All Detail Of Bioassay

1. BIOASSAY
Prepared By :- Miss Anamika Singh

2. ASSAY
ANALYSIS DONE TO DETERMINE THE PRESENCE OF A SUBSTANCE & AMOUNT OF THAT
SUBSTANCE (KNOWN OR UNKNOWN ) IN BIOLOGICAL FLUIDS IS KNOWN AS ASSAY .
DETERMINES ACTIVITY AND POTENCY OF DRUG .

3. BIOASSAY
BIOASSAY IS DEFINED AS THE ESTIMATION OF THE POTENCY OF AN ACTIVE PRINCIPAL IN A UNIT
QUANTITY OF PREPARATION .
OR
DETECTION AND MEASUREMENT OF THE CONCENTRATION OF THE SUBSTANCE IN A PREPARATION
USING BIOLOGICAL METHODS.

4. PRINCIPAL OF BIOASSAY
THE BASIC PRINCIPAL OF BIOASSAY IS TO COMPARE THE TEST SUBSTANCE WITH THE INTERNATIONAL
STANDARD PREPARATION OF THE SAME AND TO FIND OUT HOW MUCH TEST SUBSTANCE IS REQUIRED
TO PRODUCE THE SAME BIOLOGICAL EFFECT , AS PRODUCED BY THE STANDARD .

5. INDICATION OF
BIOASSAY
 MEASURE PHARMACOLOGICALACTIVITY OF NEW/ CHEMICALLY UNDEFINED SUBSTANCE .
 MEASURE CONCENTRATION OF KNOWN SUBSTANCE IN TISSUE EXTRACT OR BODY FLUID EG :-
ACH, HISTAMINE
 MEASURE ED 50/ LD 50 OF DRUG .
 BIOLOGICAL STANDARDIZATION OF NATURAL DRUGS WHICH CANNOT BE OBTAINED IN
CHEMICALLY PURE FROM EG.VASOPRESSIN, OXYTOCIN

6. PURPOSE OF BIOASSAY
 COMPARE TEST SAMPLE WITH STANDARD SUBSTANCE TO DETERMINE QUANTITY OF
TEST SAMPLE REQUIRED TO PRODUCE AN EQUIVALENT BIOLOGICAL RESPONSE TO
THAT OF THE STANDARD SUBSTANCE .
 MEASURING PHARMACOLOGICAL ACTIVITY OF NEW OR CHEMICALLY UNDEFINED
SUBSTANCE.
 TEST METHOD EMPLOYED IN MEASURING THE RESPONSE OF LIVING ANIMAL TO
TOXICITY OF CHEMICAL CONTAMINANT .CERTAIN NO. OF INDIVIDUALS OF SENSITIVE
SPECIES ARE EXPOSED TO SPECIFIC CONC. OF CONTAMINANT FOR SPECIFIC PERIOD
TO EXAMINE TOXIC EFFECT .
 INVESTIGATING FUNCTION OF ENDOGENOUS MEDIATORS.
 DETERMINE CONC. AS WELL AS POTENCY OF UNKNOWN SUBSTANCE.
 IMPROVING AND MAIN TANNING STANDARDS OF BASIC ENVIRONMENTAL CONDITION
AFFECTING WELL- BEING OF PEOPLES E.G.- POLLUTANTS RELEASED BY PARTICULAR
SOURCE.

7. CHARACTERISTICS OF A
GOOD BIOASSAY
SENSITIVITY :- ABILITY TO DETECT SMALLEST CONCENTRATION
SPECIFICITY :- THE RESPONSE WHICH IS BEING MEASURED SHOULD SPECIFIC
REPRODUCIBILITY :- SAME OBSERVATION USING DIFFERENT INSTRUMENT AND OPERATOR , OVER
LONGER TIME PERIODS.
STABILITY :- SENSITIVITY OF PREPARATION SHOULD BE CONSTANT .
AVAILABILITY :- THE PARTICULAR TISSUE SHOULD BE EASILY AVAILABLE.

8. IMPORTANCE OF BIOASSAY
BIOASSAY AS COMPARED TO OTHER METHODS OF ASSAY ( EG- CHEMICAL OR PHYSICAL ASSAY
) IS VERY IMPORTANT . BECAUSE IT IS THE ONLY METHOD OF ASSAY IF
 ACTIVE PRINCIPAL OF DRUG IS UNKNOWN OR CANNOT BE ISOLATED E.G. INSULIN ,
POSTERIOR PITUITARY EXTRACT ETC.
 CHEMICAL METHOD IS EITHER NOT AVAILABLE OR IF AVAILABLE , IT IS TOO COMPLEX
AND INSENSITIVE OR REQUIRE HIGHER DOSE EG- INSULIN, ACETYLCHOLINE .
 CHEMICAL COMPOSITION IS NOT KNOWN EG- LONG ACTING THYROID STIMULANTS .
 CHEMICAL COMPOSITION OF DRUG DIFFERS BUT HAVE THE SAME PHARMACOLOGICAL
ACTION AND VICE –VERSA E.G. CARDIAC GLYCOSIDE, CATECHOLAMINES ETC.

9. ADVANTAGES
 BIOASSAY ARE PROCEDURE THAT CAN DETERMINES THERE CONCENTRATION OF
PURITY OR BIOLOGICAL ACTIVITY OF A SUBSTANCE SUCH AS VITAMINS, HORMONE, AND
PLANT GROWTH FACTOR.
 WHILE MEASURING THE EFFECT ON AN ORGANISM , TISSUE CELLS, ENZYMES OR THE
RECEPTOR IS PREPARING TO BE COMPARED TO A STANDARD PREPARATION.
 BIOASSAY MAY BE QUALITATIVE OR QUANTITATIVE BIOASSAY ARE USED FOR
ASSENTING THE PHYSICAL EFFECT OF A SUBSTANCE THAT MAY NOT BE QUANTIFIED
SUCH AS ABNORMAL DEVELOPMENT OR DEFORMITY .
 QUANTITATIVE BIOASSAY INVOLVE ESTIMATION OF THE CONCENTRATION OR POTENCY
OF A SUBSTANCE BY MEASUREMENT OF BIOLOGICAL RESPONSE THAT IT PRODUCES.
QUANTITATIVE BIOASSAY ARE TYPICALLY ANALYSED USING THE METHOD OF
BIOSTATICS.

10.  THEY NOT ONLY HELP TO DETERMINE THE CONCENTRATION BUT ALSO THE POTENCY
OF SAMPLE.
 IT IS ESPECIALLY USED TO STANDARDIZE DRUG, VACCINE , TOXINS OR POISONS ,
DISINFECTANTS , ANTISEPTICS ETC. AS THESE ARE ALL USED OVER BIOLOGICAL
SYSTEM IN SOME OR OTHER FORMS.
 THESE ALSO HELP DETERMINES THE SPECIFICITY OF A COMPOUND TO BE USED EX;-
PENICILLIN'S ARE EFFECTIVE AGAINST GRAM+VE . TESTING OF INFECTED PATIENTS
SPUTUM HELPS DETERMINE WHICH ANTI –BIOTIC BE GIVEN FOR QUICK RECOVERY.
 CERTAIN COMPLEX COMPOUNDS LIKE VITAMIN B-12 WHICH CAN’T BE ANALYSED BY
SIMPLE ASSAY TECHNIQUES CAN BE EFFECTIVELY ESTIMATED BY BIOASSAY ,
 SOMETIMES THE CHEMICAL COMPOSITION OF SAMPLE ARE DIFFERENT BUT HAVE
SAME BIOLOGICAL ACTIVITY .
 FOR SAMPLE WHERE NO OTHERS METHOD OF ASSAY ARE AVAILABLE .

11. DISADVANTAGES
 KEY PROBLEM IS VARIABILITY IN RESPONSE .
 LARGE NUMBER OF ANIMAL TO BE USED .
 EXPERTISE IN EXPERIMENT DESIGN , EXECUTION OF ASSAY & ANALYSIS OF
DATA REQUIRED .
 LEADS TO EXPENSIVE & TIME CONSUMING . ‘
 TIME RELATED CHANGES INS SENSITIVITY OF TEST ORGAN .
 TACHYPHYLACTIC RESPONSE OF SUBSTANCE BEING ASSAYED .


12. TYPE OF BIOASSAY
QUALITATIVE BIOASSAY :-
QUALITATIVE BIOASSAY IS USED FOR ASSESSING THE PHYSICAL
EFFECT OF A SUBSTANCE THAT MAY NOT BE QUANTIFIED , SUCH AS ABNORMAL
DEVELOPMENT OR DEFORMITY.
E.G.:- ARNOLD ADOLPH BERTHOLD'S FAMOUS EXPERIMENT ON CASTRATED CHICKENS.
THIS ANALYSIS FOUND THAT BY REMOVING THE TESTS OF A CHICKENS , IT WOULD NOT
DEVELOP INTO A ROOSTER BECAUSE THE ENDOCRINE SIGNALS NECESSARY FOR THIS
PROCESS WERE NOT AVAILABLE .
QUANTITATIVE BIOASSAY :-
INVOLVE ESTIMATION OF CONCENTRATION / POTENCY OF A
SUBSTANCE BY MEASUREMENT OF THE BIOLOGICAL RESPONSE IT PRODUCES. THESE
BIOASSAY ARE TYPICALLY ANALYSED USING THE METHOD OF BIOSTATICS .

13. BIOASSAY METHOD
GRADED QUANTAL
Matching
Bracketing
Interpolation
Multiple Point
Direct and point
assay (DEPA)
LD50
Determination

14. GRADED ASSAY
 GRADED ASSAY IN THESE ASSAYS AS THE DOSE
INCREASE THERE IS AN EQUIVALENT RISE IN
RESPONSE . THE POTENCY IS ESTIMATED BY
COMPARING THE TEST SAMPLE RESPONSE WITH
THE STANDARD RESPONSE CURVE .
 CONC. OF UNKNOWN = THRESHOLD DOSE OF
STANDARD / THRESHOLD DOSE OF TEST X
CONC. OF STANDARD .
 E.G. :- ACETYL- CHOLINE PRODUCING
CONTRACTION IN THE MUSCLE OF FROG
RECTUS ABDOMINIS .

15. BIOASSAY
 IT IS THE SIMPLEST TYPE OF BIOASSAY , IN THIS TYPE OF BIOASSAY RESPONSE OF THE
TEST SUBSTANCE TAKEN FIRST AND THE OBSERVED RESPONSE IS MATCHED WITH THE
STANDARD RESPONSE .
 SEVERAL RESPONSE OF THE STANDARD DRUG ARE RECORDED TILL A CLOSE MATCHING
POINT OF THAT OF THE TEST SUBSTANCE IS OBSERVED .
 A CORRESPONDING CONCENTRATION IS THUS CALCULATED . THIS ASSAY IS APPLIED
WHEN THE SAMPLE SIZE IS TOO SMALL .
 SINCE THE ASSAY DOSE NOT INVOLVE THE RECORDING OF CONCENTRATION RESPONSE
CURVE THE SENSITIVITY OF THE PREPARATION IS NOT TAKEN INTO CONCENTRATION .
 THEREFORE , PRECISION AND RELIABILITY IS NOT VERY GOOD .

16.  FIRSTLY RESPONSE OF THE TEST OF A PARTICULAR DOSE IS TAKEN .
 IT IS MATCHED WITH THE DOSE OF STANDARD ( WHOSE STRENGTH IS KNOWN ) & ERROR
METHOD .
 DONE TILL A CLOSED MATCHING IS OBSERVED .
 CORRESPONDING CONCENTRATION CALCULATION .
 POTENCY RATIO OF THE TWO CAN BE APPROXIMATELY FOUND & STRENGTH OF THE
UNKNOWN TEST SOLUTION BE CALCULATED.

17. MATCHING ASSAY

18. BRACKETING BIOASSAY
 USED WHEN TEST SAMPLE IS TOO SMALL .
 RESPONSE OF TEST IS BRACKETED B/W TWO RESPONSE ( GREATER & SMALLER ) OF
STANDARD SUBSTANCE .
 STRENGTH OF UNKNOWN FOUND BY SIMPLE INTERPOLATION OF THIS BRACKETED
RESPONSE ON THE DOSE AXIS .
 PRECISION & RELIABILITY IS POOR .

19. BRACKETING ASSAY

20. INTERPOLATION BIOASSAY
 BIOASSAY ARE CONDUCTED BY DETERMINING THE AMOUNT OF PREPARATION OF
UNKNOWN POTENCY REQUIRED TO PRODUCE A DEFINITE EFFECT ON SUITABLE TEST
ANIMAL OR ORGAN OR TISSUE UNDER STANDARD COLLECTION ,
 THIS EFFECT IS COMPARED WITH THAT OF A SLANDERED . THUS THE AMOUNT OF TEST
SUBSTANCE REQUIRED TO PRODUCE THE SAME BIOLOGICAL EFFECT AS A GIVEN
QUANTITY THE UNIT OF A STANDARD PREPARATION IS COMPARED AND THE POTENCY OF
UNKNOWN IS EXPRESSED AS A THAT OF A STANDARD BY EMPLOYING A SIMPLE FORMULA .

21. INTERPOLATION
BIOASSAY

22. MULTI POINT
BIOASSAY
 THIS METHOD INCORPORATES THE PRINCIPAL OF INTERPOLATION AND BRACKETING.
 2+1 INDICATES – TWO RESPONSE OF STANDARD AND ONE RESPONSE OF TEST
RESPECTIVELY.
 THIS PROCEDURE OF 2+1 OR 2+2 IS REPEATED 3 TIME OR 4 TIME BASED ON THE METHOD
WITH CROSSING OVER OF ALL SAMPLE .
 IT CAN BE FURTHER DIVIDED AS 3 POINT 4 POINT AND 6 POINT BIOASSAY .
 3 POINT METHOD – 2 DOSE OF STD +1 DOSE OF TEST
 4 POINT METHOD – 2 DOSE OF STD + 2 DOSE OF TEST
 6 POINT METHOD – 3 DOSE OF STD + 3 DOSE OF TEST
LATIN SQUARE METHOD OF RANDOMIZATION TO AVOID ANY BIAS.

23. 3 – POINT ASSAY

24. LATIN SQUARE
DESIGN
CALCULATION
MEAN RESPONSE OF THESE 3 SETS
PLOTTED
LOG POTENCY RATIO (M) =
(T- S1 ÷ S2-S1 ) X LOG D
WHERE , D – DOSE RATIO = S2 / S1
STRENGTH OF UNKNOWN =
S1 / T X ANTILOG OF M

25. 4- POINT ASSAY

26. LATIN SQUARE
DESIGN
CALCULATION
MEAN RESPONSE OF 4 SETS PLOTTED
LOG POTENCY RATIO (M)
( T2 –S2) + (T1 –S1)
( S2-S1 ) + ( T2- T1 )
WHERE , D-DOSE RATIO = S2/ S1
STRENGTH OF UNKNOWN =
S1/ T1 X ANTILOG OF M
x log d

27. SIX POINT ASSAY
 3+3 DOSE ASSAY
 3 CONC. EACH OF STD & TEST DRUG ARE USED
 6 STEP OF EXPERIMENTS USING 6 DOSE IN EACH SET
 MORE TIME CONSUMING LESSER IN USE
 RELIABILITY IS EXCELLENT

28. DOSE RESPONSE
CURVE
 INCREASE CONC. OF DRUG IN BATH
FLUID STEP BY STEP WITHOUT
WASHING OUT THE PROCEEDING
DOSE .
 CONTINUE TILL SUPRAMAXIMAL
EFFECT IS SEEN .
 DOSE RESPONSE CURVE IS
PLOTTED .

29. USES OF BIOASSAY
 TO MEASURE THE PHARMACOLOGICAL ACTIVITY OF NEW / CHEMICALLY UNDEFINED
SUBSTANCE.
 TO INVESTIGATE THE FUNCTION OF ENDOGENOUS MEDIATORS .
 TO MEASURE DRUG TOXICITY AND UNWANTED EFFECT .
 TO MEASURE THE CONC. OF DRUG AND OTHER ACTIVE SUBSTANCE IN THE BLOOD
OR OTHER BODY FLUIDS .
 DETERMINATION OF POTENCY , ED-50 / LD-50 OF DRUGS.
 NEW DRUG DEVELOPMENT
 MEASURE CLINICAL EFFECTIVE .

30. BIOASSAY OF
DRUGS
Oxytocin Digitalis
D-
tubocuraine
Histamine Insulin
Vasopressin ACTH 5 HT

31. BIOASSAY OF
OXYTOCIN
PRINCIPAL :- THE POTENCY OF OXYTOCIN IS DETERMINED BY COMPARING ITS
ACTIVITY WITH THAT OF THE STANDARD PREPARATION OF OXYTOCIN UNDER THE
CONDITION OF A SUITABLE METHOD OF ASSAY .
STANDARD PREPARATION :- THE STANDARD PREPARATION IS THE 4TH INTERNATIONAL
STANDARD FOR OXYTOCIN , ESTABLISHED IN 1978 , CONSISTING OF FREEZE- DRIED
SYNTHETIC OXYTOCIN PEPTIDE WITH HUMAN ALBUMIN AND CITRIC ACID ( SUPPLIED IN
AMPOULES CONTAINING 12.5 UNITS)

32. BY DEPRESSION OF THE BLOOD PRESSURE
IN CHICKEN
 ANAESTHETIZE A YOUNG HEALTHY ADULT COCKEREL WEIGHTING 1.2 TO 2.3 KG WITH
AN ANAESTHETIC THAT WILL MAINTAIN A PROLONGED AND CONSTANT HIGH BLOOD
PRESSURE .
 EXPOSE THE GLUTEUS PRIMUS MUSCLE IN ONE THIGH AND CUT AND RETRACT IT TO
REVEAL THE POPLITEAL ARTERY AND CRURAL VEIN .
 CANNULATE THE POPLITEAL ARTERY AND RECORD THE BLOOD PRESSURE .
 CANNULATE THE CRURAL OR BRACHIAL VEIN .
 PREPARE STANDARD SOLUTION WITH SALINE . INJECT 0.1 -0.5 ML
 INJECT 2 DOSE OF STANDARD SOLUTION INTO CANNULATED VEIN AND RECORDED
BLOOD PRESSURE .
 DOSE SHOULD CAUSE DECREASED IN B.P.

33.  INTERVAL BETWEEN 2 INJECTION IS 3- 10 MINS OR DEPENDS ON RATE AT WHICH B.P.
COMES TO NORMAL
 DILUTE TEST SAMPLE WITH SALINE SAME AS STANDARD ONE.
 RATIO BETWEEN STANDARD AND TEST SHOULD BE EQUAL .
 IF ANIMAL BECOME INSENSITIVE DUE TO REPETITIVE DOSE SO ANOTHER ANIMAL IS
TAKEN .
 MEASURE ALL RESPONSE AND RESULT IS CALCULATED BY STANDARD STATISTICAL
METHOD .

34. BIOASSAY OF DIGITALS
PRINCIPAL :- POTENCY OF THE TEST SAMPLE IS COMPARED WITH THAT OF STANDARD
PREPARATION BY DETERMINING THE ACTION ON THE CARDIAC MUSCLE .
STANDARD PREPARATION AND UNITS :- THE STANDARD PREPARATION IS A MIXTURE OF
DRIED AND POWDERED DIGITALIS LEAVES ( 1 UNIT = 76 MG)
PREPARATION OF EXTRACTS :- EXTRACTS AMOUNT OF THE POWDER IS EXTRACTED WITH
DEHYDRATED ALCOHOL IN A CONTINUOUS EXTRACTION APPARATUS FOR SIX HOURS . THE
FINAL EXTRACT SHOULD CONTAIN 10 ML. ( 5 ML . ALCOHOL + 5 ML. WATER ) PER 10 G. OF
DIGITALIS POWDER . IT SHOULD BE STORED IN BETWEEN 5 DEGREE SALIYAS .

35. PIGEON METHOD :-
 MINIMUM 6 PIGEON ARE USED FOR TESTING EACH SAMPLE
 THE WEIGHT OF THE HEAVIEST PIGEON SHOULD NOT EXCEED TWICE THE WEIGHT OF
THE LIGHTEST PIGEON.
 FOOD IS WITHHELD 16-28 HOURS BEFORE THE EXPERIMENT .
 PIGEON ARE DIVIDED ON THE BASIS OF THEIR SEX , WEIGHT AND BREED INTO TWO
GROUP.
 THEY ARE ANAESTHETIZED WITH ANESTHETIC ETHER.
 ONE SIDE OF THE WING IS DISSECTED AND THE ALAR VEIN IS CANNULATED BY MEANS OF
A VENOUS CANNULA. DILUTION ARE MADE WITH NORMAL SALINE .
 THE TEST SAMPLE AND STANDARD SAMPLE IS INFUSED THROUGH CANNULA.
 IN PIGEONS STOPPAGE OF HEART IS ASSOCIATED WITH A CHARACTERISTIC VOMITING
RESPONSE CALLED EMESIS .

36.  THE MILK FROM THE CROP SAC OF PIGEONS IS BEING EJECTED OUT. THIS MAY BE TAKEN
AS THE END POINT RESPONSE OF DIGITALIS .
 THE LETHAL DOSE PER KG. OF BODY WEIGHT IS DETERMINED FOR EACH PIGEON .
 THE POTENCY OF THE TEST SAMPLE IS DETERMINED BY DIVIDING THE MEAN LETHAL
DOSE OF STANDARD BY THE MEAN LETHAL DOSE OF THE TEST SAMPLE.

37. BIOASSAY OF D-
TUBOCURARINE
RABBIT HEAD DROP METHOD
PRINCIPAL :-
D- TUBOCURARINE HYDROCHLORIDE IS INJECTED INTO THE MARGINAL VEIN
OF A RABBIT ‘S EAR TILL THE RABBIT ‘S NECK MUSCLES ARE RELAXED SUCH THAT THE
ANIMAL CANNOT HOLD ITS HEAD UP. THE TOTAL AMOUNT OF TEST SAMPLE REQUIRED TO
PRODUCE THE END POINT IS COMPARED WITH THE TOTAL AMOUNT OF THE STANDARD
SAMPLE REQUIRED TO PRODUCE SIMILAR ENDPOINTS
SELECTION OF RABBITS :-
RABBITS WEIGHTING 2 KG ARE USED . ANIMAL SHOULD BE FREE
FROM DISEASE , OBTAINED FROM A HEALTHY COLONY AND SHOULD BE ACCUSTOMED WITH
THE EXPERIMENTAL PROCEDURE .

38. EXPERIMENTAL
PROCEDURE
 RABBIT IS PLACED IN A HOLDER WITH ITS HEAD PROTRUDING OUTSIDE.
 THE HEAD SHOULD BE FREELY MOVABLE .
 MINIMUM 8 RABBIT ARE USED .
 THEY ARE DIVIDED INTO TWO GROUPS EACH CONTAINING 4 RABBITS
 FIRST GROUP WILL RECEIVE STANDARD SAMPLE AND THE SECOND GROUP WILL RECEIVE THE
SAMPLE UNDER TEST .
 D- TUBOCURARIN SOLUTION IS INJECTED AT A CONSTANT SPEED BY INFUSION APPARATUS
THROUGH THE MARGINAL VEIN .
 INJECTION SHOULD BE GIVEN AT A RATE OF 0.4 ML / MIN AND SHOULD TAKE ABOUT 10 MIN.
DOSE 0.012% W/V IN SALINE .

39.  INFUSION IS CONTINUED TILL THE RABBIT WILL NOT BE IN A POSITION TO HOLD ITS
HEAD ERECT OR THERE WILL BE NO RESPONSE BY FOCUSING LIGHT ON THE EYES .
 RABBITS RECOVER IMMEDIATELY FROM THE EFFECT OF CURARIZATION .
 DURING THE EXPERIMENT THERE IS A POSSIBILITY OF RESPIRATORY
EMBARRASSMENT , WHICH IS TREATED BY INJECTING NEOSTIGMINE , METHYL
SULPHATE (0.05 MG) AND ATROPINE SULPHATE IMMEDIATELY THROUGH THE
MARGINAL EAR VEIN .
 CROSS – OVER TEST IS CARRIED OUT TO MINIMIZE BIOLOGICAL ERROR DUE TO
ANIMAL VARIATION.
 THOSE RABBITS WHICH RECEIVED THE STANDARD SAMPLE ON THE FIRST DAY WILL
BE GIVEN TEST SAMPLE ON THE SECOND DAY OF EXPERIMENT AND VICE VERSA .
 MEAN DOSE WHICH PRODUCE HEAD DROP OF THE TEST SAMPLE IS COMPARED WITH
THE MEAN DOSE OF STANDARD PREPARATION .

40. BIOASSAY OF INSULIN
TO COMPARE TEST SUBSTANCE WITH THE INTERNATIONAL STANDARD PREPARATION. TO
FIND OUT HOW TEST SUBSTANCE IS REQUIRE TO PRODUCE THE SAME BIOLOGICAL
EFFECT AS PRODUCED BY STANDARD .
PRINCIPAL :- THE POTENCY OF TEST SAMPLE IS ESTIMATED BY COMPARING THE
HYPOGLYCAEMIC EFFECT OF THE SAMPLE WITH THAT OF STD. PREPARATION OF INSULIN
.
STANDARD PREPARATION AND UNIT :- IT IS PURE, DRY AND CRYSTALLINE INSULIN . ONE
UNIT CONTAIN 0.0482 MG . THIS UNIT IS SPECIFIED BY MINISTERY OF HEALTH .
GOVERNMENT OF INDIA IS EQUIVALENT TO INTERNATIONAL UNIT .
PREPARATION OF STANDARD SOLUTION :- ACCURATELY WEIGHT 20 UNIT OF INSULIN AND
DISSOLVE IN NORMAL SALINE ACIDIFY IT WITH HCL TO PH 2.5 ADD 0.5 % PHENOL AS
PRESERVATIVE ADD 1.4%TO 1.8% GLYCERINE .FINAL VOLUME SHOULD CONTAIN 20 UNIT /
ML STORE THE SOLUTION IN A COOL PLACE AND USES IT WITHIN SIX MONTHS .

41. PREPARATION OF TEST SAMPLE SOLUTION .
THE SOLUTION OF THE TEST SAMPLE IS
PREPARED IN THE SAME WAY AS THE STANDERED SOLUTION
RABBIT METHOD
SELECTION OF RABBITS :- THEY SHOULD BE HEALTHY WEIGHTING ABOUT 1800-3000 GMS.
THEY SHOULD THEN BE MAINTAINED ON UNIFORM DIET BUT ARE FASTED FOR 18 HRS.
BEFORE ASSAY ,WATER IS WITHDRAWN DURING THE EXPERIMENT .
STANDERED AND SAMPLE DILUTION:- THESE ARE FRESHLY PREPARED BY DILUTING WITH
THE NORMAL NACL SOLUTION SO AS TO CONTAIN 1 UNIT / ML. AND 2 UNIT / ML.
DOSE:- THE DOSE WHICH CAN PRODUCE SUITABLE FALL IN BLOOD SUGAR LEVEL IS
CALCULATED FOR THE STANDERED .
EXPERIMENTAL PROCEDURE:- ANIMAL ARE DIVIDED INTO 4 GROUP OF 3 RABBIT EACH . THE
RABBIT ARE THEN INTO AS ANIMAL HOLDER . THEY SHOULD BE HANDLED WITH CARE TO
AVOID EXCITEMENT .

42. FIRST PART OF TEST
A SAMPLE OF BLOOD IS TAKEN FROM THE MARGINAL EAR VEIN OF EACH RABBIT .
PRESENCE OF REDUCING SUGAR IS ESTIMATED PER 100 ML. OF BLOOD BY A SUITABLE
CHEMICAL METHOD. THIS CONCENTRATION IS CALLED INITIAL BLOOD SUGAR LEVEL .
THE FOUR GROUP OF RABBITS ARE THE GIVEN SC. INJECTION OF INSULIN AS FLOWS :-
12 RABBITS
3 3 3 3
STD DILUTION 1 STD DILUTION II TEST SAMPLE TEST
SAMPLE
DILUTION 1
DILUTION II

43. FROM EACH RABBIT A SAMPLE OF BLOOD IS WITHDRAWN UP TO 5 HRS. AT THE INTERVAL OF
1 HR. EACH BLOOD SUGAR IS DETERMINED AGAIN . THIS IS KNOWN AS “FINAL BLOOD SUGAR
LEVEL “ .
SECOND PART OF THE TEST ( CROSS OVER THE TEST ) THE SAME ANIMALS ARE USED FOR
THE SECOND PART .THE EXPERIMENT CAN BE CARRIED OUT AFTER ONE WEAK . AGAIN THEY
ARE FASTED AND INITIAL BLOOD SUGAR IS DETERMINED . THE GROUPING IS RESERVED ,
THAT IS TO SAY , THOSE ANIMAL WHICH RECEIVED THE TEST ARE NOW GIVEN BE
STANDERED .
THOSE ANIMALS WHICH RECEIVED THE
LESS DOSE OF THE STANDARD ARE GIVEN THE HIGHER DOSE OF TEST SAMPLE AND VICE –
VERSA THIS TEST IS KNOWN AS TWIN CROSS OVER TEST “.

44. BIOASSAY OF
VASOPRESSIN
PRINCIPAL :- POTENCY OF VASOPRESSIN INJECTION IS DETERMINED BY COMPARING TEST
ACTIVITY WITH THAT OF STANDARD PREPARATION OF VASOPRESSIN .
STANDARD PREPARATION :-
SPECIFIC PRESSOR ACTIVITY CORRESPONDING TO THAT
YIELDED BY 0.0005 GM OF STANDARD PREPARATION (20 UNITS / ML).
STANDARD UNIT : -
SPECIFIC PRESSOR ACTIVITY CORRESPONDING TO THAT YIELDED BY 0.005
GM OF STANDARD PREPARATION (20 UNIT / ML)

45. TEST
PREPARATION
 ALBINO RAT OF 300 G WEIGHT
 ANAESTHETIZE IT BY S.C. INJECTION OF ETHYL CARBAMATE .
 AFTER 40-60 MIN. CANNULATE THE TRACHEA WITH POLYETHYLENE TUBE OF 2.5 MM
EXTERNAL DIAMETER .
 DISSECT CAROTID ARTERY FOR CANNULATION .
 CANNULATE FEMORAL VEIN CLOSE TO INGUINAL LIGAMENT BY THE FOLLOWING PROCESS .
 RETRACT ABDOMINAL MUSCLE TO EXPOSE THE INGUINAL LIGAMENT AND SUPERFICIAL
PRUDENTAL VEIN TO ONE SIDE .
 DISSECT FEMORAL VEIN TOWARDS INGUINAL LIGAMENT FROM CORRESPONDING ARTERY .

46.  TIE A SHORT POLYETHYLENE CANNULA ( 1MM EXTERNAL DIAMETER ) INTO FEMORAL VEIN
BY TWO LIGATURES .
 JOINED BY SHORT PIECE OF RUBBER TO 1ML BURETTE WITH AN ATTACHED THISTLE
FUNNEL CONTAINING SALINE SOLUTION .
 FIX A WET COTTON SWAB &TIE TO COVER THE INCISION AND CANNULA .

47.  INJECT 200 U HEPARIN IN SALINE SOLUTION /100 G BODY WEIGHT
 CONNECT CAROTID ARTERY CANNULA WITH MERCURY MANOMETER (2-3MM INTERNAL
DIAMETER )
 INJECT ALL SOLUTION THROUGH VENOUS CANNULA BY 1 ML SYRINGE
 A SUITABLE HYPOTENSIVE AGENT IS GIVEN TAIL VEIN TO PRODUCE A CONSTANT BASAL
PRESSURE
OF 50 TORR.
 DILUTE STANDARD &TEST PREPARATIONS SUCH THAT VOLUME TO BE INJECTED IS
BETWEEN 0.1 -0.5 ML
 CHOOSE 2 DOSE OF STANDARD SO THAT LOWER DOSE PRODUCE 30 TORR B.P. & HIGHER
PRODUCE 50 TORR B.P.
 RATIO OF DOSE SHOULD 3.5
 SELECT TEST DOSE ACCORDING TO STANDARD DOSE .
 DOSE ARE ADDED AT INTERVALS OF 3-5 MIN. IN A RANDOM ORDER
 RECORD RISE IN B.P. IN RESPONSE TO EACH DOSE .

48. SELECTION OF DOSE
Lower dose Higher dose

49. BIOASSAY OF ACTH
 ACTH (ADRENOCORTICOTROPIC HARMONE , CORTICOTROPIN ) IS POLYPEPTIDE TROPIC
HARMONE ( 39 AMINO ACID ) SECRETED BY ANTERIOR PITUITARY GLAND .
 ACTH STIMULATE THE PRODUCTION OF CORTISOL A STEROID HARMONE IMPORTANT FOR
REGULATING GLUCOSE PROTEIN AND LIPID METABOLISM , SUPPRESSING THE IMMUNE
SYSTEM RESPONSE AND HELPING TO MAINTAIN BLOOD PRESSURE .

50. OFFICIAL
PREPARATIONS
 CORTICOTROPIN INJECTION IS A STERILE SOLUTION ,IN A SUITABLE DILUENT OF THE
POLYPEPTIDE FROM THE PITUITARY GLAND OF MAMMALS .POTENCY RANGE SHOULD
BE 80.0 -12.0 % OF USP CARTIOTROPIN UNITS .
 CORTICOTROPIN FOR INJECTION , ANTIMICROBIAL AGENT .
 REPOSITORY CORTICOTROPIN INJECTION IS CORTICOTROPIN IN A STERILE SOLUTION
OF PARTIALLY HYDROLYZED GELATINE AND IS INTENDED FOR SUBCUTANEOUS AND
INTRAMUSCULAR USE. THIS SOLUTION HAS BEEN ADOPTED AS THE REFERENCE
STANDARD FOR THE BIOASSAY.
 PACKING – PRESERVE IN SINGLE –DOSE OR MULTIPLE –DOSE CONTAINER OF TYPE-1
GLASS
 STORAGE – STORED IN COLD PLACE.
 LABELLING – INJECTION RECOMMENDS INTRAVENOUS ADMINISTRATION .

52. Estimation of ascorbic acid (flowchart )

53. BIOASSAY OF
HISTAMINE
BIOASSAY OF HISTAMINE CAN BE DONE BY RECORDING
1) CONTRACTION OF ISOLATED GUINEA PIG ILEUM
2) BP FALL IN ANAESTHETISED CAT OR DOG
BIOASSAY USING GUINEA PIG ILEUM :-
BIOASSAY OF HISTAMINE ON ISOLATED GUINEA PIG
ILEUM CAN BE DETERMINED BY :-
 MATCHING BIOASSAY
 INTERPOLATION BIOASSAY
 BREAKETING BIOASSAY
 MULTIPOINT BIOASSAY

54. ILEUM
 3/5 OF INTESTINE
 EMPTIES IN THE LARGE INTESTINE VIA ILEOCECAL VALVE
 BILE SALTS ,VITAMIN B12 ,WATER AND ELECTROLYTE ABSORPTION
 DOES NOT HAVE MYOGENIC CONTRACTION
 MORE SENSITIVE TO HISTAMINE ACTION
HISTAMINE RECEPTOR IN ILEUM :-
RECEPTOR TYPE :- G-PROTEIN COUPLED RECEPTOR
AGONIST :- HISTAMINE
MOA ++ G- PROTEIN  ++ PHOSPHOLIPASE C SPLITTING OF PIP INTO 1) DAG THAT INCREASE
THE OPENING OF CALCIUM CHANNEL . 2) IP3 WHICH INCREASE CALCIUM MOBILIZATION FROM
SARCOPLASMIC STORES .

55.  DAG & IP3 LEAD TO INCREASE IN THE INTRACELLULAR CONCENTRATION OF CALCIUM AND
SMOOTH MUSCLE CONTRACTION
REQUIREMENTS :-
INSTRUMENT : THERMO STATICALLY CONTROLLED ORGAN BATH , CYMOGRAPH, AERATOR
PHYSIOLOGICAL SOLUTION :- TYRODE’S SOLUTION
TEMPERATURE :- 32 C
ANIMAL :- GUINEA PIG
STANDARD HISTAMINE SOLUTION
PREPARING STANDARD :-
 TAKE 10 MG OF HISTAMINE + 10 ML OF WATER ( 1000 UG /ML )
 TAKE 0.1 ML AND DILUTE WITH 10 ML WATER (10 UG/ ML )

56. BIOASSAY OF HISTAMINE THREE POINT
METHOD
 2 STANDARD  1 TEST

57. THANK YOU