Bioassay of insulin
Standard preparation and unit: It is pure, dry and crystalline insulin. One unit contains
0.04082 mg. This unit is specified by Ministry of Health, Government of India and is
equivalent to international unit.
Preparation of standard solution: Accurately weigh 20 units of insulin and dissolve it in
normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative. Add
1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a
cool place and use it within six months.
Preparation of test sample solution: The solution of the test sample is prepared in the
same way as the standard solution described above.
1. Rabbit Method: Selection of rabbits: They should be healthy, weighing 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 Water is withdrawn during the experiment.
Standard and Sample Dilutions: These are freshly prepared by diluting with normal
NaCl solution so as to contain 1 unit/ml. and 2 units/ml.
Doses: The dose which can produce suitable fall in blood sugar level is calculated for the
standard.
Principle: The potency of a test sample is estimated by comparing the hypoglycemic
effect of the sample with that of the std. preparation of insulin. Any other suitable method
can also be used.
14Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each. The
rabbits are then put into an animal holder. They should be handled with care to avoid
excitement.
First part of the 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 groups of rabbits are then given sc. injections of insulin as follows:
12 RABBITS
3 3 3 3
Standard Standard Test Sample Test Sample
Dilution Dilution Dilution Dilution
(I) (II) (I) (II)
From each rabbit, a sample of blood is withdrawn up to 5 hrs.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 test) : The same animals are used for the second part.
The experiment can be carried out after one week. Again they are fasted and initial blood
sugar is determined. The grouping is reversed, that is to say, those animals which
received the standard are given the test and those which received the test are now given
the standard. Those animals which received the less dose of the standard are given the
higher dose of the test sample and vice-versa. This test is known as ‘Twin Cross Over
Test’.
Mean percentage decrease in blood sugar of the first and second part is calculated.
2. Mouse Method: Mice show characteristic convulsions after s.c. inj. of insulin at
elevated temperatures. The percentage convulsions produced by the test and standard
preparations are compared.
Experimental procedure: Minimum 100 mice weighing between 18-22 gms. of the
same strain are used. They should be maintained on constant diet. They should be fasted
18 hrs. prior to the experiment.
Standard and sample dilutions: Dilutions are prepared with sterile saline solution, so as
to contain 0.064 units/ml. (std dilution I) and 0.096 untis/ml. (std. dilution II). Similarly,
test sample solutions are also prepared.
Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as
follows:
15100 MICE
25 25 25 25
Standard Standard Test Sample Test Sample
Dilution Dilution Dilution Dilution
(0.064 units/ml) (0.096 units/ml)
Mice are put in an air incubator at 33oC and observed for one and a half hr. An air
incubator with a glass front provided with six shelves is used. The temperature is
thermostatically controlled. Two mice are kept in each of the boxes made up of
perforated sheets of metal.
The mice which convulse or die are taken out of the incubator and observed. These mice
usually convulse severely but failure of the animal to upright itself when placed on its
back, should as well be considered as convulsion. Convulsive mice may be saved by an
inj. of 0.5 ml. of 5% dextrose solution.
Percentage convulsions produced by the test sample are compared with those of the
standard sample. Those animals which survive may be used again for another expt. after
an interval of one week.
Dose S1 0.25 ml S2 0.25 ml T1 0.25 ml T2 0.25 ml
Convulsions
Percentage
Convulsions
3. Rat diaphragm method: In this method increase in glycogen content of the muscle or
increase in glucose uptake by muscle in response to insulin is taken as the index of
potency of insulin.
4. Rat epididymal fat-pad method: Here, the ability of insulin to increase CO2
production by the fat-pad is taken as the parameter for the measurement of potency of the
insulin preparation.
5. Radioimmunoassay: It is the estimation of the concentration of the substance in a unit
quantity of preparation using radiolabelled antigens. A number of drugs are estimated
now days by radioimmunoassy methods because these methods are highly specific and
highly sensitive.
The radioimmunoassay of insulin is based on the ability of human insulin (unlabelled) to
displace beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies).
The method involves the following steps:
I. Bovine insulin is injected into the sheep. After a week the serum containing
antibodies produced against bovine insulin is collected form the blood of
the sheep.
16II. The serum containing antibodies is exposed to radiolabelled insulin and the bound vs
free ratio is determined.
III. The mixture of labelled antigen-antibodies is then added in different test-tubes
labelled as standard and test. About 6 concentrations of the standards are taken. They
are then added to different tubes and the bound vs free ratio is again determined using
gamma-counter.
IV. Standard curves are determined and the concentration of test insulin is determinded
using this standard curve.
Standard preparation and unit: It is pure, dry and crystalline insulin. One unit contains
0.04082 mg. This unit is specified by Ministry of Health, Government of India and is
equivalent to international unit.
Preparation of standard solution: Accurately weigh 20 units of insulin and dissolve it in
normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative. Add
1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a
cool place and use it within six months.
Preparation of test sample solution: The solution of the test sample is prepared in the
same way as the standard solution described above.
1. Rabbit Method: Selection of rabbits: They should be healthy, weighing 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.
Standard and Sample Dilutions: These are freshly prepared by diluting with normal
NaCl solution so as to contain 1 unit/ml. and 2 units/ml.
Doses: The dose which can produce suitable fall in blood sugar level is calculated for the
standard.
Principle: The potency of a test sample is estimated by comparing the hypoglycemic
effect of the sample with that of the std. preparation of insulin. Any other suitable method
can also be used.
14Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each. The
rabbits are then put into an animal holder. They should be handled with care to avoid
excitement.
First part of the 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 groups of rabbits are then given sc. injections of insulin as follows:
12 RABBITS
3 3 3 3
Standard Standard Test Sample Test Sample
Dilution Dilution Dilution Dilution
(I) (II) (I) (II)
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 test) : The same animals are used for the second part.
The experiment can be carried out after one week. Again they are fasted and initial blood
sugar is determined. The grouping is reversed, that is to say, those animals which
received the standard are given the test and those which received the test are now given
the standard. Those animals which received the less dose of the standard are given the
higher dose of the test sample and vice-versa. This test is known as ‘Twin Cross Over
Test’.
Mean percentage decrease in blood sugar of the first and second part is calculated.
2. Mouse Method: Mice show characteristic convulsions after s.c. inj. of insulin at
elevated temperatures. The percentage convulsions produced by the test and standard
preparations are compared.
Experimental procedure: Minimum 100 mice weighing between 18-22 gms. of the
same strain are used. They should be maintained on constant diet. They should be fasted
18 hrs. prior to the experiment.
Standard and sample dilutions: Dilutions are prepared with sterile saline solution, so as
to contain 0.064 units/ml. (std dilution I) and 0.096 untis/ml. (std. dilution II). Similarly,
test sample solutions are also prepared.
Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as
follows:
15100 MICE
25 25 25 25
Standard Standard Test Sample Test Sample
Dilution Dilution Dilution Dilution
(0.064 units/ml) (0.096 units/ml)
Mice are put in an air incubator at 33oC and observed for one and a half hr. An air
incubator with a glass front provided with six shelves is used. The temperature is
thermostatically controlled. Two mice are kept in each of the boxes made up of
perforated sheets of metal.
The mice which convulse or die are taken out of the incubator and observed. These mice
usually convulse severely but failure of the animal to upright itself when placed on its
back, should as well be considered as convulsion. Convulsive mice may be saved by an
inj. of 0.5 ml. of 5% dextrose solution.
Percentage convulsions produced by the test sample are compared with those of the
standard sample. Those animals which survive may be used again for another expt. after
an interval of one week.
Dose S1 0.25 ml S2 0.25 ml T1 0.25 ml T2 0.25 ml
Convulsions
Percentage
Convulsions
3. Rat diaphragm method: In this method increase in glycogen content of the muscle or
increase in glucose uptake by muscle in response to insulin is taken as the index of
potency of insulin.
4. Rat epididymal fat-pad method: Here, the ability of insulin to increase CO2
production by the fat-pad is taken as the parameter for the measurement of potency of the
insulin preparation.
5. Radioimmunoassay: It is the estimation of the concentration of the substance in a unit
quantity of preparation using radiolabelled antigens. A number of drugs are estimated
now days by radioimmunoassy methods because these methods are highly specific and
highly sensitive.
The radioimmunoassay of insulin is based on the ability of human insulin (unlabelled) to
displace beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies).
The method involves the following steps:
I. Bovine insulin is injected into the sheep. After a week the serum containing
antibodies produced against bovine insulin is collected form the blood of
the sheep.
16II. The serum containing antibodies is exposed to radiolabelled insulin and the bound vs
free ratio is determined.
III. The mixture of labelled antigen-antibodies is then added in different test-tubes
labelled as standard and test. About 6 concentrations of the standards are taken. They
are then added to different tubes and the bound vs free ratio is again determined using
gamma-counter.
IV. Standard curves are determined and the concentration of test insulin is determinded
using this standard curve.
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