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  1. 1. prof. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000
  2. 2.  Introduction principle Schematic representation of cometetive binding inRIA Objective of RIA Instrumentation Requirements for RIA Methodology of the assay Applications of RIA in pharmaceutical analysis Novel applications of RIA-techniques Conclusion references
  3. 3. INTRODUCTION: Ria was primarili developed byberson & yalow(1959) for the quantitative measurement ofinsulin in human plasma. Ria principles have found wide application in the field ofdrug analysis,pharmacokinetic studies,immunodiagnosis. Ria is specific,sensitive &rapid. The major disadvantage of ria is health&safety risks by theuse of radiation &maintaining licensed radiation &disposalprogram is difficult.
  4. 4.  ria is competative binding assay. The antibody &labelled antigen are alwayspresent as limiting factors&the concentrationsof unlabelled antigen(sample) underexamination is increased continually. It has been observed that the % of antibody-bound labelled antigen declines progressively.
  5. 5. The most 2 vital equipments essentiall required forRIA are:i)centrifugeii)radioactive counterscentrifuge↓↓ ↓swing- bucketrotor fixedangleheadrotor↓ ↓capable of generating capable of generating1200-2500rpm 3500-4000rpm↓ ↓
  6. 6. the pellect is formed pellet is performed atat bottom of test tube an angleRADIOACTIVE COUNTERS↓↓ ↓gamma counters scintillationcounters↓ ↓Used for counting gamma- used for countingbeta-Energy emitting isotopes energy emmitingisotopesSuch as i125 such as ,3H,14c.
  7. 7. The following steps are involved in RIA:1)Radio label production2)Conjugate preparation3)Antibody production & characterization4)Separation techniques
  8. 8. Eg:the most commonly used radiolabels in RIAare 3H , I.125I125 -> for iodination drugs are coupled withtrimethylester,tryamine,tyrosine to serveas sites for iodination.-> iodination is performed by enzymaticiodination,monochloride exchange etc.
  9. 9. The combined hapten(such as drug) and carrier(aprotein,polypeptide)is called conjugate.→common carriers are human γ-globulin,albumin,syntheticpeptides etc.→several reactive groups on protein can be used forpurpose of conjugating the standard amino,carbovyl groups,phenolicgroups of tyrosine&SH group of cysteine etc.→conjugate is prepared by various conjugation,gluteraldehyde reactions etc.
  10. 10. →the conc.of antibody(called its liter)is important for the antigenbeingassayed.→general method of inducing antibody formation is to inject 0.2 to 2mg ofpure antigen mixed with “freunds adjuvant”(a mixture of mineral oil,waves,killed bacilli which enhances&prolongsthe antigenicresponse).→animals used include rabbits,sheep,guinea pigs depending on vol.ofantiserum desired.→once an animal has been imminized,it can be injected several times toobtain different lots of antisera.→antiserum collected is stored in liquid-nitrogen at -196 c.after thawingthe antiserum is stored at 4 c.
  11. 11. To separate free from bound labelled antigenseparation techniques are:a) physical methods:-filtration,chromatography,electrophoreses,charcoaldextran adsorption etc.b) Chemical methods:-organic solvents such asethanol,dioxane,PEG,ammonium sulfate forprecipitating antibody- boundhapten.
  12. 12. In RIA the sequential steps followed are:1)Mix a fixed concentration of antiserum containingspecific antibody with a constant of radiolabelledantigen.2)Incubate it for specified duration&at an appropriatetemperature usually +4°c.3)A definite volume of sample containing unlabelledantigen to be measured is added to the reactiontest tube.
  13. 13. 4)The antibody reacts with both radioactive&unlabelled antigenforming an ab-radiolabelled antigen complexes.5)Both radioactive&unlabelled antigens are more or less sameimmunochemically,they will compete for limited number ofantibody sites available.6)The radio activity falls because the unlabelled antigen dilutesit.i.e)reducing the no of labelled antigen combining withantibody.7)The counts obtained from the radioactivity are used todetermine the unlabelled antigen concentration in thesample,the interpretation being done on the standard curve.
  14. 14. RIA is used for estimation of pharmaceutical drugs like:1) morphine - narcotic analgesic2) Hydromorphone & hydrocodone in humanplasma -narcoticanalgesic,antitussive,antipyretic.3) Clonazepam - sedative&anticonvulsant4) Flurazepam - hypnotic&anticonvulsant5) Barbiturates - hypnotic&anticonvulsant6) Flunisolide - a steroid having marked anti-inflammatory activity
  15. 15. 1) Combained RIA technique – isotope dilution2) stereospeciticity
  16. 16. SYNTHESIS OF IMMUNOGEN:-sodium-p-chloroacetateMorphine > 3-0-carboxymethyl-absoluteethanol morphine→then 3-0-carboxymethyl-morphine is coupled tobovine-serum &the pH of solution maintainedto 5.5.
  17. 17. The immunogon,carboxymethyl morphine-bovine-serum-albumin is emulsified with equalvolume of complete friends adjuvant.→newzealand albino rabbits are used.
  18. 18. Various dilutions of antiserum are incubatedinpresence of fixed concentration of tritium labelledmorphine.then standard unlabelled antigen is added.incubated&saturated ammonium sulphate solutionadded↓The precipitate is sedimented by centrifugation at5000rpm↓
  19. 19. The washed with 50% ammonium sulphatesolution↓the precipitate contains antibodyboundmorphine&radioactivity counted with help ofpackrd- iri- card liquid scintillationspectrometer.
  20. 20. → combined RIA-technique &isotope dilution hasbeen successfully developed to estimateSULINDAC along with its 2 prominentmetabolites,namely:sulindac-sulphone&sulindac –sulphide,present in plasma-level.
  21. 21. Prepranolol is a racemic containsequimolecular portion of D-and L-isomers.→two antisera have been developedexperimentallya) antisera against the DL- racemicmixture&b) antisera against the L-isomers.→the DL- propranotol antiserum exhibits almostequal affintity for both D-and L-isomers
  22. 22. →by the application of two RIA- techniques,dl-and l- propronolol is quantified.→thus the concentrations of d- propranol isknown by subtracting the concentration of l-isomer from the dl-mixture.
  23. 23. → RIA have been successfully applied to a widevariety of pharmacological agents.→ RIA is an important method in the quantitativeanalysis of drugs.→the methods for preparing immunogenic drug -protien conjugates have improved during thepast few years & high-affinity antisera arebecoming more &more commonly available.
  24. 24. INTRODUCTION:-→ELISA is a biochemical technique used inimmunology to detect the presence of anantibody or antigen in a sample.→here,the tag employed is an enzyme.→ELISA is so named because the techniqueinvolvesthe use of an immunosorbent.
  25. 25. (immunosorbent is an absorbing material specificfor one of the components of the reaction,theantigen or antibody)→eg.of immunosorbents :cellulose or agarose→ELISA is actually done using 96-well microlitreplates suitable for automation.
  26. 26. ELISA are devided into 3 types:-1)competitive binding ELISA2)sandwich assays3)indirect ELISA
  27. 27. Competitive binding ELISA:-{here analyse antigen&taggedantigen compete forsites on the absorbed antibody}the antibody to the antigen analyte is absorbed on tothe solid phase by hydrophobic interactions↓Then a known amount of enzyme-labeledantigen&sample containing unlabeled antigen isadded
  28. 28. ↓Incubation is done & the wells are washed &enzyme substrate at 37°c for 30 min is added toproduce coloured product via enzymecatalysed reaction↓Maximum coloration occurs if there is noantigen in sample to compete with binding oflabelled antigen.
  29. 29. SANDWICH ASSAYS:-[these are noncompetetive]Sample is added to the wells containingabsorbed antibody↓incubation done↓Then enzyme labelled antibody added &it willbound to antigen
  30. 30. ↓the unbound labeled antibody is washed away↓The solid phase then contains antigensandwiched b/w labeled&unlabeled antibody↓The colour produced upon enzymatic reaction isdirectly preportional to the amount of antigo
  31. 31. sample antigen first adsorbed the solid phase↓Then unlabeled primary antibody is added↓Then incubated &washed↓Then a secondary labeled antibody is added↓Then incubated &washed↓the enzyme activity is proportional to the antigen
  32. 32. Indirect ELISA > noncompetitive > competitivemethod method method
  33. 33.  ELISA can be performed to evaluate thepresence of antigen or antibody in a sample.itis useful for determining serum antibodyconcentrations in HIV test. ELISA is highly sensitive &allows accuratemeasurement of very low levels of IGHsubclass.
  34. 34. 1)Analytical chemistry (sixth edition)→by GARY D.CHRISTIAN2)Practical pharmaceutical chemistry(fourth edition)→by A.H.BECKETT J.B.STENLAKE3)Pharmaceutical drug analysis(second edition)→by ASHUTOSHKAR4)Pharmaceutical analysis modern methods (partA)→by JAMES W.MUNSON
  35. 35. THANK YOU