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RADIOIMMUNOASSAY (RIA)

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

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RADIOIMMUNOASSAY (RIA)

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  1. RADIOIMMUNOASSAY (RIA)

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

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

  8. B% concentration

  9. BASIC TECHNIQUE • Standard substanceThe fundamentals of accurate quantitation: they must be as same as the antigen which will be assayed.

  10. Labeled antigen 1. Minute amount: less than the minimum of the Ag to be assayed 2. Optimal specific activity3. Radiochemical purity: >95%4. Good immunoactivity5. Stability

  11. Specific binding substanceAntibody in high binding affinityThe higher affinity, the more sensitiveThere are some specific binding globulin besides antibody.Cortical binding globulin for cortisolThyroid binding globulin for THSex binding globulin for P, E

  12. 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)

  13. 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

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

  15. 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.

  16. 结 IRMA 合 率 Ag剂量

  17. COMPARE IRMA WITH RIA RIA IRMA Labeled substance Ag Ab Principle Competing depression Noncompetitive Ab Limited Overdose Standard curve Negative Positive Balance Slow Fast Measure range Narrow Wide Measure object Large or small molecular Large molecular

  18. QUALITY INDEX • High Sensitivity • Strong Specificity • Precise in Precision • Good Accuracy

  19. DEVELOPMENT RIA CLIA ECLI TRFIA Tracer 125I 吖啶酯 Ru2+ Eu3+ Condition liquid, solid solid solid solid Procedure easy easy easy easy Automation no yes yes yes Separation centrifugation, wash wash wash wash Stability high high high high Validity short long long long Factors many few few many Incubation long short short long

  20. TUMOR MARKER Clinic application: • Assistant diagnosis. • Observe treatment response. • Monitor metastasis, recrudesce, and prognosis. • Screen in high incidence area.

  21. Tumor site Tumor marker Adenocarcinoma CEA Primary hepatoma AFP Pancreatic and bile cancer CA19-9, CA242 Gastrointestinal cancer CEA, CA19-9, CA72-4 Lung cancer CEA, CYFRA21-1, NSE, CA125 Breast cancer CA153, CEA Ovary cancer CA125 Cervix cancer SCC Prostate cancer PSA, FPSA Other CA50, Fe, β2-M

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