CN104483486A - Kit for identifying deer antlerblood slide - Google Patents

Abstract

The invention discloses an application of a deer nerve factor serving as an index for identifying an antlerblood slide, and further discloses a kit for identifying a deerantlerblood slide. Firstly, a rabbit polyclonal antibody of the deer nerve factor is prepared and taken as a capture antibody and well coats an elisa plate; a monoclonal antibodyof the deer nerve factor is further prepared and taken as a detecting antibody; and a corresponding enzyme labeled second antibody and substrate are well prepared, and a kit is prepared for standby application. The kit for identifying the deer antlerblood slide is an ELISA kit and comprises the elisa plate coated with the polyclonal antibody of the deer nerve factor, the detecting antibody, the labeled second antibody, a developing substrate, a terminating agent, a sample dissolving solution, a washing solution and a standard substance; the elisa plate, the labeling second antibody, the developing substrate, the terminating agent and the washing solution are all commercial standard products, and the polyclonal antibody of the deer nerve factor, the detecting antibody (a rat anti-deer nerve factormonoclonal antibody) and the standard product (the deer nerve factor) are specially prepared for the kit.

Description

A kit for identifying deer antler blood slices

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a kit for identifying deer antler blood slices.

Background technique

At present, due to the high price of deer antler products, especially blood antler slices with the highest quality and the highest price, there are a large number of fake and inferior products in the deer antler products market, and various fake and inferior products even occupy more than 90% of the antler decoction slices market. At present, eye observation, microscope inspection and genetic identification methods based on PCR technology are mainly used to detect various fake and inferior products. It is easier to identify non-deer source products and deer antler decoction pieces containing fake deer blood, but for real deer with fake antler matrix Blood imitations are more difficult to distinguish. In addition, some imitations are made of real blood-free antler matrix coated with deer blood. Although they are indistinguishable from blood antler tablets, their active ingredients are quite different from real blood antler. Current identification methods cannot distinguish such blood antler film pseudo-films.

In the annual regeneration of velvet antler, in addition to bone tissue, some supporting tissues such as nerves will also regenerate. The growth rate of the nerve is about 1cm/d. The mechanism that controls the rapid growth of antler nerves is currently unclear. However, Garcia et al. used semi-quantitative RT-PCR to detect the relative expression of NT-3 (neurotrophin-3) mRNA in different tissues at the top of velvet antler, and found that the expression of NT-3 (neurotrophin-3) mRNA was the highest in the epidermis layer at the top of the antler, and the lowest in the cartilage layer. The results coincided exactly with the innervation of these tissues. Huo Yushu et al. extracted and separated protein and polypeptide components with a molecular weight of more than 10,000 from freeze-dried velvet antler, and found that the components had nerve growth factor-like effects by using the method for measuring the activity of cell regulatory factors. The current scientific research has basically confirmed the spatio-temporal characteristics of the distribution of nerve growth factor, indicating that there is a relatively high level of deer nerve growth factor in velvet, and it has a trend that is directly proportional to the quality of velvet. At present, there is no report on using this index to identify the quality of deer antler products.

Contents of the invention

In the existing detection methods, identification is basically based on shape, color, smell and genetic characteristics, and most types of counterfeit and shoddy products can be detected, but the combined counterfeiting methods of deer sources cannot be identified, especially The blood antler sheet made of real blood-free antler matrix coated with deer blood is real from appearance to genetic characteristics, but compared with the real blood antler sheet, its active ingredient content is very low, which cannot reach customers Expected use effect. At present, there is no effective detection method to identify such counterfeit products. The present invention selects a core active factor of blood antler tablets—deer nerve factor as a detection index, and makes an identification kit as an effective detection method for true and false blood antler tablets. The product detection rate reaches 100%.

In order to solve the problem of identification of blood antler tablets, the present invention selects a core active factor of blood antler tablets: deer nerve factor as a detection index, and makes a kit as an effective detection method for genuine and fake blood antler tablets.

The invention discloses the application of deer nerve factor as an index for identifying deer antler blood slices.

The invention also discloses a kit for identifying velvet antler blood tablets: firstly, a rabbit-derived polyclonal antibody of deer nerve factor is prepared as a capture antibody, and coated with an enzyme plate; further, a monoclonal antibody of deer nerve factor is prepared as a detection antibody; Prepare the corresponding enzyme-labeled secondary antibody and substrate, and make a kit for use.

The kit for identifying velvet antler blood slices according to the present invention is an ELISA kit, which includes an enzyme plate coated with a deer nerve factor polyclonal antibody, an antibody for detection, a labeled secondary antibody, a chromogenic substrate, a terminator, and a sample dissolution solution, washing solution, and standard products; among them, materials for ELISA plates, labeled secondary antibodies, chromogenic substrates, terminators, and washing solutions are all commercial standard products, deer neurofactor polyclonal antibody, detection antibody (mouse anti-deer neurofactor Monoclonal antibody) and standard product (deer nerve factor) are specially prepared by the present invention.

The kit of the present invention technically solves the problem of identification of counterfeit products with similar genetic backgrounds that cannot be solved by the background technology. By selecting the deer nerve factor with strong specificity for blood antler as the detection index item, and adopting the current mainstream high-sensitivity Immunodiagnostic kits are used for detection, and the detection efficiency is also greatly improved. The same batch can detect more than 80 samples at the same time, and it takes less time. It only takes 4-5 hours to complete each batch of detection.

In the preliminary trial test, the detection rate of counterfeit products reached 100%.

Detailed ways

In the present invention, a specific core active factor of blood antler slices: deer nerve factor is selected as a detection index, and by making a conventional ELISA kit, it includes an ELISA plate coated with deer nerve factor polyclonal antibody, an antibody for detection, a labeled secondary antibody, Chromogenic substrates, terminators, washing solutions, standards, etc., have obtained effective detection methods to identify the authenticity of blood antler tablets.

The present invention first separates and purifies the deer nerve factor antigen: dilute fresh velvet blood with 1% HAc solution 1:5 → centrifuge at 2,000rpm/min at low temperature → use Sephadex G-50 column for coarse separation of the supernatant → collect the active peak at pH 6. 0 Phosphate buffer dialysis → CM-Sepharose FF column repurification → Sephacryl S-200 column chromatography separation and purification to obtain the antigen. A part of the purified deer nerve factor was diluted to make a standard. Then, rabbit-derived polyclonal antibody of deer nerve factor was further prepared: animal immunization (New Zealand white rabbit)→Elisa titer detection (or WB detection of antigen)→antiserum→recombinant protein A column separation and purification; mouse deer nerve factor was prepared at the same time Source monoclonal antibody: deer nerve factor antigen sensitized B lymphocytes (immunized animals) → preparation and screening of hybrid cell tumors → confirmation of antibody secreted by hybridoma cells → clone of hybridoma cells secreting antibodies → mass production of monoclonal antibodies. Finally, the kit of the present invention is assembled according to the collocation of conventional ELISA kits.

Specifically implement the embodiment of kit of the present invention:

1. Self-made 5 samples of blood antler slices, 5 samples of inferior velvet slices (bloodless velvet slices) coated with deer blood, cow blood, pig blood, sheep blood, and chicken blood, and 20 commercially available samples were collected. Grinding at low temperature, adding sample solution (PBST) 5mL/g, incubating at 37°C for 2 hours, centrifuging to get the supernatant;

2. Add 0.1ml of the extract of the sample to be tested to the reaction well of the coated microplate, and incubate at 37°C for 1 hour. Then wash, and set blank wells, negative control wells and positive control wells at the same time.

3. Add detection antibody to each reaction well, incubate at 37°C for 0.5-1 hour, wash, and then add 0.1ml of enzyme-labeled secondary antibody. Incubate at 37°C for 0.5-1 hour and wash. Then add 0.1ml of chromogenic substrate solution, 37°C for 10-30 minutes; finally add 0.05ml of stop solution to each reaction well.

6. Result judgment: On a white background, observe the results directly with the naked eye: compare the color of the blank and negative control wells, the darker the reaction well, the stronger the positive degree, and the lighter the color than the blank and negative control wells, it is judged as negative , according to the depth of the color, represented by "+" and "-". Further use a microplate reader to measure the OD value of each well after zeroing the blank or negative control wells. If it is greater than 2.1 times the OD value of the specified negative control, it is judged as positive. Among the 30 samples tested this time, the test results of 10 self-made samples were completely in line with the real situation of the samples, and all 5 self-made counterfeit samples were tested negative; among the 20 commercially available samples collected, 6 were positive, and the rest Negative, positive rate was 30%.

Claims (2)

1. deer neural factor is as the application differentiating antler blood sheet index.

2. differentiate the kit of antler blood sheet for one kind, it is characterized in that: be ELISA kit, comprise the how anti-ELISA Plate of quilt deer neural factor, detection antibody, mark two anti-, chromogenic substrate, terminator, sample dissolution liquid, cleansing solution, deer neural factor standard items.