Experiments on cyclic precipitation reactions

Summary

The cyclic precipitation reaction is widely used in forensic medicine for the identification of bloodstains and the determination of food adulteration. Usually these suspicious materials are used as antigens and identified with standard antisera. It has the advantage of using less material; for example, bloodstains on clothing and other items can be examined as antigens by washing them down with saline. Source: Laboratory Microbiology (Third Edition)

Operation method

basic program

Principle

Soluble antigens such as bacterial extracts, serum, viral solutions, etc. react with the corresponding antibody and, in the presence of electrolytes, produce a fine precipitate called a precipitation reaction. The principle of precipitation reaction and agglutination reaction is basically the same, the difference is that the antigen used is soluble, because it is a soluble antigen, its individual antigen molecules are small in size, and contain more antigen in the unit volume of solution, the total reaction area is large, and the amount of antibody required for the reaction is large, so the test is to dilute the antigen, not to dilute the antibody. The antigen that causes the precipitation reaction is called the precipitogen, and the corresponding antibody is called the precipitin. The cyclic precipitation reaction involves the formation of an interface between the antigen and the antibody in the precipitation tube, followed by the appearance of a ring of milky-white precipitate at this interface (Figure 1). The highest dilution of the antigen at which the cyclic reaction occurs is the potency of the precipitin. Figure 1 Cyclic precipitation test A. Positive tube with precipitation ring; B. Negative tube Ag: antigen; Ab: antibody.

Materials and Instruments

Horse serum (antigen) Rabbit anti-horse immune serum (antibody) Normal rabbit serum
Physiological saline
Sedimentation tube (inner diameter approx. 2.5-3.0 mm, length approx. 30 mm) Small test tube Capillary pipette Suction tube

Move

1. Take 1 ml of 1:25 horse serum and dilute to various concentrations in small test tubes using the multiple dilution method according to the table below.

undefined1, undefined2 means 1 ml from tube 1, 1 ml from tube 2 and so on. 2.

2. Insert 9 dry and clean precipitator tubes into the holes of the test tube rack so that they stand upright.

3. Using a capillary pipette, aspirate 1:2 rabbit anti-horse immune serum into the bottom of the precipitation tubes, approximately two drops per tube. 4.

4. Pipet the top diluted horse serum (antigen) with another capillary pipette and add to each tube according to the table below.

Starting from the highest dilution, add slowly along the wall of the tube to form an interface with the lower layer of rabbit anti-horse immune serum without shaking. Add saline to tube 8 and diluted rabbit serum to tube 9 as a control. 5.

5. Allow to stand at room temperature for 15-30 minutes before observing the results. Look for a white ring-like precipitate at the junction of the two liquid surfaces. 6.

6. Record the results as "+" if there is a white ring-like precipitate and "-" if there is no precipitate. If there is still white precipitation between the antigen and antibody interface at the maximum dilution, the dilution of the tube is the potency of the precipitin.

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