Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof
An enzyme-linked immunoassay detection and Chlamys farreri technology, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of large error, strong subjectivity, and high cost of consumables in the blood counting plate method, and achieve good repeatability and stability , Convenient prevention and control measures, high sensitivity effect
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Embodiment 1
[0026] Example 1: Preparation of Chlamys scallop blood cell ELISA detection kit
[0027] 1. Preparation of Blood Cell Standard Samples
[0028] Take 30-40 vigorous and healthy scallops with normal physique (suitable living environment: water temperature 15°C, salinity 31ppt, sufficient bait, no pathogenic microorganisms, etc.), extract 50ml of hemolymph from the adductor muscle, and the volume ratio is 1:1 with pre-cooled blood cell anticoagulant (PBS containing 0.02M EDTA: 0.02M EDTA, 0.14M NaCl, 3mM KCl, 8mM NaCl 2 HPO 4 , 1.5mM KH 2 PO 4 , pH 7.4), the mixture was centrifuged at 3000r / min, 4°C for 10min, the obtained blood cells were precipitated, resuspended with 50ml of anticoagulant, and then crushed by ultrasonic waves, which was the blood cell standard sample, aliquoted, and stored at -80°C .
[0029] 2. Preparation of monoclonal antibody against hemocytes of Chlamys farreri
[0030] (1) Animal immunization: Take 100 μl of blood cell standard sample as antigen, m...
Embodiment 2
[0042] Example 2: Specific methods of use of Chlamys scallop blood cell ELISA detection kit
[0043] 1. Pretreatment of samples to be tested: quantitatively extract hemolymph from the adductor muscle with a sterile syringe, and immediately mix it with pre-cooled blood cell anticoagulant at a volume ratio of 1:1. The mixture was centrifuged at 3000r / min, 4°C for 10min, and the obtained blood cell pellet was resuspended with the same amount of anticoagulant as the hemolymph, and after ultrasonic crushing, it was the sample to be tested;
[0044] 2. Coating antigen: the above-mentioned samples to be tested and blood cell standard samples were mixed with PBS in 2 -1 Dilute, add to the wells of the microtiter plate, 100 μl per well, and coat overnight at 4°C;
[0045] 3. Blocking: Discard the liquid in the wells, add 200 μl of washing solution (PBST) to each well, shake gently for 5 minutes, pour off the washing solution, and repeat 3 times. After washing, add 200 μl blocking sol...
Embodiment 3
[0050] Example 3. Application of this kit to detect seasonal changes in hemocytes of Chlamys farreri cultured in Qingdao area
[0051] 1. Sample collection and processing: Select the Shazikou area of Qingdao City, Shandong Province, randomly collect 100 scallops in the middle of each month from March to December 2009, and randomly select 20 scallops from the collected scallops after each sampling. The method for the pretreatment of the sample to be tested in Example 2 is used to process the sample;
[0052] 2. Sample detection: perform ELISA detection on blood cell samples collected every month according to the specific usage method of the kit provided in Example 2;
[0053] 3. Result analysis ( Figure 4 ): OD of Chlamys farreri detected by hemocyte ELISA from March to December 2009 in Shazikou breeding area 405nm The values are 0.623, 0.648, 0.598, 0.685, 0.634, 0.402, 0.385, 0.476, 0.487 and 0.542, respectively. The values from March to July were all higher than 0....
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