General principles of immunoassay.

1. • Name : Aishwarya Sadanand Teli
• Topic Name : Immunoassay
• Class : M. Pharmacy - I
• Department : Pharmacology

2. GENERAL PRICIPLES OF IMMUNOASSAY

3. Introduction to immunoassay
 Immunoassays are bioanalytical methods in which the quantitation of the analyte
depends on the reaction of an antigen (analyte) and antibody.
 immunoassays have been widely used in many important areas of pharmaceutical
analysis such as diagnosis of diseases therapeutic drug monitoring clinical
pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical
industries.
 The importance and widespread of immunoassay methods in pharmaceutical analysis are
attributed to their inherent specificity high sensitivity for the analysis of wide range of
analytes in biological samples.

4. Principle
 These methods are based on a competitive binding reaction between a fixed amount of
labelled form of an analyte and a variable amount of unlabelled sample analyte for a
limited amount of binding sites on a highly specific anti-analyte antibody.
 When these immunoanalytical reagents are mixed and incubated, the analytis bound to
the antibody forming an immune complex.
 This complex is separated from the unbound reagent fraction by physical or chemical
separation technique.
 Analysis is achieved by measuring the label activity (radiation fluorescence, or enzyme)
in either of the bounds or free fraction. A standard curve, which represents the measured
signal as a function of the concentration of the unlabeled analyte in the sample is
constructed.

5. Immunoassay methods have been widely used in many important areas of pharmaceutical
analysis such as:
 Diagnosis of diseases
 Therapeutic drug monitoring
 Clinical pharmacokinetic
 Bioequivalence studies in drug discovery and pharmaceutical industries

6. BASIC METHODOLOGY INVOLVED IN PHARMACEUTICAL
ANALYSIS
 Immunoassay methods that have been applied in pharmaceutical analysis, based on
whether the separation step is or is not required can be classified into heterogeneous or
homogeneous assay .
 These methods can be performed in either competitive or non-competitive designs.
 The choice from these designs is based on nature of the analyte labeling chemistry
available and the analytical parameter required from the assay (e.g. sensitivity, dynamic
range, and precision).

7. TYPES
1. Competitive immunoassay
2. Non-Competitive immunoassay
3. Homogenous immunoassay
4. Heterogeneous immunoassay

8. 1. Competitive immunoassay:
 Competitive design of immunoassays can be carried out in an antigen-capture or antibody-
capture format, depending on whether the sold phase is coated with antibody or antigen
(analyte), respectively.
 In the antigen-capture format, The competition reaction occurs between the analyte (in
sample) and a labelled analyte for the binding to a limited amount ofanti-analyte antibody
coated onto a solid support.
 After equilibration and separation, the label activity on the solid phase is measured, and the
measured signal is inversely correlated to the concentrations of analyte in the sample.
 In antibody-capture format the analyte (or its protein conjugate) is coated onto a solid
support.
 The competition occurs between the analyte (in sample) and the immobilized analyte for the
binding to a limited amount of labelled anti-analyte antibody.
 After equilibration and separation, the activity of the label bound to the solid support is
measured, and the signal is inversely correlated to the concentration of the analyte.

9. 2. Non-competitive immunoassay:
 Non-competitive immunoassay (two-site" or "sandwich" assay).
 The non-competitive design is used for large analytes possessing more than one
recognition epitopes on the molecule. It requires two antibodies that bind to non-
overlapping epitopes on the analyte molecules .
 One of the two antibodies is bound to the sold phase and the second one is labelled and
used for detection.
 The sample analyte is allowed to bind to an immobilized antibody. After washing the
sold support (contains the formed analyte antibody complex) is incubated with an excess
of the second labelled antibody, which binds to the remaining epitope on the analyte

10. 3.Homogenous immunoassay
 Homogeneous are generally applied to the measurement of small analytes such as
therapeutic drugs.
 Since homogeneous methods do not require separation of the bound Ab-Ag from the
free Ag+, they are generally much easier and faster to perform.

11. Heterogeneous immunoassays
• Heterogeneous immunoassays require separation of the analyte‐antibody complex from
the remaining sample prior to final analysis. This can be accomplished using precipitating
chemicals, cross‐linking with other antibodies or use of an antibody bound to a solid
phase. Once the remaining matrix components are washed away, the remaining assay
components are added for final detection molecule.

12. Immunoassay Development, Optimization and Validation Flow Chart

13. Methodology for immunoassay
 Isotopic immunoassay
 Non-isotopic immunoassay
ISOTOPIC IMMUNOASSAY
 Based on competition for antibody between radioactive indicator antigen and unlabelled
antigen in test sample.
 Increase in count of unlabeled antigen in test sample decrease the labeled antigen in
bound.
 The concentration of the test antigen can be determined by comparison with a standard
calibrated curve with known concentration of purified antigen.

14. Non-isotopic immunoassay
Two types of nonisotopie immunoassay are:
1. Fluor immunoassay
2. ELISA (Enzyme-Linked Immunosorbent assay)

15. ELISA (Enzyme-Linked Immunosorbent assay)
• • ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or
drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.
• • ELISA has many of the advantages (e.g., sensitivity, ease of handling multiple samples) without the
disadvantages of dealing with radioactivity (like in RIA).
• The test requires:
1. the antibodies fixed to a solid surface, such as the inner surface of a test tube;
2. a preparation of the same antibodies coupled to an enzyme. This is one (e.g., B-galactosidase) that produces
a colored product from a colorless substrate.

16. Procedure
 The tubes are filled with the antigen solution (e.g.,urine) to be assayed. Any antigen
molecules present bind to the immobilized antibody molecules. The antibody-enzyme
conjugate is added to the reaction mixture. The antibody part of the conjugate binds to
any antigen molecules that were bound previously, creating an antibody-antigen-
antibody. After washing away any unbound conjugate, the substrate solution is added.
 After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the
concentration of colored product formed is measured in a spectrophotometer. The
intensity of color is proportional to the concentration of bound antigen.
 detecting allergens in food and house dust.
 measuring toxins in contaminated food.

17. Fluor immunoassay
 Radioisotopes and enzymes were replaced by new fluorescent compound in labeling the
antibodies and antigen.
 Substances used such as chelate of Europium which fluoresces when it excite by light in
the presence of a solution
 Emission duration is short but less than that of the background noise. can be measured
by special instrument which is unfortunately very expensive.
 It is 10 times more sensitive than RIA.
 A modern fluorescent based immunoassay uses as the detection reagent a fluorescent
compound which absorbs light or energy (excitation energy) at a specific wavelength and
then emits light or energy at a different wavelength.
 The difference between the wavelength of the excitation light and the emission light is
called the Stokes shift.

18. WHAT IS LUMINESCENCE?
 Luminescence -is a general term for the emission of light from a cool object. (In
contrast to, e.g., incandescence - the emission of light from a hot piece of metals such as
the filament in a light bulb.) There are many examples of naturally occurring
luminescence. Most of these fall into one of 3 categories:
 Phosphorescence- Absorption and slow re-emission of light. Most commonly observed
in minerals.
 Fluorescence- Absorption and fast re-emission of light.. Seen in deep sea organisms and
some insects and plants.
 Chemiluminescence- Emission of light driven by a chemical reaction. The most
common form of natural luminescence (often called "bioluminescence"). Seen in aquatic
organisms, insects and plants.