Lecture 5 Bioassay of Insuline

Bioassay of Insulin
Injection
Index
• Insulin
• Principle of insulin bioassay
• Rabbit Method
• Mouse Method
Insulin
• Insulin was discovered in 1921, which helped millions suffering from
type-1 diabetes
• It is a hormone made in pancreas, special cells call “beta cell” produce
insulin
• When a person suffer from type-1 diabetes the capability of these
cells is lost
• Most people now a days use human insulin or insulin analogs.
• Its is produced by bacteria (lilly) or by yeast (Novo-Nordisk) by using
genetic engineering
Principle of Insulin Bioassay
• The potency of the sample of insulin injection is estimated by
comparing the hypoglycemic affects it produces with that produced
by the standard preparation of insulin under the condition of suitable
method of assay.
Standard Preparation and Unit
• The standard preparation is the international standard established in
1958 and consists of recrystallized (purified) insulin from ox (52%) and
pig (48%).
• It is supplied in ampoules containing approximately 110 to 125 mg.
• The unit is contained in 0.04167 mg of standard preparation and 1 mg

contains 24 units.
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.
• The solution of the test sample is prepared in the same way as the
standard solution
1. Rabbit Method
• Selection of rabbits: They should be healthy, weighing about 1800-3000
g. 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 Water is withdrawn during the experiment.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.
Experimental 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. Sugar level 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
subcutaneous injections of insulin as follows:
• 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. By Cutaneous Injection into mice
• Mice show characteristic convulsions after subcutaneous injection of
insulin at elevated temperatures. The percentage convulsions produced
by the test and standard preparations are compared.
• 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. Dilutions are such that respective doses are contained in equal
volumes not greater than 0.5 ml
• Suitable doses for mice weighing about 20 grams are 0.015 units and
0.030 units
Experimental Procedure
• At least 96 mice weighing between 18-22 g are used. They should be
maintained on constant diet. They should be fasted 18 hrs. prior to
the experiment.
• They are divided into four equal groups. Two groups are injected with
subcutaneous injection of two different dilutions of the standard
preparation and other two groups with two dilutions of the sample.
Continued…
• After injection the mice are kept at a uniform temperature between
29 to 35oC in an air incubator with a transparent front. The mice are
observed for 1.5 hours.
• After injection, the number of mice which are dead or convulsive or

which lie still for more than two or three seconds when placed on
their back is recorded.
• 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.

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Bioassay of Insulin

• It is supplied in ampoules containing approximately 110 to 125 mg.

• The unit is contained in 0.04167 mg of standard preparation and 1 mg

• Standard and Sample Dilutions: These are freshly prepared by diluting

• After injection, the number of mice which are dead or convulsive or

• These mice usually convulse severely but failure of the animal to

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