Identification method of deer blood

According to the theory of forensic serological antibody precipitation reaction, deer blood dried powder was quickly and accurately identified by using anti-deer Hb (hemoglobin) serum as antibody, which provided a new, accurate and stable method for raw material quality control of this valuable traditional Chinese medicine.

1. Experiments and methods

1.1 preparation of anti-deer Hb serum

Antigen: after aseptic operation, several milliliters of deer blood are put in a container with an appropriate amount of anticoagulant and stored in the refrigerator. Take out the components of plasma centrifuged with normal saline, remove several milliliters, add sterilized cold distilled water to wash into 20% Hb solution of deer blood, remove the matrix, add the same amount of hypertonic saline, and then add the same amount of incomplete Freund's adjuvant emulsified for immune injection. Immune injection: healthy rabbits were used as experimental objects.

It was injected subcutaneously on the back of the muscle in the thigh of rabbits. 4-7ml was injected once every 6 days for 18 days. After the first test of cardiac puncture was satisfactory, all blood samples were collected to separate serum by neck A hanging line bloodletting method. Determination of titer and specificity: the titer of 1-5 anti-deer Hb serum was determined with deer blood HbV1/1000,1/2000,1/5000,1/10000,1/30Q0 prepared in advance. The results showed that the titer of each anti-deer Hb serum was higher than 1x1000, and the highest titer of No. 5 anti-deer Hb serum was 30000 times. Other pig, bovine and sheep animal hemoglobin lx1000 was used as precipitation reagent, the results were negative, indicating that the specificity of anti-deer Hb serum was good.

1.2 Deer blood dried powder was qualitatively identified by anti-deer Hb serum. The deer blood dried powder came from Liaoning far East Pharmaceutical Group, and the sample number was lumb5. All the five samples were dark purple dried blood powder. Extraction and treatment of blood samples: 0.1 g of each sample was loaded into the test tube, and the appropriate amount of normal saline was added to the test tube. After soaking for several hours, the serum solution was centrifuged to determine its concentration and the following experiments were done with a thousand times dilution. Precipitation test: the diluent of the blood sample 1tick 1000 was slowly injected into the micro precipitating tube with half amount of anti-deer Hb serum in the capillary wall, and the results showed that the precipitation reaction was observed by sunlight at room temperature, and the white precipitation ring appeared on the interface of the two liquids in the miniature precipitation tube 15 minutes later, which was an anti-deer positive reaction. The blood sample showed anti-negative reaction after 60 minutes, and was identified as pig blood by anti-porcine Hb serum test. That is, the blood sample is deer blood.

2. Results and discussion

In this paper, an anti-deer Hb serum with high titer and specificity was prepared by subcutaneous injection of incomplete adjuvant and antigen into rabbit ventral muscle and back at an interval of three times in six days. The application of anti-decay serum Hb can conveniently and accurately identify the authenticity of dried deer blood powder, which provides a good identification method for the identification of this valuable traditional Chinese medicine and the quality control of the acquisition of medicinal materials. At the same time, it can also provide a reliable experience for the species identification of other animal products.