WS/T 630-2018Detection of antibody against Schistosoma japonicum - Indirect haemagglutination test (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard was proposed by the National Health Standards Committee Parasitic Diseases Standards Committee.

This standard was drafted. Anhui Institute of Schistosomiasis Control, Chinese Center for Disease Control and Prevention, Institute of Parasitic Diseases Prevention and Control, Zhejiang Institute of Parasitic Diseases, Provincial Academy of Medical Sciences, Jiangsu Institute of Schistosomiasis Control, Jiangxi Institute of Parasitic Diseases.

1 Scope

This standard specifies an indirect red blood cell agglutination test for the detection of Schistosoma japonicum antibodies.

This standard applies to the detection of Schistosoma japonicum antibodies in human serum by disease prevention and control institutions and medical institutions at all levels.

2 Terms and definitions

The following terms and definitions apply to this document.

Schistosomiasis A disease caused by schistosomiasis parasitic on humans and other mammals, specifically referred to as schistosomiasis in Japan (schistosomiasis) Japonica).

Note. Rewrite GB 15976-2015, definition 3.1 2.2 Indirect haemagglutination test the antigen is coated on the surface of the red blood cells to become a sensitized carrier, and then binds to the corresponding antibody, and is bridged by the antibody, and the naked eye can be seen.

See the agglutination phenomenon to detect the corresponding antibodies in the sample. The most commonly used red blood cells are healthy human "O" type red blood cells.

Soluble egg antigen

The supernatant of the schistosomiasis eggs is treated by homogenization and sonication, and the supernatant is collected by centrifugation, containing various components such as protein, glycoprotein and polysaccharide.

Compound.

3 Instruments

4 Reagent materials

5 Detection steps

6 Result determination

6.1 See Table C for the criteria for the determination of results. Negative control serum did not show agglutination, and the positive control serum showed agglutination.

It is considered valid.

6.2 The highest dilution of serum agglutinated with "+" or above as the serum titer, with a titer of >=1.10 as the positive criterion quasi.

WS/T 628-2018

Appendix A

(informative appendix) Main reagent material preparation method A.1 freeze-dried sensitized red blood cells A.1.1 soluble egg antigen Take the freeze-dried eggs, prepare a suspension of 0.01% thimerosal chloride, stir the tissue homogenizer and freeze and thaw for 3,000 °C for 3 times, 10 000 G centrifuge for 30 min and take the supernatant. After measuring the protein content, it was diluted to 0.75 mg/mL with thiomersal physiological saline and dispensed into a vial.

Store at -20 °C for later use.

A.1.2 Aldolized red blood cells A.1.2.1 Take healthy human "O" type red blood cells, wash them with physiological saline for 3 times, transfer them to a graduated centrifuge tube, centrifuge at 800 g for 10 min, and record the pressure.

Red blood cell volume.

A.1.2.2 Take the packed red blood cells, mix with 25 mL of pH 7.2PBS per 1 mL, then slowly add 2 mL of 2.5% glutaraldehyde, and shake while shaking.

Shake for 1 h at room temperature, wash 3 times with PBS, and wash twice with distilled water. Finally, 10% hydroformylated red cell suspension was prepared with 0.01% thimerosal chloride normal saline.

The solution is set at 2 °C ~ 8 °C for use.

A.1.3 Deuterated red blood cells A.1.3.1 Prepare normal saline containing 0.01% citric acid and mix with an equal amount of 0.1 mol/L pH 7.2 PBS.

A.1.3.2 Take the pressured aldolized red blood cells and pour into the above solution to prepare a concentration of 1%, and shake it in a constant temperature water bath shaker at 37 °C for 15 min.

Wash 3 times with pH 7.2 PBS.

A.1.3.3 Pour the washed red blood cells into 0.1 mol/L pH 6.4 PBS solution to make a concentration of 1%, mix well, that is, reddened red Cell.

A.1.4 Sensitized red blood cell suspension A.1.4.1 Add a soluble egg antigen with a final concentration of 0.25% to the red blood cell suspension, mix well, and shake at 37 °C in a constant temperature water bath.

Shake for 45 min in the device.

A.1.4.2 Centrifugation and sedimentation The supernatant was discarded and washed with pH 7.2 PBS containing 1% normal rabbit serum (sera after inactivation at 56 °C for 30 min).

The polyester was precipitated to remove unbound soluble antigen, and a 4% concentration of sensitized red blood cell suspension was prepared in pH 7.2 PBS containing 1% normal rabbit serum.

A.1.5 freeze-dried sensitized red blood cells Sucrose (10% final concentration) and normal rabbit serum (2% final concentration) were added to the sensitized red blood cell suspension. Shake well and dispense in 2 mL Within the ampoule, each 0.5 mL. The dispensed sensitized red blood cells were placed in a freeze dryer and lyophilized, and sealed with a sealing machine. freeze the appearance of dry sensitized red blood cells is brown or light red loose body. The indirect hemagglutination method determines the serum titer of the positive control is not less than 1.640, and the determination is negative.

The control serum titer was not higher than 1.5.

A.2 positive control serum WS/T 628-2018 Healthy rabbits weighing 2.5 kg to 3.0 kg were selected, each infected with 800 to 1 000 Schistosoma japonicum cercariae, after 60 days of feeding, Whole blood of the heart, serum was separated, mixed, and then lyophilized in a microcentrifuge tube in 0.1 mL each, as a positive control serum. Positive control serum the test titer is not less than 1.640.

A.3 negative control serum Healthy rabbits weighing 2.5 kg to 3.0 kg were selected, whole blood was taken, serum was separated, and serum without hemolysis was selected to inactivate complement (56 ° C).

Inactivated for 30 min), and then lyophilized according to each 0.1 mL microcentrifuge tube as a negative control serum. Negative control serum test titer not higher than 1.5.

A.4 diluent

Weigh 35.8 g of disodium hydrogen phosphate dodecahydrate, 8.5 g of sodium chloride, add deionized water (or distilled water) to 1 000 mL, and weigh it into A solution;

Take 6.8 g of potassium dihydrogen phosphate, 8.5 g of sodium chloride, add deionized water (or distilled water) to 1 000 mL, and weigh B. Press liquid A. liquid B is 72.28 the ratio of the mixture is pH 7.2 phosphate buffer.

WS/T 628-2018

Appendix B

(informative appendix) Operating procedure B.1 Schematic diagram of the operation steps is shown in Figure B.1.

a) sample number

b) Negative, positive control serum dilution

c) blood clot plate plus diluent

d) Dilution of the sample to be tested

e) Freeze-dried sensitized red blood cell dilution and detection

Figure B.1 Schematic diagram of the operation steps

WS/T 628-2018

B.2 Operation Precautions B.2.1 Sample collection and preservation. The peripheral blood is generally collected by polyethylene plastic tube. The plastic tube has a hole diameter of 2 mm and a length of 10 cm. Venous blood Generally, disposable vacuum blood collection tubes are used for collection. Serum separation should be performed within 4 h after whole blood collection. Serum samples should not be mixed with red blood cells or be Bacterial contamination, serum should be no less than 25 µL. If the serum sample cannot be detected in time, it can be stored at 2 °C ~ 8 °C for 3 d ~ 5 d for a long time.

Storage should be stored frozen below -20 °C.

B.2.2 Reagent preparation. If the test reagent is taken out from the refrigerator, it should be equilibrated for 30 min at room temperature before use.

B.2.3 Loading. Use a pipette to load the sample. The nozzle should be replaced every time one sample is tested. In the diluted serum to be tested, avoid mixing.

Air bubbles are generated (mixing and squeezing are performed in the first gear) to affect the accuracy of the liquid absorption.

B.2.4 Sensitized red blood cell suspension. The prepared red blood cell suspension should be thoroughly mixed before use. In the case of multi-sample operation, it is not necessary to add the suspension.

The suspension is mixed to avoid the erythrocyte sinking in the suspension and affect the accuracy of the results.

B.2.5 After the sample is added, the hemagglutination reaction plate should be sealed (with a glass plate or a disposable sealing film or placed in a sealed wet box) before being placed at 37 °C.

Electric heated water bath.

B.2.6 Observation of results. The results should be observed within 10 minutes; when the results are observed, it is not appropriate to shake the reaction plate to avoid agglutination and dispersion, affecting the knot Judging.

B.2.7 Cleaning of the blood coagulation reaction plate. The 96-well "V" type plexiglass plate should be used for the blood coagulation reaction plate, and should be cleaned in time after use. Not when cleaning it can be scrubbed with sharp objects or soaked in acid and alkali solution. It can be washed with a washer or high-pressure tap water, and then washed once with distilled water for 1 or 2 times.

After drying, it is placed in a 37 ° C incubator for drying.

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Appendix C

(normative appendix) Result criteria C.1 Negative reaction. All red blood cells sink at the bottom of the hole, forming small dots with tight edges and smooth edges. The reaction intensity is indicated by "-".

C.2 Positive reaction. agglutination reaction with different degrees of red blood cell appearance. The reaction intensity is expressed by "+" "++" and "+++".

Most of the "+" red blood cells are collected at the bottom of the hole, forming a dot, surrounded by a small amount of agglutinated red blood cells, and the naked eye sees the surrounding blur (or in the middle Now more obvious blank spots).

Most of the "++" red blood cells formed a thin layer of agglutination around the bottom of the well, and a small number of red cells sinking in the center of the bottom of the well showed small dots.

The "+++" red blood cells form a thin layer of agglutination, which fills the entire bottom of the hole and has irregular wrinkles at the edges.

C.3 The agglutination pattern of different reaction intensity is shown in Figure C.1.

Figure C.1 Different reaction intensity agglutination pattern

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references

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