GB/T 21870-2008Medical gloves made from natural rubber latex -- Determination of water-extractable protein using the modified Lowry method (English PDF)

5.1 Protein-free Gloves

Gloves that are made from synthetic latex or plastic, powder-free and free of other substances that may transfer to the specimen or extracting solution.

5.2 Centrifuge

It shall reach at least 60,000 m/s2 (6,000 g).

NOTE. temperatures may rise with prolonged centrifugation time; a refrigerated centrifuge is preferred.

5.3 Centrifuge Tubes

Polypropylene or polyethylene (if applicable) centrifuge tubes with low protein adsorption, with a capacity of 200 mL, 50 mL, 10 mL, 2 mL or 1.5 mL.

5.4 Conical Flasks

With a capacity of 250 mL.

5.5 Micropipette.

5.6 Test tube vibrator

With a vibration frequency of 3 Hz ~ 6 Hz.

5.7 Vortex mixer or sonicator.

5.8 Disposable filter membranes

Filter membranes with low protein adsorption and a pore size of 0.45 µm or smaller.

5.9 Clamp holder

A device used to hold the gloves and maintain a watertight seal during the extraction process.

The clamp holder can be a pair of aluminum strips connected to foam rubber and can be fastened together by screws, or a 170 mm long plastic clamp for hemodialysis.

5.10 Spectrophotometric apparatus

5.10.1 Spectrophotometer

Equipped with disposable transparent polystyrene cuvettes (quartz is acceptable but must be very clean).

5.10.2 Microplate reader

A 96-well polystyrene flat-bottom microplate reader with a capacity of 0.25 mL ~ 0.5 mL.

NOTE. a 0.5 mL well plate is preferred; smaller well plates may be used, but this will reduce analytical sensitivity.

5.11 Balance

Accurate to 0.0001 g.

6.3 Modified Lowry protein analytical reagent

6.3.1 Reagent A. alkaline copper citrate solution. Mix 10 portions of Reagent C and 0.2 portions of Reagent D. Prepare on the day of testing.

Alkaline copper tartrate may also be used and shall also be prepared on the day of testing. The complete reagent kit may contain unspecified preservatives that may affect the testing.

6.3.2 Reagent B. a dilute solution obtained by adding 72 mL of 2 mol/L Folin reagent to 28 mL of water.

NOTE. 2 mol/L Folin reagent is commercially available, for example, from Sigma Chemical

Company (Box 14508, St. Louis, MO 63178, USA) (Catalog No. F 9252). Some high- concentration Folin reagents used in industry may not reach 2 mol/L.

6.3.3 Reagent C. 6 g of sodium carbonate dissolved in 100 mL of water.

6.3.4 Reagent D. 1.5 g of copper sulfate and 3 g of sodium citrate dissolved in 100 mL of water.

6.3.5 Sodium hydroxide solution. c(NaOH) = 0.2 mol/L.

6.3.6 Sodium deoxycholate solution (DOC). use water to dissolve 0.15 g of sodium

deoxycholate and dilute to 100 mL. Perform refrigerated storage, and it shall remain valid for

4 weeks.

6.3.7 Trichloroacetic acid (TCA) solution. use water to dissolve 72 g of trichloroacetic acid, dilute to 100 mL, and thoroughly mix it. Refrigerated storage allows for stable preservation for a long period of time.

6.3.8 Phosphotungstic acid (PTA) solution. use water to dissolve 72 g of phosphotungstic acid and dilute to 100 mL. Thoroughly mix it and store it in a refrigerating chamber. It shall remain valid for 4 weeks. For convenience, the TCA and PTA solutions can be pre-mixed simultaneously in equal volumes in accordance with the step of 7.4.2.However, there is no shelf life data for this mixed solution, so it can only be mixed on the day of testing.

6.4 Ovalbumin stock solution. ovalbumin is obtained by fractionation with ammonium sulfate and repeated crystallization at pH = 4.5.For example, it can be obtained from Sigma Chemical

Company (Box 14508, St. Louis, MO 63178, USA) (Catalog No. A 5503).

Dissolve 100 mg of ovalbumin in 100 mL of the extracting solution (6.2) to prepare a stock solution with a concentration of 1 mg/mL. Filter the solution through a 0.45 µm (or smaller pore size) low-protein-adsorption filter membrane (5.8). Use a UV spectrophotometer and a cuvette (with a 1 cm optical path), and at a wavelength of 280 nm, determine the protein absorbance. Use the extracting solution (see 6.2) as a blank. Then, divide the absorbance by

0.641) to obtain the precise concentration of the ovalbumin stock solution. At or below 7 °C, the solution can be stably stored for 48 hours, or frozen at -10 °C for 2 months. Thawing requires heating to 45 °C and holding for 15 minutes.

NOTE. refrigeration time is cumulative. To avoid repeated freezing and thawing, it is recommended to store the protein stock solution in several portions, each sufficient for preparing a calibration curve or for use in the validation test procedure (see Appendix A).

7.1 Principle

The test procedures include extraction of the entire glove, purification and concentration of the extracting solution. Use diluent of the protein stock solution (see 6.4 and 7.3) concentrated using the same method to draw the standard curve. Determine the protein concentration in the extracting solution against the standard curve. The analytical skills of the analyst must be validated in accordance with Appendix A.

Take 3 gloves or 3 pairs of gloves from a given batch to perform parallel determination by 3 portions. Each extracting solution is independently purified, concentrated, and finally determined.

7.2 Extraction

7.2.1 Overview

At (25 ± 5) °C, fully unfold the glove surface and extract in the extracting solution for (120 ±

5) min. Extraction can be performed using either the “cut-glove” or “glove-in-glove” extraction method. The extraction method used must be stated in the test report. All samples of the same type shall be extracted using the same method. During extraction, 3 parallel tests shall be performed, and each extracting solution shall be separately determined.

During the extraction process, wear protein-free gloves (see 5.1) to handle the test glove samples.

NOTE. sampling and the distinction between left and right gloves are beyond the scope of this

1) The exact value of the extinction coefficient of ovalbumin has been confirmed.

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