Blood clotting inhibition test

1 The scope of application
this standard provides for blood clotting tests for avian influenza and blood clotting inhibition tests.
this standard is applicable to the detection
avian influenza
antibody testing (mainly for
serum
samples).
2 Blood coagulation test
2.1 material and
reagents
2.1.1 equipment: normal
balance
, analytical balance, ordinary
centrifuge
, micro oscillator, syringe, refrigerator, autoculator, trace

and 96-well V blood clotting plate.
2.2.2 pH7.2 phosphate buffer (PBS), the preparation method is shown in Appendix A.
2.2.31% chicken red blood cell suspension, the preparation method is shown in Appendix B.
2.2.4 A's liquid, the preparation method can be found in Appendix C.
2.2.5 Standard avian influenza virus
antigen
, the purchased standard avian influenza virus should test its blood clotting price and carry out sterile testing to check whether it is consistent with the marked HA effect price. If not, repeat the test and go to the relevant laboratory for verification.
2.2 operator
A. Take the 96-hole V-shaped micro-reaction plate and add 25 μL PBS per hole in 1 to 12 holes with a micro-shifter, with a total of 8 rows of drops and 25 μL PBS added to the first column hole in the last four rows.
B. Absorb the standard avian influenza antigen of 25 μL and add it to the first hole, blowing 3 to 5 times to mix well.
C. The antigen fluid after mixing from the first column hole is added to the 2nd column hole, 25 sl is added to the 3rd column hole after mixing, the series ratio is diluted to the 11th column hole in turn, and finally 25 sl is discarded from the 11th column hole, and the 12th column hole is set as a red blood cell control.
D. Add 25 μL 1% of the chicken red blood cell suspension to each hole from right to left.
E. The reaction plate is placed on a trace oscillator oscillating 1 min, room temperature (20-25 degrees C) after 45 min observation results, if the ambient temperature is too high, can be set at 4 degrees C static 60 min, red blood cell control hole into a distinct button-like sink to the bottom of the hole can determine the results.
2.3 result determines the
A. With the correct results in the red blood cell control hole, tilt the reaction plate by 45 degrees to see if the red blood cells are fully condensed. The maximum dilution of the virus with full coagulation is the blood coagulation titration of the antigen. The maximum dilution multiply of the virus with full coagulation is 1 blood clotting unit (HAU).
B. The final result was taken by 2 times and 3 times the geometric mean of the blood coagulation titration of diluted antigens.3 Blood coagulation inhibition test
3.1 materials and reagents
3.1.1 equipment: ordinary balance, analytical balance, ordinary centrifuges, micro oscillators, boiling sterilisers, refrigerators, autoculsive sterilizers, micro-transfusions and 96-well V-shaped blood clotting plates.
appendix A for the preparation method of 3.1.2 pH7.2 phosphate buffer (PBS).
3.1.31% chicken red blood cell suspension preparation method can be found in Appendix B.
3.1.4 A's liquid, the preparation method can be found in Appendix C.
3.1.5 4 units of antigen, the preparation method can be found in Appendix D.
can continue to increase the number of diluted holes when the 3.1.6 HA effect price is higher than 10log2.
3.2 Operator
A. 4 units of antigens are rationed according to the results of the blood clotting test. The maximum dilution of the virus that can cause 100% blood coagulation represents 1 blood clotting unit, and the preparation method of 4 units of antigen is as follows: Assuming that the blood coagulation titration of antigen is 1:256 (28), then the dilution multiplied of 4 units of antigen should be 1:64 (256 divided by 4), and when diluted, 1 mL antigen is added to 63 mL PBS. However, the 4-unit antigen must be validated (e.g. appendix D 4 unit antigen calibration) before its dilution multiply can be determined for the determination of the effect of blood clotting inhibition.
B. Take the 96-well V-shaped micro-reaction plate, add 25 μL PBS each in the 1st to 11th holes with the shifter, and add 50 μL PBS in the 12th hole.
C. Add 25 μL of the serum in the 1st hole, mix well and move out 25 μL to the 2nd hole, and so on, multiply to the 10th hole, discard 25 μL in the 10th hole, set the 11th hole as a virus control, and the 12th hole as a red blood cell control.
D. Add 25 μL 4 units of antigens to each of the 1st to 11th holes, gently tap the reaction plate, so that the reactants mix evenly and set aside 30 min at room temperature of 20-25 degrees C.
E. Add 25 μL 1% of the chicken red blood cell suspension to each hole from right to left.
F. The reaction plate is placed on a trace oscillator oscillating 1 min, room temperature (20-25 degrees C) after 40 min observation results, if the ambient temperature is too high, can be set at 4 degrees C static 60 min, red blood cell control hole into a distinct button-like sink to the bottom of the hole can determine the results.
3.3 result determines the
A. In the case of complete precipitation of red blood cell control and complete condensation of viral control, the reaction plate is tilted by 45 degrees and observed from the back side of the reaction plate. The maximum dilution multiply of the total suppression of red blood cell coagulation was judged to be the blood coagulation inhibition titration of the serum.
B. When the serum tested had an EFFECT of 4log2 or greater than or equal to 4log2, it was found to be positive for avian influenza antibodies.
Appendix A
pH7.2 Phosphate Buffer (PBS) Preparation
Sodium chloride (NaCl)
Potassium chloride (KCl)
Sodium Phosphate (Na2HPO4)
Potassium Phosphate (Phosphate) KH2PO4)
add distilled water to
to dissolve the above ingredients in turn, with hydrochloric acid (HCl) pH to 7.2, divided, 121 degrees C, 15 min autocultension, once used, stored at 4 degrees C for no more than 3 weeks. Appendix B
1% chicken red blood cell suspension preparation
collect at least 3 2 to 6 month-old SPF cocks or non-immune chickens without avian influenza and avian influenza antibodies antibody anticoagulant mixed with the same amount of sodium citrate solution mixed, placed in the centrifuge tube, add 3 to 4 times the volume of PBS mixed, with 1500 r/min centrifugation 5 to 10 min, remove the plasma and white blood cell layer, repeated washing three times, and finally absorb the pressure-accumulated red blood cells with PBS to make a volume fraction of 1% of the suspension, at 4 degrees C to save spare. Glucose

(C6H12O6. H2O)
sodium citrate (Na3C6H5O7.2H2O)
citric acid (C6H8O7.H2O)
sodium chloride (NaCl)
add distilled water to 100 ml, dissolved,
filtration
, divided, 121 degrees C, 30 min autocultension, 4 degrees C to save standby. preparation of appendix D 4 units of antigen
According to the blood coagulation price determined by the blood coagulation test, the dilution multiply of 4 blood coagulation units was determined, and the re-entry test was carried out. For example: the virus 2 times the dilution of the HA efficiency price of 9log2, 3 times the HA efficiency of 8log2, its 4 blood coagulation units of 6log2 to 7log2, the virus in 96 to 128 times dilution.
can be assumed that 90××, 100×, 120×, 130× are 4HAU, 2 × , 3×, 4×, 5×, 6× dilution, each 4 holes, and red blood cells and virus control, according to this to do a blood clotting experiment, so as to determine 4 units of antigen. Half of the 100% coagulation of 4× at the time of determination is 4 units of antigen, 1 100% coagulation indicates that the dilution multiply is large and the dilution multiplied of 3 100% condensation is small.