According to the principles of forensic serological antibody precipitation, the use of anti-deer hemoglobin (Hb) serum as an antibody offers a fast and accurate method for identifying deer blood dry powder. This technique provides a new, reliable, and stable approach for quality control of this valuable traditional Chinese medicine raw material.
1. Experiments and Methods
1.1 Preparation of Anti-Deer Hb Serum
The antigen was prepared by aseptically handling several milliliters of deer blood, which was then placed in a container with an appropriate anticoagulant and stored in the refrigerator. After centrifugation using a saline solution, the plasma components were removed. A few milliliters of the sample were taken, mixed with sterile cold distilled water to create a 20–40% deer blood Hb solution, and then treated to remove the matrix. An equal volume of hypertonic saline was added, followed by an incomplete adjuvant, and the mixture was emulsified for immunization purposes. Healthy rabbits were used as test subjects for the immune injection. The serum was injected subcutaneously on the back muscles of the rabbits, with each dose of 4–7 ml given every six days over an 18-day period. After successful heart rate blood tests, blood was collected via neck bleeding to avoid serum separation.
Titer and specificity were determined using pre-prepared deer blood Hb at dilutions of 1/1000, 1/2000, 1/5000, 1/10000, and 1/30000. The titer of the anti-deer Hb serum was found to be higher than 1/1000, with the highest being 1/30000. When tested against hemoglobin from pigs, cattle, and sheep at a 1/1000 dilution, the results were all negative, confirming the high specificity of the serum.
1.2 Application of Anti-Deer Hb Serum for Qualitative Identification of Deer Blood Dry Powder
Five samples of deer blood dry powder from Liaoning Far East Pharmaceutical Group (sample numbers 1–5) were analyzed. Each sample was 0.1g and placed into separate test tubes. Appropriate amounts of physiological saline were added, and the mixtures were stirred and left to soak for several hours before centrifugation. The supernatant was collected, and the concentration was measured. Then, each sample was diluted 1000 times for testing.
Precipitation tests were conducted by placing the 1/1000 diluted samples into capillary tubes containing pre-loaded anti-deer Hb serum. Observations were made under sunlight at room temperature. After 15 minutes, a white precipitate ring formed at the interface of the two liquids in the micro-precipitate tubes for samples 1, 2, 4, and 5, indicating a positive reaction for deer blood. Sample 3 showed no precipitation after 60 minutes and was identified as pig blood when tested with anti-pig Hb serum. Thus, samples 1, 2, 4, and 5 were confirmed as deer blood.
2. Results and Discussion
This study successfully produced a high-titer, specific anti-deer Hb serum within 18 days by immunizing rabbits with incomplete adjuvant and antigen. The method proved to be efficient and precise. The use of anti-deer Hb serum enables quick and accurate identification of deer blood dry powder, offering a practical tool for quality control of valuable traditional Chinese medicines. Additionally, this technique can be applied to other animal-derived products for species identification, providing a reliable and repeatable method for authenticity verification in the herbal medicine industry.
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