ImmunoAssay Protocol

8/6/2019 ImmunoAssay Protocol

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Introduction of Immunoassay


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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


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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


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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 areglycoproteins 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.


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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.


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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 .


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Monoclonal Antibodies

Monoclonal antibodies differ from polyclonal antibodies in

that they are highly specific for a single epitope on a

multivalent antigen .


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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.


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Immunoassay methods evaluation

Introduction

Selection of an analytical methods which meets the clinical

laboratorys objectives of reliable,accurate,timely and cost-

effective service is essential.

Evaluation of Immunoassays requires an efficient protocol and

objective criteria for method selection.


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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.


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A. Specific Laboratory Objectives for

M

ethod 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


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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.


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Assay characteristics described above are analytical goals

shared by all clinical laboratories.

How well on assay achieves them depends on the unique

characteristics of the reagents and on experimental

optimization.


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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)


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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.


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1.Precision

Compare claims of precision between kits.

Surprisingly,you may have to check the calculations.


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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.


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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.


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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.


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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.


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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.


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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.


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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


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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 .


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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 orbinned into dose ranges and the

mean concentration and mean CV used for profile.


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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.


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The effect of this choice is felt by the laboratory.

Source of variation(Random and /or systematic)

Source of error effect on assay

1. Pipetting Accuracy

Precision

Carry over


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2. Separation Reagents

Reaction

Timing

Stripping

NSB effects

3.Chemistry Reagents

Reaction(EquilibriumVs non equilibrium )


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4. Counting Total Counts

Instrumental QC

Geometry

Quench

5. Color reaction Interference

Stability


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Sources of Error: Non isotopic assays

Source of error Effect on assay

1.Interferences Add color or reduce color etc.,

2.Color reaction Stability of substrate

Colored product

3.Precision Timing

Washing

Separation

Spectrophotometric error

Binding reaction


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Thank you

Documents

ImmunoAssay Protocol