specified on the y-axis and the standard curve is drawn



Applications for Immunoassays:

Tumor Markers, e.g. AFP, CEA, hCG, PSA …

Cardiac Markers, e.g. CK-MB, CRP, Digoxin, Myoglobin …

Cell based Assays, e.g. cell cytotoxicity …

Allergy, e.g. histamines, egg, milk, allmonds …

Growth Deficiency, e.g. hGH

Enzyme acivity

Hormone and Steroid Screening, e.g. T4, fT3, TSH …

Drug Abuse Screening, e.g. amphetamines, cocaine, LSD …

Immunological Screening

Infectious Diseases, e.g. Chlamydia, CMV, Hepatitis, Rubella …

Veterinary, e.g. bacterial infection, fertility, drugs, BSE …

Food and Beverages, e.g. pathogens, toxins…

Water Analysis, e.g. bacterial contamination, toxins, heavy metalls …

Agriculture, e.g. endotoxins, pesticides …

Environment, e.g. industrial chemicals, pesticides, surfactants …

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