Avian Influenza Virus (H5 Subtype, Strain H5-Re13) Hemagglutination Inhibition Test Antigen, Positive Sera and Negative Sera

【Product Name】
Avian Influenza Virus (H5 Subtype, Strain H5-Re13) Hemagglutination Inhibition Test Antigen, Positive Sera and Negative Sera
Chinese pinyin: Qinliuganbingdu H5 Yaxing (H5-Re13 zhu) Xueningyizhishiyan Kangyuan,Yangxingxueqing Yu Yinxingxueqing
【Ingredients and Contents】
The antigen is an inactivated H5 subtype avian influenza virus (H5-Re13 strain), and the hemagglutination (HA) titer is not less than 1:128. When measuring chicken serum, use the chicken positive serum of the H5 subtype of avian influenza virus (H5-Re13 strain) and the chicken negative serum in the HI test of avian influenza virus as control serum. The hemagglutination inhibition (HI) antibody titer of the positive serum should not be less than 1 ∶128, and the HI antibody titer of negative serum is not higher than 1:4. When measuring duck and goose serum, the duck positive serum of avian influenza virus H5 subtype (H5-Re13 strain) and the duck negative serum of avian influenza virus HI test are used as control serum, and the HI antibody titer of duck positive serum after treatment is not less than 1:80, and the HI antibody titer of negative serum is less than 1:10.
【Description】The antigen is a white or light yellow spongy loose mass; the positive serum and negative serum are a light yellow or light red spongy loose mass. It is easy to separate from the bottle wall and dissolves quickly after adding diluent.
【Function】Used in HI test to detect antibodies against avian influenza virus H5 subtype (H5-Re13 strain)
【Usage and Determination】
1.2 pH 7.0~7.2 0.01mol/L PBS Prepare as follows or purchase commercial products. Once used, PBS should be stored at 2-8℃ for no more than 3 weeks. PBS preparation method: weigh 80g NaCl, 2g KCl, 36.3g disodium hydrogen phosphate dodecahydrate, and 2.4g potassium dihydrogen phosphate, dissolve in ddH2O and make up to 10L, adjust the pH to 7.0-7.2 with sodium hydroxide or hydrochloric acid, and finally sterilize at 121℃ for 30 minutes or filter before use.
1.3 Alsevers solution: weigh 2.05g glucose, 0.8g sodium citrate, 0.055g citric acid, and 0.42g sodium chloride, add distilled water to 100ml, heat to dissolve and adjust the pH to 6.1, sterilize at 69Kpa for 15min, and store at 2-8°C for later use.
1.4 1% chicken red blood cell suspension: Blood from 2 to 4 SPF chickens aged 2 to 6 months was collected and mixed with an equal amount of Alsevers solution. The blood was then washed 3 to 4 times with PBS (0.01 mol/L, pH 7.0 to 7.2, the same below), each time centrifuged at 1500 r/min for 5 to 10 minutes. The deposited red blood cells were prepared into a 1% suspension with PBS.
1.5 Antigen dissolution: The freeze-dried antigen and serum were dissolved in PBS according to the amount indicated on the bottle label.
2. Operation
2.1 HA Test
2.1.1 In the V-type microreaction plate, 0.025mIPBS was added to each well.
2.1.2 Add 0.025 ml of antigen to the first well and repeatedly pump 3 to 5 times to mix.
2.1.3 Pipette 0.025 ml of antigen from well 1 and add it to well 2. After mixing, pipette 0.025 ml and add it to well 3. Repeat this process of 2-fold serial dilution to well 11. Pipette 0.025 ml from well 11 and discard it.
2.1.4 Add 0.025ml PBS    to every well.
2.1.5 Add 0.025 ml of 1% (V/V) chicken red blood cell suspension to each well.
2.1.6 To judge the results, shake the reaction plate on the oscillator for 1 to 2 minutes or lightly press the reaction plate to mix the reactants, then place it at room temperature for 20 to 30 minutes or at 2-8°C for 45 to 60 minutes. When the red blood cells in the control well become obviously button-shaped, time to determine the result. When judging, tilt the reaction plate 60 degrees and observe whether the red blood cells have teardrop-like flow. The highest dilution factor with no teardrop-like flow (100% agglutination) is judged as the hemagglutination titer.
2.2 HI Test
2.2.1 According to the titer determined by the HA test, if the antigen agglutination titer is 1:1024 (for example), 4 hemagglutination units (4 HAU) = 1024/4 = 256 (1:256). Take 9.0ml of PBS, add 1.0ml of antigen, that is, a 1:10 dilution, add 1.0ml of the 1:10 dilution to 24.6ml PBS, so that the final concentration is 1:256.
2.2.2 Four-unit hemagglutination unit test: To check whether the hemagglutination value of 4 HAU is accurate, the prepared 1:256 dilution should be serially diluted to make the final dilutions 1:2, 1:3, 1:4, 1:5, 1:6 and 1:7. Then, take 0.025ml from each dilution, add 0.025ml PBS, and then add 0.025ml 1% chicken red blood cell suspension and mix well. Place the hemagglutination plate at room temperature for 20-40 minutes or at 2-8°C for 40-60 minutes. If the prepared antigen solution is 4 HAU, a 1:4 dilution will give the agglutination endpoint; if the 4 HAU is higher than 4 units, 1:5 or 1:6 may be the endpoint; if it is lower, 1:2 or 1:3 may be the endpoint. The antigen dilution should be appropriately adjusted according to the test results to ensure that the working solution is indeed 4 HAU.
2.2.3 Add 0.025mI PBS to wells 2 to 11 and 0.05mI PBS to well 12.
2.2.4 Add 0.025ml of serum to the first or second well, mix the serum with PBS in the second well, then inhale 0.025ml into the third well, and dilute the serum to the 10th well, then absorb 0.025ml from the 10th well and discard.
2.2.5 Add 0.025 ml of 4HAU antigen to wells 1 to 11 and let stand at room temperature (20-25°C) for at least 20 minutes or at 2-8°C for at least 60 minutes.
2.2.6 Add 0.025ml of 1% (V/V) chicken red blood cell suspension to each well, shake and mix, and let stand at room temperature (20-25°C) for 20 to 40 minutes or 2-8°C for 40 to 60 minutes. Red blood cells in the control hole. The result is judged when the button shape is obvious.
3 Result determination: The highest serum dilution multiple that completely inhibits 4 HAU antigens is used as the HI antibody titer of the serum. The test is valid only when the HI antibody titer of the positive control serum is within 1 titer of the known antibody titer and the chicken negative control serum antibody titer is not higher than 1:4 (if the treated duck negative serum is used, the antibody titer should be lower than 1:10). When testing chicken serum, the HI antibody titer of the tested serum is not higher than 1:8 and is judged as negative, and not lower than 1:16 and is judged as positive; when testing treated duck and goose serum, the HI antibody titer of the tested serum is lower than 1:10 and is judged as negative, and not lower than 1:10 and is judged as positive.
【Warnings】
(1)There are many factors that affect the HA and HI tests, so the test conditions should be strictly controlled. The pipette tip should be replaced after each reagent or sample is added, and the temperature and time of the reaction should be strictly controlled.
(2) Prepare the red blood cell suspension accurately and shake it frequently during use.
(3) Use PBS (0.01 mol/L) with a pH value of 7.0-7.2 as the diluent.
(4) Antigens and negative and positive sera should be discarded if contaminated.
(5) Accurately prepare 4HAU antigen and titrate it before use. The titrated antigen should be used up within 2 hours.
(6) Antigens and serum should be stored as specified. Freeze-dried reagents should be dissolved in PBS according to the specified volume. After dissolution, store at 2-8°C for no more than 1 month.
(7) If the HI test antigens of different strains of the same subtype have different antigenicity, the HI antibody titers of the same serum will be different.
(8) Duck and goose sera generally need to be treated with nonspecific agglutination inhibitors, and duck positive serum is used as the positive control serum for the HI test. The following treatment methods can be used according to the actual situation of the laboratory.
① Pancreatic enzyme-heat-periodate method
A. Take 0.3ml serum, add 0.15ml pancrease solution (8mg/ml:200mg P-250 pancrease dissolved in 25ml 0.01mol/L PBS with pH value of 7.0-7.2, mix well, filter and remove bacteria, pack and store at -15℃ for 6 months), and mix well. Inactivate in 56℃ water bath for 30 minutes and cool to room temperature.
B. Add 0.9ml potassium periodate (use 230mg KIO4 with 0.01mol/L PBS, PH7.0-7.2, constant volume to 100ml, filter and remove bacteria, and store at room temperature away from light for 1 week), mix, and incubate at room temperature for 15 minutes.
C. Add 0.9ml of glycerol salt solution (add 1ml of glycerol into 99ml of 0.01mol/L PBS with pH value of 7.0-7.2, mix well, filter and remove bacteria, store at room temperature), mix and incubate at room temperature for 15 minutes.
D. Finally add 0.75ml normal saline, mix well, and store at 4℃ for later use. The treated serum ended up being 10 times diluted serum.
② Receptor-destroying enzyme (RDE) treatment
A. Take 1 volume of serum (50ul), add 4 volumes of RDE (200ul), and bathe in 37℃ water for 18 hours (overnight).
B. Add another 5 volumes (250ul) of 1.5% sodium citrate, mix well, and place in a 56℃ water bath for 30 minutes (to destroy residual RDE activity).
C. Add 1 volume of 50% red blood cells to 10 volumes of RDE treated serum (50ul red blood cells +500ul serum), shake and mix, and place at 4°C for 1 hour, during which you can gently shake the suspended red blood cells several times.
D. Then centrifuge 1000g for 10 minutes, carefully absorb the upper supernatant for detection. The treated serum ended up being 10 times diluted serum.
【Specification】(1)1ml/bottle (2)2ml/bottle
【Packaging】10bottles/box
【Storage and Validity】Store below -15℃. Valid for 24 months.
【Approval Number】Shouyaoshengzi 080018907
【Manufacturer】 Harbin Weike Biotechnology Co., Ltd.