A method for characterization of residual α-gal antigen in enzymatic hydrolysis of bovine-derived bio-bone material
A bovine-derived, bone material technology, applied in the direction of analysis of materials, biological testing, material inspection products, etc., to achieve the effect of ensuring accuracy, simple operation, and avoiding experimental errors
Active Publication Date: 2016-08-03
山东省医疗器械产品质量检验中心
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Problems solved by technology
Comparing the final color development results with the results of bovine bone material samples without α-Gal antigen removal, the clearance rate of α-Gal antigen in the bovine bone material samples to be tested is obtained, and the clearance rate is used to evaluate the bovine bone material There are few reports on the effectiveness of α-Gal antigen clearance process
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preparation example Construction
[0046] 2.1 Preparation of enzymatically hydrolyzed bovine-derived bio-bone material
[0047] Aseptically thaw the bovine bone (5cm×1cm) at room temperature, put the bovine bone into the recombinant α-galactosidase citrate buffer (pH6.0) containing 30% polyethylene glycol and 400mg / L cephalosporin V ), incubated at room temperature for 12 h. Then the bovine bone was fully washed with PBS and placed in PBS containing 0.1% glutaraldehyde, soaked and incubated at 4°C for 12h. Then the bovine bone was fully washed with PBS and placed in PBS containing 0.1M aminoacetic acid, soaked and incubated at 4°C for 24 hours to block the active groups of the remaining glutaraldehyde molecules. Finally, the bovine bone was put into PBS containing 30% propylene glycol and 0.1M glycine, stored at -80°C, and sterilized by radiation (18kGy) for later use.
[0048] 2.2 Optimization of sample pretreatment: One of the key factors affecting this method is to ensure sufficient exposure of the α-Gal a...
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The invention discloses a characterization method of residual alpha-Gal antigen in an enzymolytic bovine-derived biological bone material. The method comprises the following steps: grinding a bovine-derived biological bone material subjected to enzymolysis by utilizing a high-speed low-temperature homogenization process and adding the ground bovine-derived biological bone material into BSA solutions to obtain homogenate fluid with certain concentration; then, detecting and calculating an IC50 value of combination inhibition rate of an alpha-Gal antigen corresponding to M86 antibody and an IC50 value of a positive reference product in the bovine-derived biological bone material under the concentration through an inhibition ELISA method to obtain the clearance rate of the alpha-Gal antigen in the bovine-derived biological bone material to be detected, thus characterizing the residual alpha-Gal antigen in the enzymolytic bovine-derived biological bone material by the clearance rate. According to the method, samples can be prepared and processed easily, the cost is low, and a clearing effect of the alpha-Gal antigen in the enzymolytic bovine-derived biological bone material can be accurately determined.
Description
technical field [0001] The invention relates to a characterization method for enzymolysis of residual α-Gal antigen in bovine-derived biological bone materials, in particular to the establishment of a characterization method for α-Gal antigen clearance rate in bovine-derived biological bone materials. Background technique [0002] Bovine-derived biological bone is a bone repair material made from bovine bone material after a series of decellularization, degreasing, and freezing treatments, and it retains a certain amount of collagen components. Bovine-derived biological bone is more and more used in clinical bone graft repair operations due to its advantages of wide sources and easy preservation, such as: for bone defects and various depressions that need to be performed in orthopedics, stomatology, and plastic surgery repair etc. However, as animal-derived tissue, bovine-derived biological bone has strong antigenicity, which may cause immune rejection of the host after tra...
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IPC IPC(8): G01N33/68
CPCG01N33/6887
Inventor 刘成虎侯丽孙晓霞施燕平吴世福
Owner 山东省医疗器械产品质量检验中心