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1、Inhibition of coagulationAdd 0.025 mL volume fraction to 1 per cent of the chicken red RBC suspension (add the chicken RBC to the top and then add) to appendix B.3.6, oscillation and mixed under room temperature (20 25 ) stand for 40 min observations (if the environment temperature is too high, can
2、buy 4 environment response 1 hour). In contrast, the red blood cells will appear to be buttoned to the bottom of the hole.3.7 results determined to tilt the board, observe the blood clot, and read the results (see table below).Table 1: criteria for interpretation of blood coagulation testcategoryfor
3、amenThe results ofone2345The total concentration of red blood cells is spread evenly over the bottom of the hole, which is 100 percent red blood cellsThe hemagglutinate is basically the same, but the bottom has a large circleThe red blood cells formed a medium large circle at the bottom of the hole,
4、 surrounded by small clotsThe red blood cells form a small dot in the bottom of the hole, with a small set of clumps aroundThe red blood cells are small dots at the bottom of the hole, and the edges are smooth and neat, the red blood cells completely uncollected+ + + + + + +-The maximum dilution of
5、the antigen, which is a complete set of red blood cells (100% agglutination, + + + +), is the blood coagulation of the antigen, which is a unit of blood coagulation (HAU). Note that the control hole should be completely unset (-), otherwise the test will be void.4 blood clotting inhibition (HI) test
6、 (trace method)The 4HAU virus antigen was prepared according to the results of the three trials. The maximum dilution of the virus with a full blood clot is the end point, and the end dilution is divided by 4, the dilution of the antigens containing 4HAU. For example, if the end of the blood coagula
7、tion is 1:25, the dilution of the 4HAU antigen should be 1:6, 4 (256 divided by 4).Add 0.025 mL PBS to the 1 hole in the microreaction plate, and the 12th hole to the 0.05 mL PBS.4.4.025 mL of serum added to the first hole, fully mixed and then absorbed 0.025 mL in the second hole, and then diluted
8、to the 10th hole, from the 10th hole to the 025 mL.4.4 bore 1 11 with resistance to concentrate virus contains 4 hau blending of 0.025 mL, room temperature (20 ) let stand at least 30 min.4.5 0.025 mL per hole to join the volume fraction of 1% chicken red blood cell suspension blending, blending, ge
9、ntly let stand for about 40 min (room temperature 20 , if the ambient temperature is too high to buy 4 conditions), the comparison of red blood cells will render button sink on the bottom of the hole.4.6 decisionThe highest dilution of the serum from four HAU antigens was treated as a HI drop.The te
10、st results were valid only if the serum drops of negative control were no greater than 21og2 and the serum error of the positive control was no more than one. The HI price is less than or equal to 21og2 and the HI test is negative. The price of HI is 31og2. The HI price is greater than or equal to 4
11、1og2 positive. Greater than or equal to 11og2 with strong infection, combined with epidemiological investigation.Many researchers believe that the red blood cells of different individual chickens have different sensitivity to the new towns disease, with a difference of 1 2 drops in the average HI dr
12、oplet. So the best way to do this is by three to four red blood cells. The concentration of red blood cells also had a significant effect on the results of the experiment. The concentration of erythrocytes increased (0.5% 1%) and HA drops declined, and HI drops increased.In the blood clotting and bl
13、ood coagulation test, when the red blood cells appear in agglutination,Each row of the first hole in each row is added to the sample 25 mu L, and then the left to right is twice the continuous dilution to the seventh hole (vertical plate) or the 11th hole (cross plate). For each row the final hole i
14、s reserved for the diluent.2.2.3 and standard positive serum: 25 mu L for each hole in the first and third rows. In the second row, each hole was added 25 mu L to 1:20 for the foot-and-mouth disease O standard positive serum; In the fourth row, each hole was added 25 mu L diluted to 1:10. Set on the
15、 micro mixer oscillation 1 minute to 2 minutes, sealed by the role of 37 for 30 minutes.2.2.4 drip-sensitized erythrocyte diagnostic fluid: in the first and second row, each entry was added to the disease. In the third and fourth row, each hole was added to the pigs blister. Set on the micro mixer o
16、scillation 1 minute to 2 minutes, with 20 35 placed determination result after 2 hours.3 the result judgementTo determine the level of red blood cells by the following criteria: + + + + - 100% complete agglutination, with a uniform distribution of red blood cells around the bottom of the hole; + + +
17、 -75% of the agglutination, and the red blood cells are evenly distributed around the bottom of the hole, but in the center of the pore, there is a small point of the needle of red blood cells. + + -50% agglutination, with an uneven red blood cell distribution around the bottom of the hole, a small
18、dot of a red blood cell at the bottom of the hole. + -25% of the agglutination, with an uneven distribution of red blood cells around the bottom of the hole, but most of the red blood cells have been deposited at the bottom of the hole; - - in uncondensed, red blood cells are completely deposited in
19、to a dot in the bottom of the hole.The results of the 3.2 operation method: the non-condensate and standard antigen-positive pore agglutination test are established.If only the first row of holes were collected, the other four rows were not collected, and the samples were tested as foot-and-mouth di
20、sease type A. If only the second row of holes were collected, and the other four rows were not collected, the samples were tested for the foot-and-mouth disease. And so on. If only the fifth row of holes were collected, the other four rows were not collected, and the samples were tested as a pigs bl
21、ister diseaseThe maximum dilution of the sample of the 3.2.2 agglutination was the agglutinate effect of the sample.3.2.3 if appear above 2 row of holes agglutination, with a hole agglutination titer higher than that of the rest of the hole agglutination titer 2 logarithm (at the bottom with a 2) co
22、ncentrations above can be jailed for positive, the rest of the sentence to negative.The results of the 3.3 operation method are determined: the non-condensate II control test can be established.3.3.3.1 if the first row of a 2 hole above the agglutination of (+ + +), there are two hole corresponding
23、to the hole and the second row above agglutination inhibition, the third and fourth row not agglutinate sentence to foot-and-mouth disease type O positive. If the third row appear above 2 hole agglutination (+ + +), and the fourth row corresponding to the hole more than 2 hole agglutination inhibiti
24、on, the first and second row does not appear agglutination positive for swine vesicular disease.The maximum dilution of the sample of the 3.3.3.2 agglutination was the agglutinate effect.The attachment 3Positive indirect blood coagulation test (IHA)1 the principleAn experiment with known blood clott
25、ing antigen to detect unknown serum antibodies is called the forward indirect blood coagulation test (IHA).Antigens meet their corresponding antibodies, forming an antigen complex under certain conditions, but the molecular mass of the compound is small and invisible to the naked eye. If the antigen
26、 is adsorbed (sensitized) on the surface of a specially treated red blood cell,A small amount of antigen can greatly increase the response sensitivity of antigen and antibody. The red blood cells, which are sensitized by foot-and-mouth disease, meet with the foot-and-mouth disease, and the red blood
27、 cells appear to be clearly visible.2 the applicable scopeIt is mainly used to detect the antibody effect of the serum antibody of the type O FMD.Test equipment and reagents3.1 96 hole 110 V type medical blood clotting, and blood clotting plate glass plate with the same size3.2 micropipettes (50 mu
28、L 25 mu L) take liquid plastic mouth3.3 microoscillatorType 3.4 O FMDType 3.5 O FMD negative control serum3.6 O FMD positive control serum3.7 diluent3.8 waiting for serum (around 0.5 ml per head serum) 56 water bath inactivated for 30 minutes4 test method4.1 the diluent1-9 holes in 1-6 rows on blood
29、 clotting. Row 7, 1-4 hole 6-7. 1-12 holes in row 8 are 50 mu L.4.2 dilute the serumTake 1 influenza virus serum 50 mu L add 1 # 1 hole, and insert the plastic nozzle hole bottom, right hand thumb gently press spring 1-2 second blending, avoid to produce too much bubbles), from the hole 50 mu L move
30、d to 2 hole, take out after blending 50 mu L moved to the third hole. Until the ninth hole is mixed, 50 mu L is discarded. The first row 1-9 hole influenza virus serum dilution degrees (diluted multiples) in the order: (1), and 2, 8 1:16, 1:32 5, 4 yenshan 1:6 4 6, once 1:12 8, 6 being supplied, and
31、 behold 2 levies.Add the serum to the 2nd row of the serum. Take serum no. 3 to the 3rd row. All press the method to dilute, notice! When you take a serum, you must replace the plastic mouth.4.3 diluted negative control serumIn the 7th row of the blood clotting board, the first hole plus negative se
32、rum 50 mu L, which is diluted to the fourth hole, then the 50 mu L discarded from the hole. The diluted multiple of the negative serum is 1:2, 1:4, 1:8, 1:16. The 6-7 hole is controlled by the diluent.4.4 diluted positive control serumIn the eighth row of the blood clot, the positive serum 50 mu L i
33、s diluted to the 12th hole, and then the 50 mu L is removed from the hole. The current dilution of the positive serum is 1:2-1:40 96.4.5 plus blood clotting antigenChecked each hole, negative control serum of each hole, positive control serum each hole and diluent control hole with type O blood clot
34、 antigen (fully shake, the bottle should be no blood sedimentation) 25 mu L.4.6 oscillationPuts plate of coagulation trace oscillator on 1 to 2 minutes, if no oscillator, with handle gently pat blender can be, and then put the plate of coagulation on the white paper to observe whether each red blood
35、 cells of blending, not appear blood sedimentation for qualified. Cover glass plate, or 37 let stand at room temperature determination results 1.5 2 hours, can also be extended to the next day.4.7 criterionRemove the glass plate, put the plate of coagulation on the paper, first observed negative con
36、trol serum 1:16 hole, diluent control hole, shall be no agglutination (blood all sink into the form at the bottom of the bore is neat little dots), or only a + agglutination (blood most sink on the bottom of the hole, margin slightly small amounts of blood cell suspension).Positive serum control 1:2-1:256 each hole should appear + + - + + + agglutination (a small amount of blood ball sink to the bottom of the hole,Otherwise, the sediment will be prone to the effects of the use.