What methods are there for identifying the authenticity of deer blood?
Based on the theory of serological antibody precipitation reactions in forensic medicine, using anti-deer Hb (hemoglobin) serum as the antibody, deer blood powder was rapidly and accurately identified, providing a new, accurate and stable method for the raw material quality control of this precious traditional Chinese medicine.
1. Experiment and Methods
1.1 Preparation of anti-deer Hb serum
Antigen: Several milliliters of deer blood were collected under aseptic conditions and placed into a container with an appropriate amount of anticoagulant, then stored in a refrigerator. The sample was taken out and centrifuged with physiological saline to wash away the plasma components; several milliliters were then taken and washed with sterilized cold distilled water to prepare a 20-40% deer blood Hb solution. After removing the matrix, an equal volume of hypertonic saline was added, and then an equal volume of incomplete Freund's adjuvant was added and emulsified for immunizing injections. Immunization injections: Healthy domestic rabbits were used as the test subjects.
The popliteal fossa of the rabbit's thigh and the back muscles were used as injection sites. Each injection was 4-7 ml at 6-day intervals, one injection each time, over a total of 18 days. After preliminary testing of blood obtained by cardiac puncture was satisfactory, whole blood was collected by the rabbit carotid artery ligation exsanguination method and serum was separated. Determination of titer and specificity: Using pre-prepared deer blood Hb at 1/1000, 1/2000, 1/5000, 1/10000, and 1/30Q0, the titers of anti-deer Hb sera No. 1-5 were determined respectively. The results showed that the titers of all anti-deer Hb sera were higher than 1/1000, with the highest, No. 5, reaching 1/30000. Using hemoglobin from other pigs, cattle, and sheep at 1/1000 as precipitation reagents, the results were all negative reactions, indicating that the anti-deer Hb serum had good specificity.
1.2 Qualitative identification of deer blood powder using anti-deer Hb serum Deer blood powder was obtained from Liaoning Yuandong Pharmaceutical Group, with sample numbers 1-5#; the appearance of all five samples was dark purple dried blood powder. Blood sample extraction and processing: 0.1 g of each blood sample 1-5# was taken and placed separately into test tubes, an appropriate amount of physiological saline was added, stirred thoroughly, and after soaking for several hours, the serum liquid was obtained by centrifugation. Its concentration was measured, and a thousand-fold dilution was used for the following test. Precipitation test: The 1/1000 dilutions of blood samples 1-5# were slowly injected along the capillary tube wall into micro precipitation tubes previously containing half a volume of anti-deer Hb serum, and observed under sunlight at room temperature. The precipitation reaction results showed that in the micro precipitation tubes of blood samples 1, 2, 3, 4, and 5#, a white precipitation ring appeared at the interface of the two liquids after 15 minutes, which was a positive anti-deer reaction. Blood sample 3#, after 60 minutes, showed a negative anti-deer reaction, and was later identified as pig blood by testing with anti-pig Hb serum. That is, blood samples 1, 2, 4, and 5# were deer blood.
2. Results and Discussion
In this paper, incomplete adjuvant plus antigen was used for intramuscular injection in the rabbit popliteal region and subcutaneous injection in the back, three times at 6-day intervals, and in only 18 days a high-titer anti-deer Hb serum with good specificity was prepared. This immunization method is rapid and accurate. The use of anti-deer serum Hb can conveniently and accurately identify the authenticity of deer blood powder, providing a good identification method for the authentication of this precious Chinese medicinal material and the quality control of medicinal material purchasing. At the same time, it can also provide reliable experience for the species identification of other animal products.