RADIOIMMUNOASSAY (RIA)

INTRODUCTION

RIA is a nuclear technique widely used for measuring minute substances with IN VITRO Procedures.

It combined the technology of nuclear medicine (tracer technique) and immunology (antigen-antibody binding) so that the name RIA is designated. Someone called it was a hybrid technique.

This technique has been developed in 1959 by

Solomen Berson and Rosalyn Yalow in Bronx,New

York. For her contribution of this important analytical

technique ot medical science, R. Yolow shared the

1977 Nobel price in Medicine and physiology.

In earlier days RIA only limited to certain

hormone, but now the scope greatly expanded

to the field of reproductive physiology,

oncology, immunology, hematology,

pharmacology and parasitology etc. It makes a

great contribution to the diagnostic laboratory

and scientific research works.

PRINCIPLE

• Ag, Ab, and *AgAg --- Antigen to be assayed in serum. (unknown or standard) Variant*Ag --- Labeled antigen added in minute amount of radioactivity equally in each tube Nonvariant (constant)Ab --- Antibody added in minute amount of dilution equally in each tube Nonvariant (constant) and limited

• Competing depression reactionLabeled antigen (*Ag) possesses the same properties of unlabeled antigen (Ag). It can also bind to the correlated specific antibody (Ab) with the formation of labeled antigen-antibody complex or called bound antigen (B), leave the unbound one as free labeled antigen (F). The more Ag is present, the less likely is the *Ag bound to the Ab, thus the amount of B formed is inversely proportional to the Ag originally present in serum, this is so called competing depression reaction.

Ag+Ab Ag.Ab+Ag + *Ag

* Ag.Ab (B)+*Ag (F)

B%

concentration

BASIC TECHNIQUE

• Standard substance

The fundamentals of accurate

quantitation: they must be as same as

the antigen which will be assayed.

• Labeled antigen

1. Minute amount: less than the

minimum of the Ag to be assayed

2. Optimal specific activity

3. Radiochemical purity: >95%

4. Good immunoactivity

5. Stability

• Specific binding substanceAntibody in high binding affinityThe higher affinity, the more sensitive

There are some specific binding globulin besides antibody.Cortical binding globulin for cortisolThyroid binding globulin for THSex binding globulin for P, E

• Separating reagent (SR)Separation of B and F1. Non specific SR: (absorbing F) DCC( 葡聚糖包裹的活性碳 ), PEG( 聚乙 二醇 )2. Specific SR: (absorbing B) second antibody3. Solid phase SR: (absorbing Ab on solid material)

IRMA

• Ag, and *AbAg --- Antigen to be assayed in serum.( unknown or standard) Variant*Ab --- Labeled antibody added in minute amount of radioactivity equally in each tube Constant and overdose

Ag+*Ab Ag.*Ab+*Ab

(B) (F)

• Ag can bind to specific *Ab to form Ag*Ab

complex (B), and leave free *Ab (F).

• The amount of B formed is proportional to

the Ag originally present in serum.

• Separate B and F.

结 IRMA

合 率

Ag 剂量

RIA IRMALabeled substance Ag AbPrinciple Competing depression NoncompetitiveAb Limited OverdoseStandard curve Negative PositiveBalance Slow FastMeasure range Narrow WideMeasure object Large or small molecular Large

molecular

COMPARE IRMA WITH RIA

QUALITY INDEX

• High Sensitivity

• Strong Specificity

• Precise in Precision

• Good Accuracy

DEVELOPMENT

RIA CLIA ECLI TRFIATracer 125I 吖啶酯 Ru2+ Eu3+Condition liquid, solid solid solid solidProcedure easy easy easy easyAutomation no yes yes yesSeparation centrifugation, wash wash wash washStability high high high highValidity short long long longFactors many few few manyIncubation long short short long

TUMOR MARKER

Clinic application:

• Assistant diagnosis.

• Observe treatment response.

• Monitor metastasis, recrudesce, and prognosis.

• Screen in high incidence area.

Tumor site Tumor markerAdenocarcinoma CEAPrimary hepatoma AFPPancreatic and bile cancer CA19-9, CA242Gastrointestinal cancer CEA, CA19-9, CA72-4Lung cancer CEA, CYFRA21-1, NSE, CA125Breast cancer CA153, CEAOvary cancer CA125Cervix cancer SCCProstate cancer PSA, FPSAOther CA50, Fe, β2-M