RADIO IMMUNO ASSAY(Ria)&ENZYME LINKED IMMUNO SORBENT ASSAY

1. prof. Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000

2.  Introduction
 principle
 Schematic representation of cometetive binding in
RIA
 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. INTRODUCTION:
 Ria was primarili developed by
berson & yalow(1959) for the quantitative measurement of
insulin in human plasma.
 Ria principles have found wide application in the field of
drug analysis,pharmacokinetic studies,immunodiagnosis.
 Ria is specific,sensitive &rapid.
 The major disadvantage of ria is health&safety risks by the
use of radiation &maintaining licensed radiation &disposal
program is difficult.

4.  ria is competative binding assay.
 The antibody &labelled antigen are always
present as limiting factors&the concentrations
of unlabelled antigen(sample) under
examination is increased continually.
 It has been observed that the % of antibody-
bound labelled antigen declines progressively.

7. The most 2 vital equipments essentiall required for
RIA are:
i)centrifuge
ii)radioactive counters
centrifuge
↓
↓ ↓
swing- bucketrotor fixedangleheadrotor
↓ ↓
capable of generating capable of generating
1200-2500rpm 3500-4000rpm
↓ ↓

8. the pellect is formed pellet is performed at
at bottom of test tube an angle
RADIOACTIVE COUNTERS
↓
↓ ↓
gamma counters scintillation
counters
↓ ↓
Used for counting gamma- used for counting
beta-
Energy emitting isotopes energy emmiting
isotopes
Such as i125 such as ,3H,14c.

9. The following steps are involved in RIA:
1)Radio label production
2)Conjugate preparation
3)Antibody production & characterization
4)Separation techniques

10. Eg:the most commonly used radiolabels in RIA
are 3H , I.125
I125 -> for iodination drugs are coupled with
trimethylester,tryamine,tyrosine to serve
as sites for iodination.
-> iodination is performed by enzymatic
iodination,monochloride exchange etc.

11. The combined hapten(such as drug) and carrier(a
protein,polypeptide)is called conjugate.
→common carriers are human γ-
globulin,albumin,syntheticpeptides etc.
→several reactive groups on protein can be used for
purpose of conjugating the standard drug.
eg:terminal amino,carbovyl groups,phenolic
groups of tyrosine&SH group of cysteine etc.
→conjugate is prepared by various conjugation methods.
eg:diazotization,gluteraldehyde reactions etc.

12. →the conc.of antibody(called its liter)is important for the antigenbeing
assayed.
→general method of inducing antibody formation is to inject 0.2 to 2mg of
pure antigen mixed with “freunds adjuvant”
(a mixture of mineral oil,waves,killed bacilli which enhances&prolongs
the antigenicresponse).
→animals used include rabbits,sheep,guinea pigs depending on vol.of
antiserum desired.
→once an animal has been imminized,it can be injected several times to
obtain different lots of antisera.
→antiserum collected is stored in liquid-nitrogen at -196 c.after thawing
the antiserum is stored at 4 c.

13. To separate free from bound labelled antigen
separation techniques are:
a) physical methods:-
filtration,chromatography,electrophoreses,charcoal
dextran adsorption etc.
b) Chemical methods:-
organic solvents such as
ethanol,dioxane,PEG,ammonium sulfate for
precipitating antibody- boundhapten.

14. In RIA the sequential steps followed are:
1)Mix a fixed concentration of antiserum containing
specific antibody with a constant of radiolabelled
antigen.
2)Incubate it for specified duration&at an appropriate
temperature usually +4°c.
3)A definite volume of sample containing unlabelled
antigen to be measured is added to the reaction
test tube.

15. 4)The antibody reacts with both radioactive&unlabelled antigen
forming an ab-radiolabelled antigen complexes.
5)Both radioactive&unlabelled antigens are more or less same
immunochemically,they will compete for limited number of
antibody sites available.
6)The radio activity falls because the unlabelled antigen dilutes
it.
i.e)reducing the no of labelled antigen combining with
antibody.
7)The counts obtained from the radioactivity are used to
determine the unlabelled antigen concentration in the
sample,the interpretation being done on the standard curve.

16. RIA is used for estimation of pharmaceutical drugs like:
1) morphine - narcotic analgesic
2) Hydromorphone & hydrocodone in humanplasma -
narcoticanalgesic,antitussive,antipyretic.
3) Clonazepam - sedative&anticonvulsant
4) Flurazepam - hypnotic&anticonvulsant
5) Barbiturates - hypnotic&anticonvulsant
6) Flunisolide - a steroid having marked anti-inflammatory activity

17. 1) Combained RIA technique – isotope dilution
2) stereospeciticity

18. SYNTHESIS OF IMMUNOGEN:-
sodium-p-chloroacetate
Morphine > 3-0-carboxymethyl-
absoluteethanol morphine
→then 3-0-carboxymethyl-morphine is coupled to
bovine-serum &the pH of solution maintained
to 5.5.

19. The immunogon,carboxymethyl morphine-
bovine-serum-albumin is emulsified with equal
volume of complete friends adjuvant.
→newzealand albino rabbits are used.

20. Various dilutions of antiserum are incubated
inpresence of fixed concentration of tritium labelled
morphine.then standard unlabelled antigen is added.
incubated&saturated ammonium sulphate solution
added
↓
The precipitate is sedimented by centrifugation at
5000rpm
↓

21. The washed with 50% ammonium sulphate
solution
↓
the precipitate contains antibodybound
morphine&radioactivity counted with help of
packrd- iri- card liquid scintillation
spectrometer.

22. → combined RIA-technique &isotope dilution has
been successfully developed to estimate
SULINDAC along with its 2 prominent
metabolites,namely:sulindac-sulphone
&sulindac –sulphide,present in plasma-level.

23. Prepranolol is a racemic mixture.it contains
equimolecular portion of D-and L-isomers.
→two antisera have been developed
experimentally
a) antisera against the DL- racemicmixture&
b) antisera against the L-isomers.
→the DL- propranotol antiserum exhibits almost
equal affintity for both D-and L-isomers

24. →by the application of two RIA- techniques,dl-
and l- propronolol is quantified.
→thus the concentrations of d- propranol is
known by subtracting the concentration of l-
isomer from the dl-mixture.

25. → RIA have been successfully applied to a wide
variety of pharmacological agents.
→ RIA is an important method in the quantitative
analysis of drugs.
→the methods for preparing immunogenic drug -
protien conjugates have improved during the
past few years & high-affinity antisera are
becoming more &more commonly available.

26. INTRODUCTION:-
→ELISA is a biochemical technique used in
immunology to detect the presence of an
antibody or antigen in a sample.
→here,the tag employed is an enzyme.
→ELISA is so named because the technique
involves
the use of an immunosorbent.

27. (immunosorbent is an absorbing material specific
for one of the components of the reaction,the
antigen or antibody)
→eg.of immunosorbents :cellulose or agarose
→ELISA is actually done using 96-well microlitre
plates suitable for automation.

28. ELISA are devided into 3 types:-
1)competitive binding ELISA
2)sandwich assays
3)indirect ELISA

29. Competitive binding ELISA:-
{here analyse antigen&taggedantigen compete for
sites on the absorbed antibody}
the antibody to the antigen analyte is absorbed on to
the solid phase by hydrophobic interactions
↓
Then a known amount of enzyme-labeled
antigen&sample containing unlabeled antigen is
added

30. ↓
Incubation is done & the wells are washed &
enzyme substrate at 37°c for 30 min is added to
produce coloured product via enzyme
catalysed reaction
↓
Maximum coloration occurs if there is no
antigen in sample to compete with binding of
labelled antigen.

31. SANDWICH ASSAYS:-[these are non
competetive]
Sample is added to the wells containing
absorbed antibody
↓
incubation done
↓
Then enzyme labelled antibody added &it will
bound to antigen

32. ↓
the unbound labeled antibody is washed away
↓
The solid phase then contains antigen
sandwiched b/w labeled&unlabeled antibody
↓
The colour produced upon enzymatic reaction is
directly preportional to the amount of antigo

33. 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

34. Indirect ELISA > noncompetitive > competitive
method method method

35.  ELISA can be performed to evaluate the
presence of antigen or antibody in a sample.it
is useful for determining serum antibody
concentrations in HIV test.
 ELISA is highly sensitive &allows accurate
measurement of very low levels of IGH
subclass.

36. 1)Analytical chemistry (sixth edition)
→by GARY D.CHRISTIAN
2)Practical pharmaceutical chemistry(fourth edition)
→by A.H.BECKETT J.B.STENLAKE
3)Pharmaceutical drug analysis(second edition)
→by ASHUTOSHKAR
4)Pharmaceutical analysis modern methods (partA)
→by JAMES W.MUNSON

37. THANK YOU