1、 Reference number ISO 12243:2003(E) ISO 2003INTERNATIONAL STANDARD ISO 12243 First edition 2003-10-01 Medical gloves made from natural rubber latex Determination of water-extractable protein using the modified Lowry method Gants mdicaux base de latex de caoutchouc naturel Dtermination des protines e
2、xtractibles par leau par la mthode modifie de Lowry ISO 12243:2003(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and instal
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6、ng from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2003 All rights reservedISO 12243:2
7、003(E) ISO 2003 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 Principle . 1 4 Terms and definitions. 2 5 Apparatus. 2 6 Reagents 3 7 Procedure. 4 8 Calculation of results 7 9 Precision 8 10 Test report 9 Annex A (normative) Verification 11 Ann
8、ex B (normative) Protein adsorption on polypropylene and polyethylene tubes. 13 Annex C (informative) Alternative methods of analysis . 14 Annex D (informative) Background subtraction 15 Bibliography . 17 ISO 12243:2003(E) iv ISO 2003 All rights reservedForeword ISO (the International Organization f
9、or Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has th
10、e right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Internat
11、ional Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication
12、 as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
13、 ISO 12243 was prepared by Technical Committee ISO/TC 45, Rubber and rubber products, Subcommittee SC 3, Raw materials (including latex) for use in the rubber industry. ISO 12243:2003(E) ISO 2003 All rights reserved vIntroduction There have been problems of allergic reactions experienced by some use
14、rs of medical gloves manufactured from natural rubber latex. ISO 12243 specifies a method for the determination of the water-extractable protein in such gloves. INTERNATIONAL STANDARD ISO 12243:2003(E) ISO 2003 All rights reserved 1Medical gloves made from natural rubber latex Determination of water
15、-extractable protein using the modified Lowry method WARNING Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to es
16、tablish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. 1 Scope This International Standard specifies a method for the determination of the amount of water-extractable protein in natural rubber (NR) gloves for medical use. The method is poten
17、tially suitable for the determination of extractable protein in other articles made from NR latex; however the extraction procedures and times have not been validated and will vary with the type of article to be tested. Other methods for the determination of specific proteins in medical gloves exist
18、 (see Annex C) but they are not of general applicability. This International Standard is concerned solely with the method of assay. It is not concerned with sampling nor does it purport to address the safety implications of the values obtained or requirements for labelling. 2 Normative references Th
19、e following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 10282:2002, Single-use sterile rubber surgical
20、gloves Specification ISO 11193-1:2002, Single-use medical examination gloves Part 1: Specification for gloves made from rubber latex or rubber solution 3 Principle Water-soluble proteins are extracted into a buffer solution and then precipitated to concentrate them and separate them from other water
21、-soluble substances which may interfere with the determination (see Annexes A and D). The precipitated protein is redissolved and quantified colorimetrically by the modified Lowry method using a protein standard (for a general review of the method, see reference 1 in the Bibliography). ISO 12243:200
22、3(E) 2 ISO 2003 All rights reserved4 Terms and definitions For the purposes of this document, the following terms and definitions apply. 4.1 concentration factor F extent to which a protein extract is concentrated by precipitation followed by redissolution in a smaller volume of sodium hydroxide sol
23、ution NOTE Thus if the protein in 4 cm 3of solution is precipitated and redissolved in 0,8 cm 3 , then the concentration factor F would be 4/0,8 (= 5). 4.2 protein proteins and protein-like substances (e.g. polypeptides) occurring in articles made from NR latex and which are extractable with water 4
24、.3 modified Lowry method modification of the original Lowry assay method, which involves the precipitation and isolation of the proteins to reduce the level of other water-extractable substances that may interfere in the determination 5 Apparatus Unless otherwise stated, all laboratory equipment (i.
25、e. flasks, tubes, etc.) shall be made of polypropylene or polyethylene. NOTE Polypropylene or polyethylene equipment is specified rather than glass to minimize surface adsorption. A method for the determination of protein-binding capacity is described in Annex B. 5.1 Protein-free gloves, made from s
26、ynthetic rubber latex or plastic and that are free of powder and other materials capable of being transferred to the test samples or extractant solutions. 5.2 Centrifuge, capable of reaching not less than 60 000 m/s 2(6 000 g). NOTE A refrigerated centrifuge is preferred as it is possible for the te
27、mperature to rise considerably when centrifugation is carried out for prolonged periods. 5.3 Centrifuge tubes, capacity 200 cm 3 , 50 cm 3 , 10 cm 3 , 2 cm 3and 1,5 cm 3 , made of polypropylene or polyethylene (if available) with a low protein-binding capacity. 5.4 Conical flasks, capacity 250 cm 3
28、. 5.5 Micropipettes. 5.6 Test tube shaker, operating at between 3 Hz and 6 Hz. 5.7 Vortex mixer or ultrasonic bath. 5.8 Disposable filter, with a low protein-binding capacity and a pore size of 0,45 m or less. 5.9 Clamps, for sealing gloves watertight during extraction. Pairs of aluminium bars lined
29、 with foam rubber which can be screwed together, or 170-mm-long plastic clips as used for haemodialysis, are suggested. ISO 12243:2003(E) ISO 2003 All rights reserved 35.10 Spectrophotometric equipment. 5.10.1 Spectrophotometer, with disposable polystyrene cuvettes (quartz cuvettes may be used but r
30、equire careful cleaning). Or 5.10.2 Microplate reader, with flat-bottom polystyrene microtitre plates having 96 wells of 0,25 cm 3to 0,5 cm 3capacity. NOTE Wells with a capacity of 0,5 cm 3are preferred. Wells with a smaller capacity may be used but will reduce the sensitivity of the assay. 5.11 Bal
31、ance, accurate to 0,000 1 g. 6 Reagents During the assay, use only reagents of recognized analytical grade and distilled or deionized water. 6.1 Dye solution: Bromophenol blue, sodium salt, prepared by dissolving 0,1 g of bromophenol blue in 1 l of water. Discard the solution after four weeks. 6.2 E
32、xtractant solution: A buffer solution capable of maintaining pH 7,4 0,4 throughout the extraction. NOTE 1 Suitable buffers include phosphate buffer saline (PBS) solution (0,01 mol/l) and N-tris-(hydroxymethyl)-methyl- 2-amino-ethanesulfonic acid hemisodium salt (TES) solution (0,1 mol/l). The PBS bu
33、ffer is prepared by dissolving a PBS tablet in distilled water in accordance with the manufacturers instructions. In the event that, at the conclusion of the extraction, pH 7,4 0,4 is not achieved, it would be necessary to use a more concentrated buffer solution. The TES solution is prepared by diss
34、olving 24 g of TES in 500 cm 3of water and making the volume up to 1 l. NOTE 2 PBS tablets and TES are widely available. 6.3 Modified Lowry protein assay reagents 6.3.1 Reagent A: Alkaline copper citrate, prepared fresh daily by mixing 10 parts of reagent C with 0,2 parts of reagent D. Alkaline copp
35、er tartrate is also considered to be suitable. It shall also be prepared fresh daily. The material available in kits can contain undeclared preservatives which may affect the determination. 6.3.2 Reagent B: Dilute Folin reagent prepared by diluting 72 cm 3of 2 N Folin reagent with 28 cm 3of water. N
36、OTE 2 N Folin reagent is available commercially. It can, for example, be obtained from Sigma Chemical Co. (Catalogue No. F 9252), Box 14508, St Louis, MO 63178, USA. The concentration of some commercial Folin reagents may not be 2 N. 6.3.3 Reagent C: A solution of 6 g of sodium carbonate in 100 cm 3
37、of water. 6.3.4 Reagent D: A solution containing 1,5 g of copper sulfate and 3 g of sodium citrate in 100 cm 3of water. 6.3.5 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l. 6.3.6 Sodium deoxycholate (DOC) solution, prepared by dissolving 0,15 g of sodium deoxycholate in water and diluting with wate
38、r to 100 cm 3 . Store the solution in a refrigerator, discarding it after 4 weeks. 6.3.7 Trichloroacetic acid (TCA) solution, prepared by diluting 72 g of trichloroacetic acid to 100 cm 3with water and mixing thoroughly. Store the solution in a refrigerator. The solution is stable over a long period
39、. ISO 12243:2003(E) 4 ISO 2003 All rights reserved6.3.8 Phosphotungstic acid (PTA) solution, prepared by diluting 72 g of phosphotungstic acid to 100 cm 3with water and mixing thoroughly. Store the solution in a refrigerator, discarding it after 4 weeks. It may be convenient to premix the TCA and PT
40、A solutions in equal volumes and to add them simultaneously in 7.4.2. Such a mixture shall be prepared daily in the absence of data on its storage life. 6.4 Ovalbumin protein stock solution. Use ovalbumin prepared by ammonium sulfate fractionation and repeated crystallization at pH 4,5 such as Sigma
41、 A 5503 from Sigma Chemical Co., Box 14508, St Louis, MO 63178, USA. Prepare a solution of 100 mg of ovalbumin in 100 cm 3of the preferred extractant (6.2) to give a concentration of 1 mg/cm 3 . Filter the solution through a low-protein-binding filter of 0,45 m or smaller pore size and determine the
42、 absorbance at 280 nm using a UV spectrophotometer with a 1 cm path length cuvette and employing extractant solution (6.2) as a blank. Divide the absorbance by 0,64 1)to obtain the precise concentration of the ovalbumin stock solution. The solution is stable for 2 days when stored at a temperature o
43、f not more than 7 C or for 2 months frozen at 10 C. Thawing requires heating to 45 C for 15 min. NOTE The length of time under refrigeration is cumulative. In order to avoid repeated thawing and freezing, it is recommended that the stock solution be stored as aliquot portions each sufficient for the
44、 preparation of a single calibration curve or for use in the verification procedure (see Annex A). 7 Procedure 7.1 Principle The procedure involves the extraction of a whole glove followed by purification and concentration of the extract. The concentration of protein in the extract is determined by
45、reference to a standard calibration curve prepared using dilutions of the protein stock solution (6.4 and 7.3) which has been concentrated in the same manner. The analytical technique of the analyst must previously have been verified as described in Annex A. The extraction is run in triplicate using
46、 three gloves or pairs of gloves from a given lot; the purification and concentration of each extract and the subsequent determination are run singly. 7.2 Extraction procedure 7.2.1 General The entire surface of the glove shall be exposed to the extractant at 25 C 5 C for a period of 120 min 5 min.
47、Two extraction procedures are permitted, the so-called “cut-glove” procedure and also the “glove-in-glove” procedure. The procedure used shall be noted in the test report and all samples in a given series shall be extracted by the same procedure. The extraction shall be carried out in triplicate and
48、 single determinations run on each extract. Use protein-free gloves (5.1) to handle the glove samples used for the extraction. NOTE The frequency of sampling and left- or right-handedness of gloves are outside the scope of this document. 7.2.2 Procedure A Cut-glove procedure 7.2.2.1 Record the mass
49、of the glove (m) to an accuracy of not less than 0,001 g. 1) The precise value of the extinction coefficient of ovalbumin is subject to confirmation. ISO 12243:2003(E) ISO 2003 All rights reserved 57.2.2.2 Cut the glove along the periphery. To facilitate the extraction, it is permissible to cut the glove into smaller pieces (but see 7.2.2.3). 7.2.2.3 If the result is to be reported in micrograms per unit area of the glove (e.g. g/dm 2 ), determine the surface area of the glove as follows: Cut a rectangular
