Introduction of Immunoassay

Immuno & Assay

Immuno refers to an immune response that causes the body to generate antibodies, Assay refers to a test. Thus, an immunoassay is a test that utilizes immunocomplexing when antibodies and antigens are brought together

 Immunoassays are a group of sensitive analytical tests that utilize very specific antibody/antigen complexes to produce a signal that can be measured and related to the concentration of a compound in solution. Immunoassays also produce qualitative data in terms of the presence (+) or absence (-) of a compound in the body. They are used in a lot of laboratories, including hospitals labs, and have been widely used in the special area of Forensic Toxicology to screen for drugs and other chemicals in the body

Definition of antibody
Know that antibodies are proteins that are found in blood or other bodily fluids that are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Antibodies are produced by the B lymphocytes. They are glycoproteins belonging to the immunoglobulin supergene family that are produced in response to a foreign substance in the body. Antibodies have a generally common structure, but have regions that vary among them to accommodate the specific antigens.

Definition of antigen
Know that an antigen is any substance that causes your immune system to produce antibodies against it. An antigen may be a foreign substance from the environment such as chemicals, bacteria, viruses, or pollen; they may also be produced inside the body. An antigen is a substance with the ability to induce an immunological response. They typically enter the body from an infection. They are recognized at their epitopes by B cells or by the T cell receptor on T cells.

 Proteins or glycoproteins make the best antigens because they are the best at stimulating antigen recognition molecules. Some immunoassays test for antigens, rather than antibodies .

Monoclonal Antibodies
Monoclonal antibodies differ from polyclonal antibodies in that they are highly specific for a single epitope on a multivalent antigen .

Polyclonal Antibodies
Antiserum usually contains a mixture of antibodies that recognize and bind to the same antigen, but they may attach to different epitopes. An antigen that has multiple sites for antibodies to bind is called a multivalent antigen. These types of antibodies, present as a diverse mixture, are called Polyclonal antibodies.

Immunoassay methods evaluation

Introduction Selection of an analytical methods which meets the clinical laboratorys objectives of reliable,accurate,timely and costeffective service is essential. Evaluation of Immunoassays requires an efficient protocol and objective criteria for method selection.

 Protocol can easily be modified, depending on the specific questions being asked . Appropriate application of the tests described here depends on a thorough understanding of the analytic and bio chemical principles involved in the use of the antigen antibody reaction.

A. Specific Laboratory Objectives for Method Evaluation

Before evaluating a new method or starting a new test number of requests expected to establish clinical need. New method with convenient timing may result in inefficient turn and around affect overall service by reducing the number of other laboratory tests ordered or repeated. Some methods offer increased sensitivity and specificity highly purified or unique standards or easy to use formats

B.Analytical Objectives
The analytical terms precision accuracy and sensitivity specificity have well defined meanings in anlytical and

clinical chemistry. But because of the unique characteristics of immunoassays, these concepts can be complex. Eg. Both accuracy and assay sensitivity may be limited by precision. In the current context analytical sensitivity and specificity are distinguished from the concepts of clinical sensitivity and specificity.

 Assay characteristics described above are analytical goals shared by all clinical laboratories. How well on assay achieves them depends on the unique characteristics of optimization. the reagents and on experimental

Protocol Outline
A. Objectives and preparation for evaluation. B. Precision. C. Standard curves : shape, data reduction ,matrix ,interferences. D. Tracer- immunoreactivity, specific activity. E. Sensitivity. F. Accuracy: Parallelism ,recovery,cross-reactivity. G. Seatchard plot. H. Clinical validation. (OPSTSASC)

Establishing objectives and preparation

A.Protocol Review
The first step is to read the manufacturers claim carefully. The statement is equivalent to ones mothers reminder to galoshes. But a great deal of trouble can be avoided and questions answered before a single reagent is pipetted. A thorough review of the protocol includes careful attention to timing,temperature and centrifuge.

1.Precision
Compare claims of precision between kits. Surprisingly,you may have to check the calculations.

2.Standard Curves
Standard curves are usually illustrated . Sensitivity,range and shapes can be compared by plotting data from different kits on the same scale. This simple procedure may uncover strengths or weaknesses of one kit or another which may then be investigated further. Precision and sensitivity of the curve in the region of the upper limit of normal are extremely important.

3.Reagents
Information on the source and chemical structure of the antigens,antibodies and tracers can be related to the specificity expected and can direct attention to certain problems which may be anticipated. Antibody. Antigen. Tracer. Standard.

B.Information and Planning

Consultation with other users of a method and review of its performance in proficiency survey and quality control programs provide moral support and consensus of its popularity , precision and any systematic bias. To evaluate a kit efficiently and to make objective comparisons between methods. Adequate quality control

materials ,standards and previously assay samples shold be obtained and stored in aliquots.

Evaluation of Precision
Precision is a statistical index of the ability of an assay to yield the same result when the assay is repeated on the same sample. A.Definition 1. Within run precision : It is defined as the precision of the same sample run several times in the same assay. 2. Between-run precision : It is an index of the ability of the assay to reproduce a result on the same sample from day to day, or the confidence interval about a single result.

B. Protocol for precision studies

1. Materials : Fresh samples are often more stable and results are more precise than those obtainable on reconstituted serum. 2. Concentration to use : Samples for precision studies are usually used in atleast three concentrations. Generally the levels selected represent low (80% Bo). Midrange (50% Bo or ED50) and high (20% Bo) doses on the standard curve.

3.Calculations:

Average deviation : It is the measurements of a set is the mean of the difference of the individual measurements without regard to sign .

Standard deviation : Selected test samples are assayed in two or more replicates in every run. Results are obtained from standard curve and tabulated.

4. Acceptable performance:

A question often asked What is acceptable precision? High precision is particularly important in the concentration range of critical clinical decisions. Certainly single laboratory should get standard deviations as low as or less than those stated by the reagent manufacturer

C.Precision profile
The response (bound label or some function of bound label) measured in immunoassays is nonlinear and the variance is nonuniform : that is, the precision is different at different analyte concentrations. This is called heteroscadasticity. A device for expressing overall performance is the precision profile a graph of coefficient of variation against

concentration of analyte .

 In its simplest form the precision profile is created by assaying many replicates of standards and plotting the CV against the known concentrations . The performance over the entire curve and any existing heteroscadasticity can be visualized. To calculate the CV for the CV versus those graph obsevations on patients are grouped or binned into dose ranges and the mean concentration and mean CV used for profile.

D.Sources of Imprecision
Aside from assessing precision it is important during evaluation to reduce the both random and systematic errors to the minimum by adherence to the manufacturers procedures and by careful calibrations and quality control of all instruments in the laboratory. 1. Kit components and protocol: Although the selection of antibody , separation method and method design and optimization are out of the control of the user.

 The effect of this choice is felt by the laboratory. Source of variation(Random and /or systematic) Source of error 1. Pipetting effect on assay Accuracy Precision Carry over

2. Separation

Reagents Reaction Timing Stripping NSB effects

3.Chemistry

Reagents Reaction(EquilibriumVs non equilibrium )

4. Counting

Total Counts Instrumental QC Geometry Quench

5. Color reaction

Interference Stability

Sources of Error: Non isotopic assays

Source of error 1.Interferences 2.Color reaction Effect on assay Add color or reduce color etc., Stability of substrate Colored product 3.Precision Timing Washing Separation Spectrophotometric error Binding reaction

Thank you