Experimental principle
If the soluble nasal sputum antigen is adsorbed on the surface of red blood cells which is ten million times larger than its volume, the red blood cells can produce a new serological property and make so-called sensitized red blood cells, which can be used for diagnosing nasal discharge. As long as a small amount of the corresponding antibody is encountered, the red blood cells can be passively combined by the antigen-antibody reaction, showing a macroscopic agglutination phenomenon, and the negative red blood cells are rounded due to natural deposition on the bottom of the hole. This can significantly increase the sensitivity of the reaction.
Indirect hemagglutination test, indirect red blood cell agglutination test, also has some shortcomings. For example, different batches of prepared antigen or red blood cells may be inconsistent in sensitivity and stability. It is easily affected by external factors and non-specific agglutination occurs. Therefore, the utensils to be applied should be cleaned, and no dirt or impurities should be used. When non-specific agglutination is encountered, an indirect hemagglutination inhibition test is performed to eliminate non-specific reactions.
Experimental reagent
1. Preparation of phosphate buffered saline (PBS):
Liquid A: Na2HPO4 21.3 g (or Na2HPO4•12H2O 53.72 g) NaCl 8.5 g
Liquid B: KH2PO4 20.42 g NaCl 8.5 g
The above-mentioned A and B liquid formulations were each added with distilled water to 1000 ml, dissolved, filtered, and separately stored. When using, mix the A and B liquids, and add the same amount of distilled water to prepare 0.15mol/L PBS with different pH values, shake and sterilize for use.
pH liquid: 0.15mol/L Na2HPO4, NaCl
Liquid B: 0.15mol/L KH2PO4 NaCl
2. Preparation of 1% rabbit serum buffered saline: After inactivated the newly isolated rabbit serum at 56 ° C for 30 min, take the required amount (such as 10 ml) and add about half (such as 0.5 ml) of washed packed sheep red blood cells, and Add rabbit serum 4 times (such as 4ml) pH 7.2 PBS, and put it in a 37 ° C water bath for 10 min to absorb the heterophilic lectin in rabbit serum, centrifuge at 1500 r / min for 10 min, absorb the supernatant, add pH The value of 7.2 PBS 9.5 ml, which is 1% rabbit serum PBS, can be stored in a refrigerator at 4 ° C for 2 days.
3. Preparation of Alsever's solution: Prepare according to the following formula, adjust the pH value to 6.1, filter and disperse the vial, sterilize it at 113 °C for 15 min, then place it in a refrigerator at 4 °C for use.
Sodium citrate (Na3C6H5O7•5H2O) 0.8 g
Glucose 2.05 g
Tannic acid 0.0325 g
Sodium chloride 0.42 g
Distilled water to 100 ml
4. Red blood cells: Aseptically take the sheep's blood in the Aristotle solution and store in a refrigerator at 4 ° C for 3 weeks.
5. Fixing agent: 25% glutaraldehyde solution.
6. Tannic acid solution: The tannic acid used must be high-quality pure product. Prepare 1/200 solution with double-distilled water before use (use the pH 7.2 PBS to prepare the required concentration), and store in a refrigerator at 4 °C. It can be used within one week.
7. Snoring antigen and tested horse serum (inactivated at 56 ° C for 30 min).
Laboratory equipment
1. Serum reaction plate: made of plexiglass (polymethyl methacrylate), currently used is a 96-well plate, 132 cm long, 84 cm wide and 12 cm thick. There are basically two types of plate holes, namely, a pointed bottom (V-shaped) and a round bottom (U-shaped), wherein the V-shaped plate is available for blood coagulation test, which has a more typical agglutination end point than the U-shaped plate; The shape plate is better for the complement binding test. The 96-well V-shaped plate has a pore size of 0.6 cm and a total capacity of 0.25 ml per well.
The used serum plate can be soaked for 0.1 to 2 hours with 0.1% benzalkonium chloride, 3% to 5% sodium chlorite or 5% formaldehyde solution, or disinfected with ultraviolet light, but it can not be used for sage, because it can change the serum plate. If it is a non-infectious material to be inspected, it can be washed directly with water for 3 to 4 times, and then dried for use. Because the hardness of the serum plate is not enough, it is easy to rub the hair. Therefore, when scrubbing, it can be lightly wiped with gauze. Do not rub the surface with a finger or a hard cloth to avoid hairiness. When cleaning, the water temperature should not exceed 90 °C to avoid deformation.
2. Micro dropper: There is no stereotyped product in China, which can be processed by itself. The glass dropper used is about 20cm long. There is a spherical inflated part at 1/4, and the lower end of the needle is ground. The 16G needle is smoothed (ie, the needle tip is properly blunt to adjust the droplet size so that each milliliter Just 40 drops), each drop is 0.025ml, and the upper end is connected to a small nipple. When adding liquid, make the dropper and the serum plate in a vertical position so as to make the amount of liquid per drop as much as possible.
3. Dilution rod: metal products. The length is about 20cm, the head is made of alloy steel, it is grayish white (oxidized metal), and the surface is slightly rough to achieve the consistency of effectively sucking liquid and sucking liquid. In order to maintain the oxide layer of the stick for a long time, it is sucked. Even if it is not a liquid with bacteria, every time it is used 20 times, the head should be overheated once, but it can be burnt to dark red, but it cannot be burnt to bright red. The head can suck, pipette and mix liquids, and the liquid can be absorbed due to the action of the capillary and the adhesion of the surface. At present, there are 8 longitudinal grooves on the head made in China, and the liquid absorption is 0.025ml.
4. Micro blood oscillator: It is specially designed to shake the serum plate, which makes the elements in the hole mix more evenly.
Experimental procedure
1. Sensitization method of red blood cells
1) Red blood cell hydroformylation: Indirect blood coagulation with fresh red blood cells, not only the model of red blood cell agglutination is fresh, typical, but also sensitive, but the time of sensitized red blood cells preservation is short, and because the cells of different batches or different animal individuals have Certain differences can cause inconsistent test results and affect the analysis of the results. In order to overcome this shortcoming, red blood cells are currently fixed by a hydroformylation method (such as formaldehyde, glutaraldehyde or pyruvaldehyde) to prepare sensitized red blood cells. The hydroformylated red blood cells are stored in an ordinary refrigerator for more than one year, and there is no obvious hemolysis phenomenon. The erythrocyte sensitivity after sensitization is generally not significantly different from that of fresh red blood cells.
Take the blood preserved in the arsenic solution, wash it 3 times with pH 7.2 PBS, each time is 3000r/min 3min, the last time go to the supernatant, take 2.5ml of deposited red blood cells, add 1% glutaraldehyde (take 25% cold Glutaraldehyde 10ml and pH 7.2 PBS 240ml mixed to make) 250ml, hydroformylation in a refrigerator at 4 ° C for 50min, shaking 4 to 5 times in the middle, still dark red after removal, very viscous deposition on the bottom of the centrifuge tube, Must be strong enough to stir, and then washed 3 times with pH 7.2 PBS, each time 3000r / min 3min, the last time to clear, add 0.01% thimerosal pH 7.2 PBS 22.5ml, which is 10% The glutaraldehyde red blood cell solution can be stored in a refrigerator at 4 ° C for a minimum of 1 year.
2) Aldehyde red blood cell citrate: 12ml of 10% hydroformylated red blood cells, add pH 7.2 PBS 8ml, centrifuge at 3000 r / min for 3min, so wash twice, remove the supernatant, add pH 7.2 PBS 4ml, that is 3% red blood cell suspension, add 4ml 1:20 000 citric acid (1/200 citrate diluted with pH 7.2 PBS), warm in a 37 ° C water bath for 15 min, during which time shaking constantly, so that it fully acts, heating is completed Wash twice with pH 7.2 PBS for 3000 min/min for 3 min, decanted the supernatant, and add 4 ml of pH 7.2 PBS, which is 3% citrated red blood cell solution.
3) sensitized red blood cell sensitization: take the antigen supplemented with nasal sputum, dilute it 1:100 with pH 6.4 PBS, add 4ml diluted antigen to 4ml of citrated red blood cell solution, and act for 30min in 37°C water bath. Centrifuge at 3000 r/min for 3 min, remove the supernatant, and add 8 ml of 1% rabbit serum buffered saline, which is a 15% sensitized red blood cell diagnostic solution for indirect hemagglutination (addition of 0.01% thimerosal antiseptic). Save for 0.5 to 1 year. The red blood cell diagnostic solution was used to treat the acidified red blood cells at pH 6.4 PBS as described above.
Method of operation
2. Method of carrying out the reaction: Pipette 1% normal rabbit serum pH 7.2 PBS with a micropipette and drop it into the adjacent two columns of the serum plate, one drop of 0.025ml per well, then take 2 pre-wet bars Head (first through the flame to burn red, cool, put distilled water or physiological saline on the surface of the pre-wet for 1min, be careful not to immerse the entire head in the liquid, otherwise there will be residual liquid, resulting in incorrect results, then use water Paper moisture absorption, calibration) Take 1:100 dilution of the test serum (heating at 56 ° C for 30 min) 0.025 ml, each into the first hole dilution of the upper and lower two columns, carry out the double dilution, twirling The speed of the dilution rod should be appropriate, too slow to mix unevenly, too fast to cause foam, so that the dilution is not accurate, usually 2 to 3 times per second is appropriate, the dilution of the first to seventh holes of the serum after dilution is 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 8th well without serum, was 1% rabbit serum PBS control. Then use a micropipette to absorb the sputum antigen-sensitized red blood cell diagnosing solution and add 0.025 ml to each well of the first column, and then use another micropipette to draw the control and add the citrate cell droplets to the second column. Each well was also 0.025 ml per well. Then place the hemagglutination reaction plate on a micro blood shaker, shake for 3 to 5 minutes, mix well, remove the reaction plate, place it in an enamel plate previously covered with wet gauze, and place it in a 37 ° C incubator. ~ 15h (can also be placed at room temperature in summer), take out the observation results, if necessary, leave it at room temperature overnight, observe the reaction results again, and make a final judgment.
3. Result determination
When judging, the control well should be seen first. Only when the control wells are completely correct, the reaction conditions are normal, and the results of the tested serum columns can be determined.
Precautions
1. Erythrocytes can also be fresh, but the agglutination model is unstable and cannot be preserved for a long time. If it is used now, the consistency of the results of each test cannot be guaranteed.
2. Types of red blood cells used In addition to sheep cells, human O-type red blood cells, and red blood cells of chickens and horses can be used.
3. The dilution stress of the specimen to be inspected should be accurate. Especially when diluting with a dilution rod, the number of rotations or time of the dilution rod should be relatively fixed. Generally, each dilution can be rotated and diluted 50 to 100 times. The dilution rod should try not to touch. And the edge of the serum plate.
4. In the hemagglutination test, the concentration of sensitized red blood cells used has a great influence on the sensitivity of the test results. Generally speaking, when the concentration of red blood cells is high, the end point titer is low, and the red blood cell concentration is low, the end point titer is high, so the red blood cell concentration is high. Strive for accuracy and stability.
5. Normal rabbit serum in the dilution must be extinguished at 56 ° C for 30 min and fully absorbed by sheep red blood cells with the same sensitization number to remove non-specific lectins.
6. After the dilute stick is used, rinse it with water, blot the water with filter paper, burn the stick on the flame, and use it after cooling.
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