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

Inactive Publication Date: 2010-09-29
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hemocytometer method has large errors and strong subjectivity, and is not suitable for large-scale sample detection; the flow cytometry method is expensive, the cost of consumables is high, and requires specialized technical personnel to operate, and the pertinence of the sample is not strong, and impurities are easily removed. Debris mistaken for blood cells

Method used

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  • Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof
  • Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof
  • Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an enzyme-linked immunoassay kit for chlamys ferreri blood cells and a preparation method thereof. The kit comprises an enzyme labeled plate, confining solution, washing solution, enzyme labeled antibody, pNPP substrate developing solution, phosphate buffer solution, blood cell anticoagulant, chlamys ferreri blood cell monoclonal antibody and a blood cell standard sample. The preparation method for the kit comprises the following steps of: preparing the chlamys ferreri blood cell standard sample, preparing the blood cell monoclonal antibody, optimizing an optimal using proportion of the antigen and the antibody, and establishing a detection result assessment criteria. Detection results can assess the possibility that the chlamys ferreri to be detected is subjected to pathogenic infection or environmental stress and the like by judging the established assessment criteria so as to provide basis for monitoring the health condition of the chlamys ferreri.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent (ELISA) detection kit for quantitatively detecting blood cells of Chlamys farreri by using a monoclonal antibody and a preparation method thereof, belonging to the technical field of shellfish molecular immunology. Background technique [0002] Shellfish lack immunoglobulin and specific immunity, and their immune defense process is completed by humoral factors in blood cells and hemolymph. Blood cells achieve the purpose of identifying, encapsulating, and removing foreign matter through autolysis, aggregation, phagocytosis, encapsulation, exocytosis, and oxidative killing; blood cells can also regulate auxiliary body fluids by releasing lysins, lectins, and opsonins. Factor immunity, which plays a key role in the process of shellfish resisting external environmental stimuli and foreign pathogenic microorganisms. [0003] Chlamys farreri is an important economic species for shellfish culture in no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 战文斌林听听邢婧绳秀珍
Owner OCEAN UNIV OF CHINA
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