Method for detecting content of heparin in plasma
A technology of heparin and detection label, applied in the field of detection of heparin content in plasma, can solve the problem of insufficient detection methods of heparin, and achieve the effects of good application value, good sensitivity and good specificity
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Embodiment 1
[0028] Embodiment 1 heparin preparation
[0029]Add 100 g of isolated lung tissue to distilled water at a ratio of 1:10 (g:mL), crush it sufficiently in a tissue grinder until it becomes minced meat, and transfer it to a 1000 mL beaker for later use. Use 2mol / L NaOH solution to adjust the pH of the minced meat in the cup to 8.5, add 2% protease for 3 hours, add 5% NaCl of the sample amount, stir for 30 seconds every 10 minutes at 60°C, continue this operation for 2 hours, and immediately raise the temperature to 100 °C, let stand for 10 minutes to precipitate protein, filter through 100 mesh sieve, and collect the filtrate. The filtrate is cooled to 50°C, adjust the pH to 8.5 with 2mol / L NaOH, add 8% Dow AMBERLITETM FPA98CL food-grade special polymer (resin) resin (resin is rinsed to neutral with distilled water), in a constant temperature magnetic stirrer at 50°C , continuously adsorbed for 8 hours, filtered through a 100-mesh sieve, and collected the resin for later use. P...
Embodiment 2
[0030] Example 2 Preparation of Heparin-specific Monoclonal Antibody
[0031] Dissolve 1 mg of antigen absorbed by heparin prepared in Example 1 into 1 ml of physiological saline, and mix thoroughly to prepare an antigen solution with a concentration of 1 mg / ml.
[0032] The immunogen emulsified repeatedly was used to immunize 6-week-old female Balb / c mice for 6 times in total. The specific scheme was as follows: for the first immunization, the immunogen was added with complete Freund’s adjuvant, and the volume ratio of the two was 1:1. Mixed, multi-point injection of mouse foot pads and abdomen subcutaneously, 60ug / mouse, next immunization after 2 weeks; for the 2nd to 5th immunization, after fully emulsifying the immunogen and Freund’s incomplete adjuvant, mouse foot pads , Subcutaneous multi-point injection, 30ug / mouse, each time at an interval of 2 weeks; take serum, measure the titer, select the mouse with the best titer and inject 100ul, 50ug of immunogen intraperitoneal...
Embodiment 31B4
[0043] Example 3 Identification of 1B4 antibody affinity
[0044] Using the AMC sensor, the purified 1B4 antibody was diluted to 10ug / ml with PBST, and the heparin was serially diluted with PBST: 444.4nmol / ml, 222.2nmol / ml, 111.1nmol / ml, 55.6nmol / ml, 27.8nmol / ml, 0nmol / ml; operation process: equilibrate in PBST for 60s, immobilize antibody in antibody solution for 300s, incubate in PBST for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69GLY solution for sensor regeneration, output data , (KD represents the equilibrium dissociation constant or affinity), the results show that the Kd value of the 1B4 antibody is 2.37E-09M, which has good binding properties.
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