Immunoassay Theory

 What is Immunoassay?

 Define the reagents used.

 About EIA method used by Tosoh.

 What types of testing?

 About Tosoh analyzers.

 What is Immunoassay
 Immunoassay is a biochemical test that measures the
concentration of a substance in a biological liquid (serum)
using the reaction of an antibody to its antigen.

 The presence of antigen or antibodies can be measured.

 Detecting the quantity of antibody or antigen can be achieved

by a variety of methods.

 There are different types of Immunoassay's.

 Antibody-Antigen Reactions

 Anything that can trigger an immune response is called

an antigen.
 Antibodies are proteins that can recognize specific
antigens.
 Glossary, Key Terms
 Homogenous, (all the same) single step no pretreatment

 Heterogeneous, Different requires an extra step to remove unbound

antibody or antigen.

 Antibody-A protein that is produced by the body in response to

invasion by a foreign agent (antigen).

 Antigen-Any substance capable of triggering a specific immune

response. Not normally present, when introduced it triggers the
production of antibodies.
 Conjugate-Enzyme labeled antibody or antigen depending on assay
type (present in the test cup)

 EIA- Enzyme ImmunoAssay

 Lyophilized- freeze dried reagent, requires reconstitution before use.

 Immunoassay Methods
Commercial Immunoprecipitation
Immunoassay Methods
Particle immunoassay

Immunonephelometry

Radioimmunoassay

Enzyme Immunoassay

Fluorescent Immunoassay

Chemiluminescent
Immunoassay
 Tosoh Reagents
 All Tosoh analyzers use the
same common reagents
 Common reagents are;
 Wash solution
 Diluent solution
 Substrate solution

 Test Cup Format;

 Single dose test per cup
 Lyophilized reagent (conjugate)
 Conjugate and antibody or
antigen bound to beads
 Immunoassay Types
 Tosoh assays can be classified into 3 types

 1. One step SANDWICH method

 2. Two step SANDWICH method

 3. COMPETITIVE Binding method

 Sandwich methods have a DIRECT relationship for rate/analyte

 The higher the rate (flourescence) the greater concentration.
 Competitive methods have an INVERSE relationship for the
rate/concentration of the analyte being measured.
 Sandwich Method, One step
 Used for analytes with a high molecular weight (TSH,CEA,PSA)
 Antibody is bound to the beads
 A second antibody labeled with alkaline phosphate is in the
conjugate
 Enzyme labeled antibody binds to a different site than the
immobilized antibody on the beads.
 Both antibodies bind to the antigen creating the
antibody/antigen SANDWICH.
 The formation of antibody/antigen complexes is not limited by
the concentration of the sample analyte
 The amount of enzyme activity detected is directly proportional
to the amount of antigen in the sample.
Assay Principle
 Sandwich Method, Two Step
 In the first step, the sample( with patient antibody) is incubated
with antigen bound to the beads.

 In the second step, the enzyme labeled antibody in the

conjugate is dispensed.

 Enzyme labeled antibody binds to a different site on the

antibody (analyte).

 Both form the antigen antibody sandwich.

 The amount of enzyme activity detected is directly proportional

to the concentration of analyte (B12,Folate).
 Competitive Binding Method
 This method is used for smaller molecule sizes such as
Thyroxine and Estradiol.

 A limited amount of antibody binding sites are available on the

beads.

 Antigen in the test sample and enzyme labeled antigen in the

conjugate COMPETE for antibody binding sites on the beads.

 The rate in this type assay is INVERSELY proportional to the

analyte concentration in the sample.
Assay Principle
 Analysis Flow

 1. Seal break to open top of cup

 2. Sample and diluent are added to the cup
 3. Cup is incubated and agitated for a fixed time
 4. Bound Free washing is performed
 5.Substrate is added
 Detection process performed to measure fluorescence rate over time
 Detector Details

 The same detector is used on all Tosoh AIA analyzers

 Detector is a Top-Top multichannel
 Light source is fluorescent at wavelength of 365nm
 Excitation wavelength is 325-385
 Emission wavelength is 440-500nm
 Detection Method
 Detection is done by enzymatic substrate reaction.
 Tosoh substrate contains 4-methylumbelliferyl phosphate.
4-MUP
 After bound free separation (washing) takes place, 220u/l of
substrate solution is dispensed into the cup.
 4-MUP is decomposed by the bound alkaline phosphate labeled
antibody.
 4-methylumbelliferone is produced and fluorescent
measurements are made.
 Six fluorescent measurements are made within 297 seconds.
 Slope is calculated based on the increase per second
 Raw data is expressed in NM/sec.
Detection Timing
Tosoh Test Menu
 Tosoh AIA Analyzers
 Tosoh AIA analyzers all use the
same test cup format.

 All Tosoh analyzers use the

same common reagents.

 All analyzers are Random

Access.
 AIA-360

 Smallest system sold by Tosoh.

 Used at lower volume accounts, POL’s or STAT lab, Mobile PTH
 Throughput of 36 test Per Hour
 Single steps assays only
 Dilutions/and 2 step assays must be done off line
 AIA-600II

 Targeted for Mid-volume accounts, larger POL’s, smaller Hospital

 Random access
 Direct tube sampling
 Onboard dilutions and 2 step assays are automated
 Throughput of 60 tests per hour
 Tabletop installation
 AIA-1800

 Larger floor model system

 Designed for high volume accounts
 Fully automated system
 Throughput of 180 tests per hour
 Onboard reagent storage
Thank You!