ISO 12243:2003
(Main)Medical gloves made from natural rubber latex — Determination of water-extractable protein using the modified Lowry method
Medical gloves made from natural rubber latex — Determination of water-extractable protein using the modified Lowry method
ISO 12243:2003 specifies a method for the determination of the amount of water-extractable protein in natural rubber (NR) gloves for medical use. The method is potentially 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 (these are described in an annex) 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.
Gants médicaux à base de latex de caoutchouc naturel — Détermination des protéines extractibles par l'eau par la méthode modifiée de Lowry
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INTERNATIONAL ISO
STANDARD 12243
First edition
2003-10-01
Medical gloves made from natural rubber
latex — Determination of
water-extractable protein using
the modified Lowry method
Gants médicaux à base de latex de caoutchouc naturel —
Détermination des protéines extractibles par l'eau par la méthode
modifiée de Lowry
Reference number
©
ISO 2003
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ii © ISO 2003 — All rights reserved
Contents 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
Annex 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
Foreword
ISO (the International Organization for 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 the 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.
International 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 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.
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.
iv © ISO 2003 — All rights reserved
Introduction
There have been problems of allergic reactions experienced by some users 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)
Medical gloves made from natural rubber latex — Determination
of water-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 establish 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 potentially 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 (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
The 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 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-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).
4 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 solution
3 3
NOTE Thus if the protein in 4 cm of solution is precipitated and redissolved in 0,8 cm , 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
4.3
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.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 synthetic 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 (6 000 × g).
NOTE A refrigerated centrifuge is preferred as it is possible for the temperature to rise considerably when
centrifugation is carried out for prolonged periods.
3 3 3 3 3
5.3 Centrifuge tubes, capacity 200 cm , 50 cm , 10 cm , 2 cm and 1,5 cm , made of polypropylene or
polyethylene (if available) with a low protein-binding capacity.
5.4 Conical flasks, capacity 250 cm .
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 µm or less.
5.9 Clamps, for sealing gloves watertight during extraction. Pairs of aluminium bars lined with foam rubber
which can be screwed together, or 170-mm-long plastic clips as used for haemodialysis, are suggested.
2 © ISO 2003 — All rights reserved
5.10 Spectrophotometric equipment.
5.10.1 Spectrophotometer, with disposable polystyrene cuvettes (quartz cuvettes may be used but require
careful cleaning).
Or
5.10.2 Microplate reader, with flat-bottom polystyrene microtitre plates having 96 wells of 0,25 cm to
0,5 cm capacity.
NOTE Wells with a capacity of 0,5 cm are preferred. Wells with a smaller capacity may be used but will reduce the
sensitivity of the assay.
5.11 Balance, 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 Extractant 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 buffer is prepared by dissolving a PBS
tablet in distilled water in accordance with the manufacturer’s 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 dissolving 24 g of TES in 500 cm of 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 copper 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.
3 3
6.3.2 Reagent B: Dilute Folin reagent prepared by diluting 72 cm of 2 N Folin reagent with 28 cm of water.
NOTE 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 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 of 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 water to 100 cm . 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 with
water and mixing thoroughly. Store the solution in a refrigerator. The solution is stable over a long period.
6.3.8 Phosphotungstic acid (PTA) solution, prepared by diluting 72 g of phosphotungstic acid to 100 cm
with water and mixing thoroughly. Store the solution in a refrigerator, discarding it after 4 weeks.
It may be convenient to premix the TCA and PTA 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 A 5503 from Sigma Chemical Co., Box 14508, St Louis, MO 63178, USA.
Prepare a solution of 100 mg of ovalbumin in
...
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