Presentation onImmunoassay Systems

T.Manoj Kumar

INTRODUCTION:-

• An immunoassay is a test that uses antibody and antigen

complexes as a means of generating a measurable result.

• An antibody:antigen complex is also known as an immuno-

complex.

• “Immuno” refers to an immune response that causes the

body to generate antibodies, and “assay” refers to a test.

Thus, an immunoassay is a test that utilizes

immunocomplexing when antibodies and antigens are

brought together.

TYPES:-

• Competative immunoassays.

• Non-competative immunoassays.

• Homogenous immunoassays.

• Heterogenous immunoassays.

Competitive Format

• In competitive formats, unlabelled analyte (usually antigen) in

the test sample is measured by its ability to compete with labeled

antigen in the immunoassay.

• The unlabeled antigen blocks the ability of the labeled antigen to

bind because that binding site on the antibody is already

occupied.

• Thus, in a competitive immunoassay, less label measured in the assay

means more of the unlabeled (test sample) antigen is present. The

amount of antigen in the test sample is inversely related to the amount

of label measured in the competitive format.

One step competitive format

• In the one step competitive format (see Figure 1-8), both the labeled

antigen reagent (Ag*) and the unlabeled specimen (or test sample

analyte) compete for a limited amount of antibody.

Two step competitive format

• In the two step competitive format, the antibody concentration of

the reaction solution is present in excess in comparison to the

concentration of antigen.

• Antibody reagent is first incubated with specimen containing

antigens of interest; then in the second step, labeled antigen is

added.

• Remember that in the competitive format, less bound labeled

antigen indicates more antigen present in the test sample. assay

formats provide several fold improved assay sensitivity compared

to one step assay formats.

Noncompetitive (Sandwich) Method

• Noncompetitive assay formats generally provide the highest level of

assay sensitivity and specificity and are applied to the measurement of

critical analytes such as cardiac and hepatitis markers. This format is

referred to as a “sandwich” assay because analyte is bound

(sandwiched) between two highly specific antibody reagents.

• Noncompetitive assay formats can also utilize either one step or two

step methods, as with the competitive assay.

• The two step assay format employs wash steps in which the sandwich

binding complex is isolated and washed to remove excess unbound

labeled reagent and any other interfering substances.

• The two step noncompetitive format usually offers the highest

specificity and sensitivity of all the assay formats discussed here.

Homogeneous and Heterogeneous Immunoassay Methods

• Immunoassay methods that require separation of bound Ab-Ag*

complex are referred to as heterogeneous immunoassays. Those that do

not require separation are referred to as homogeneous immunoassays.

• Homogeneous methods have been generally applied to the

measurement of small analytes such as abused and therapeutic drugs.

Since homogeneous methods do not require the separation of the

bound Ab-Ag* from the free Ag*, they are generally much easier and

faster to perform.

Heterogeneous

Technique principle

Non specific adsorbents Low molecular weight ligands are adsorbed by particles such as charcoal and removed by centrifugation

Specific adorbents Antibodies to the ligand or to the ligand binding antibody are immobilized on the surface of a Solid matrix such as glass fibres,latex micro particles. The immobilized antibody ligand complex are separated from unbound ligand by washing,filtration,diffusion

chromatography The protein bound ligand moves at a rate through the chromatographic medium different from that of free ligand

Precipitation by ammonium sulfate The antibody bound ligand is ppt. by ammonium sulfate

• It can measure both small and large molecules.

• Heterogeneous immunoassay such as the popular EMIT assay

on the other hand measures only small molecules such as drugs.

HETEROGENOUS IMMUNOASSAYS

Immunometric assay with radiolabelled

Enzyme immunoassay with enzyme labelled

ELISA

Flouroscence immunoassay

Partical concentration flouroscence (PCFIA)

Time resolved flouroimmuno assay

EMIT-homogeneous

As [Test antigen] is

increased it occupies more

of the antibody molecules,

so fewer of the enzyme

labels are blocked.

Substrate ---> product

response increases

ELISA-heterogeneous

• The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be

used both qualitatively and quantitatively to measure antigen-

antibody binding. Depending on what variation you use, it will

detect antigen (hormones, enzymes, microbial antigens, illicit

drugs) or antibody (anti-HIV in the screening test for HIV

infection) in body fluids or tissue culture supernatents.

• Purified antigen (to detect or quantify antibody).

• Purified antibody (detect or quantify antigen).

• Standard solutions (positive and negative controls).

• Sample to be tested.

• Microtiter dishes: plastic trays with small wells in which the

assay is done.

• Wash fluid (buffer).

• Enzyme-labeled antibody and enzyme substrate.

• ELISA reader (spectrophotometer) for quantitative

measurements.

To detect antibody

• Coat the microtiter plate with purified antigen by letting an

antigen solution sit in the wells for 30-60 minutes. Wash

away unbound antigen with buffer and cover any sites that

might nonspecifically bind antibody with unrelated protein

(such as solution of powdered milk), again washing away

unbound protein.

• Add serum sample to be tested for specific antibody to plate

and allow specific antibody to bind to the antigen. Wash off

unbound antibody.

• Add anti-Ig that will bind to Fc region of specific antibody (for

example, anti-human gamma chain that will bind human IgG).

The Fc region of the anti-Ig is covalently linked with enzyme.

Wash off unbound antibody-enzyme complex.

• Add chromogenic substrate: colorless substrate that the

enzyme will convert to a colored product. Incubate until color

develops; measure color in a spectrophotometer.

• The more color that is detected, the more specific antibody is

present in the unknown sample.

• Negative controls include

• omit the antigen

• omit the test antiserum or substitute with an antibody that

will not bind the antigen.

• Positive control substitutes known positive serum for unknown

serum.

• Coat the microtiter plate with purified antibody to the

antigen. Wash away unbound antibody and cover any sites

that might nonspecifically bind with unrelated protein.

• Add sample to be tested for antigen to plate and allow

antigen to bind antibody. Wash off unbound antigen.

• Add enzyme-labeled specific antibody to a different epitope

of the antigen to make a "sandwich"; wash away unbound

antibody.

TO DETECT ANTIGEN

• Add chromogenic substrate for enzyme that will be

converted to a colored product.

• Negative control omits unknown antigen; positive control

uses known antigen.

How to interpret the results

• The amount of colored product is proportional to the amount

of enzyme-linked antibody that binds, which is directly

related to the amount of antibody that was present to bind

antigen or antigen that was present to bind antibody.

• If known amounts of antigen or antibody are added, a

standard curve can be constructed which will allow the

amount of unknown antigen or antibody to be determined.

Fluorescence Immunoassay:

• In fluorescence, a photon of an appropriate energy excites the

molecule from its ground state to a higher electronic state.

• When the molecule returns to the ground state, energy is

released as light emitted at a longer wavelength.

• The difference between the excitation wavelength and the

emission wavelength is the stocks shift.

• A large stokes shift in nanometer means that there is a large

difference between the excitation and emission wavelengths.

• For example, fluorescein, a common flurophore has small

stocks shift at 30 min. It has a maximal absorption at 490nm

and emission at 520 nm.

• In contrast a rare earth chelate such as europium has a large

stocks shift of 270 nm.

• It has a maximal absorption at 340 nm and an emission at

610 nm.

• In principle, a fluoroimmunoassay using flurometry has an

advantage over an enzyme immunoassay using colorimetry.

• In colorimetry, the light absorbed by a sample is related directly to

the concentration of the absorbing molecule and is independent of

the intensity.

• In fluorometry the intensity of the fluorescence emission is directly

proportional to the intensity of the incident light.

• There are problems associated with fluorescence measurements.

1) Endogenous fluorophores , such as bilirubin and proteins, can

increase the non specific background fluorescence and reduce the

sensitivity of FIA.

2) Light scattering by high concentrations of protein, lipid and

other particles in serum will reduce the fluorescence signal.

3) The inner filter effect of hemoglobin and albumin will absorb

part of the excitation or emission beam.

4) Quenching duo to the non specific binding of albumin and

interaction with other specific quenching species may change the

quantum yield of the fluorescence.

• An important function of this step is the removal of

endogenous fluorescent compounds and interfering

substances from the sample prior to the detection step.

• This also allows large sample sizes to be used and improves

sensitivity as well as sensitivity.

• Solid phase FIA is a convenient approach either as

competitive FIA or sandwich immunometric assay.

Fluorescence Polarization Assay

• Fluorescence Polarization (FP) assays are homogeneous, single-

step assays ideally suited for high-throughput screening (HTS)

of large numbers of samples.

• All FP assays employ a large molecular species, or binding

partner (BP), in conjunction with a small, low molecular weight

fluorophore-labeled analyte (FA).

• When the large BP molecule is an antibody, the assay is referred

to as a fluorescence polarization immunoassay (FPIA).

• FPIAs are based on the competition of FA with free (i.e.

unlabeled) analyte in the samples or standards for the high

affinity binding site an antibody.

• A microplate well filled with the FA:antibody complex will

give a high FP reading.

• Addition of a increasing amounts of unlabeled analyte will

result in a competition between the unlabeled FA for the

antibody.

• As the competition happens, some of the FA will be released

from the antibody, and will resume its intrinsic, rapid rate of

rotation. This will cause a detectable loss of FP in the well.

• The addition of large amount of analyte will result in a much

larger reduction in the mP of the well (well Plotting mP versus

analyte concentration allows the construction of a standard

curve with a broad dynamic range. This is similar to, but not

strictly analagous to, the sigmoidal dose-response curve in a

traditional solid phase EIA.

RIA-heterogeneous

• A mixture is prepared of radioactive antigen

• Because of the ease with which iodine atoms can be

introduced into tyrosine residues in a protein, the

radioactive isotopes 125I or 131I are often used.

• Known amounts of unlabeled ("cold") antigen are added to

samples of the mixture. These compete for the binding sites of

the antibodies.

• At increasing concentrations of unlabeled antigen, an

increasing amount of radioactive antigen is displaced from

the antibody molecules.

• The antibody-bound antigen is separated from the free

antigen in the supernatant fluid, and the radioactivity of each

is measured from these data, a standard binding curve.

• The samples to be assayed (the unknowns) are run in parallel.

• After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.

Summary

• Immunoassays can be either competitive or noncompetitive.

• In competitive immunoassays, the amount of antigen is indirectly

proportional to the amount of signal.

• In noncompetitive immunoassays, the amount of antigen is directly

proportional to the amount of signal.

• Homogeneous immunoassays do not require separation of

unbound complexes from the bound complexes, and thus are

faster and easier to perform then heterogeneous immunoassays.

• Heterogeneous immunoassays require the separation of

unbound complexes, often utilizing a solid phase reagent such

as a magnetic particle or plastic bead.

REFERENCES:-

• Wild, David (Ed.). (2005).The Immunoassay Handbook.

Kidlington, Oxford: Elsevier.

• Evans, Susan (2004, June 15). Retrieved January 19, 2008,

from SACB Online Web site: http://sacb.org.sg/

• Bell, Suzanne (2006). Forensic Chemistry. Upper Saddle

River, New Jersey: Pearson Prentice Hall.

• Moody, David E. (2006).Immunoassay in Forensic Toxicology.

Encyclopedia of Analytical Chemistry.