YY/T 0616.7-2020Medical gloves for single use - Part 7: Test method for the immunological measurement of antigenic protein (English PDF)

7.1 Reagents

7.1.1 Buffer

7.1.1.1 Carbonate buffer

Weigh with a balance and combine 0.795g sodium carbonate (Na2CO3), 1.465g sodium bicarbonate (NaHCO3), and 0.100g sodium azide (NaN3) is dissolved in distilled water and the volume is set to 500mL. Check the pH and adjust if necessary.

Note. The carbonate buffer can be stored at (4 ± 3) °C for at least 1 month.

7.1.1.2 PBS buffer

Use a balance to weigh 5.125g sodium dihydrogen phosphate (NaH2PO4·H2O), 45,000g disodium hydrogen phosphate (Na2HPO4·7H2O) and Dissolve in 1.5L distilled water and adjust the pH to 7.4. Add 175.3g of sodium chloride (NaCl) and dilute to 2L, which is 10 times the stock solution.

Use distilled water to dilute the 10-fold stock solution to PBS working solution before use.

7.1.1.3 T-PBS washing solution

Add 0.5mL Tween 20 (Tween 20) to 1LPBS working solution and mix well.

7.1.2 Blocking solution and antibody diluent

7.1.2.1 Sealing fluid

T-PBS containing 3g/mL skimmed milk powder, 100mL (used for the sealing treatment of the measuring plate and the dilution plate).

7.1.2.2 Diluent

T-PBS with 0.2g/mL skimmed milk powder, 100mL (used for blocking in antibody dilution and competitive inhibition steps).

7.1.3 Reference reagents

A NRL protein solution with a concentration of 1 mg/mL prepared with distilled water. The polypropylene tube is stored in (-20±10)°C, after thawing Store at (4±3)°C.

Note. The commercially available StAg standard solution is optional for IRM-913.

7.1.3.2 Coating antigen

The StAg solution was formulated into a solution with a concentration of 3µg/mL with carbonate buffer, which was used to coat the assay plate.

7.1.3.3 Reference standard

The StAg solution was formulated into a solution with a concentration of 2µg/mL with the diluent for competitive inhibition. In order to reduce possible protein loss, all steps involving protein-containing solutions should be performed in polypropylene tubes or containers. Avoid using polystyrene or glass containers.

7.1.4 Antibodies

7.1.4.1 Primary antibody

The commercially available anti-NRL antibody IRM-914 can be used.

7.1.4.2 Secondary antibodies

Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG is used to detect the primary antibody bound to NRL protein on the solid phase. Recommended diluent Dilute 1.5 and store in aliquots at (-20±10)°C.

7.1.5 Substrate color developing solution

O-phenylenediamine (OPD) and hydrogen peroxide (H2O2) can be used for yellow color reaction. Dissolve commercially available OPD tablets in distilled water, And add an appropriate amount of H2O2 according to the operating instructions. For example, dissolve 10mgOPD tablets in 10ml distilled water and add it before use 30µL30% H2O2.

Note. TMB kit can also be used for color development.

7.2 Consumables

96-well microassay plate; dilution plate, 96-well plate with low protein binding for sample dilution and antibody reaction; multi-channel pipette; ELISA Plate sealing tape or plastic cover.

8.1 Sample preparation

8.1.1 It is advisable to use the PBS working solution of 7.1.1.2 as the extract.

8.1.2 Weigh the natural rubber latex gloves, and record the mass and surface area; 5 mL of extraction medium should be added to each gram of natural rubber latex gloves. Extraction temperature The temperature is (25 ± 5) °C; the extraction time is (120 ± 5) min. The extraction process should be agitated continuously to ensure that the sample is in full contact with the extraction medium.

Or oscillate 3 times at the beginning, middle and end stages of the extraction process, with an interval of 15s/time.

Filter the low-protein binding 0.45µm filter to collect the extract into a polypropylene tube.

Note 1.The extraction is carried out in a polypropylene container to reduce the possibility of protein loss due to adsorption to the inner surface of the container wall.

Note 2.Generally, the obtained extract can be used immediately, but it can be stored at (4 ± 3) °C for up to 2 days.

8.2 Test procedure

8.2.1 Closure of the dilution plate

Add 300µL of blocking solution to each well of the 96-well dilution plate, and incubate overnight at (4±3)°C.

8.2.2 Coating of the measuring plate

Add 100µL of coating antigen (StAg, 3µg/mL) to each well of the 96-well assay plate, and seal the plate with ELISA plate sealing tape or plastic lid.

Incubate at (37±1)°C for (120±5)min. Wash the plate once with T-PBS and discard the supernatant. Or coat the antigen at (4±3)°C overnight, and in the follow-up T-PBS washes the plate once before the start of the test. Add 300µL of blocking solution to each well and seal the plate, and incubate at (37±1)°C in a thermostat for 1h.

8.2.3 Inhibition steps

8.2.3.1 In the 96-well dilution plate, wash the plate twice with T-PBS; as shown in Table 1, except for row A, add 100µL of dilution buffer to each well; add wells A1 and A2 to row A Add.200 µL of antigen standard (StAg, 2 µg/mL) to each well, and add.200 µL of test sample to wells A3 to A12 (at least 2 replicate wells for each sample); by taking 100 µL/well from row A to each well of row B, and then Sequentially from row B to row G, perform 7 serial dilutions of the antigen standard or sample to be tested (mix by pipetting up and down 5 times each time, and finally discard it after mixing in row G)

100µL/well); H behavior has no inhibition, and each well contains 100µL diluent.

Note. Each sample to be tested has 3 dilution multiples (at least 2 dilution multiples), and each 1 dilution multiple has 2 replicates.

8.2.3.2 Prepare an appropriate dilution of the primary antibody so that the maximum OD value of the "uninhibited" well is 0.8 to 2.0. According to Table 1, add the primary antibody 100µL/hole, none...