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
General Information
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Standards Content (Sample)
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 12243:2003(E)
©
ISO 2003
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ISO 12243:2003(E)
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ISO 12243:2003(E)
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
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ISO 12243:2003(E)
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.
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ISO 12243:2003(E)
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.
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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).
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ISO 12243:2003(E)
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.
2
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.
3
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.
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ISO 12243:2003(E)
5.10 Spectrophotometric equipment.
5.10.1 Spectrophotometer, with disposable polystyrene cuvettes (quartz cuvettes may be used but require
careful cleaning).
Or
3
5.10.2 Microplate reader, with flat-bottom polystyrene microtitre plates having 96 wells of 0,25 cm to
3
0,5 cm capacity.
3
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
3
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.
3
6.3.3 Reagent C: A solution of 6 g of sodium carbonate in 100 cm of water.
3
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
3
water and diluting with water to 100 cm . Store the solution in a refrigerator, discarding it after 4 weeks.
3
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.
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ISO 12243:2003(E)
3
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.
3
Prepare a solution of 100 mg of ovalbumin in 100 cm of the preferred extractant (6.2) to give a concentration
3
of 1 mg/cm . Filter the solution through a low-protein-binding filter of 0,45 µm or smaller pore size and
determine the absorbance at 280 nm using a UV spectrophotometer with a 1 cm path length cuvette and
1)
employing extractant solution (6.2) as a blank. Divide the absorbance by 0,64 to obtain the precise
concentration of the ovalbumin stock solution. The solution is stable for 2 days when stored at a temperature
of 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 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 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 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. 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 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 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.
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ISO 12243:2003(E)
7.2.2.2 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).
2
7.2.2.3 If the result is to be reported in micrograms per unit area of the glove (e.g. µg/dm ), determine the
surface area of the glove as follows:
Cut a rectangular piece from the back of the glove of about 0,5 dm by 0,5 dm and measure its dimensions
accurately. Calculate the area A .
1
Determine the mass (m ) of the rectangular piece to the nearest 0,001 g.
p
The total surface area A of both sides of the glove is given by A = 2A × m/m .
1 p
7.2.2.4 Transfer all the pieces of the glove to a suitable conical flask (5.4).
7.2.2.5 Add accurately a volume V of extractant (6.2). The total volume V of extractant used shall be
3 3
between 10 cm and 15 cm per gram of glove and sufficient to cover the pieces.
7.2.2.6 Extract the test sample at 25 °C ± 5 °C for 120 min ± 5 min, shaking for 15 s initially and
thereafter at intervals not greater than 30 min. If practical, continuous slow shaking is desirable.
7.2.2.7 Decant off the extract and remove any particulate matter by centrifuging at not less than
2
20 000 m/s (2 000 × g) for 15 min. The extract is preferably used immediately but may be stored for up to
48 h at a temperature of not more than 7 °C or frozen for up to 15 days at below −10 °C.
7.2.3 Procedure B — Glove-in-glove procedure
7.2.3.1 Take two gloves and determine the mass of each one to an accuracy of not less than 0,001 g (m
1
and m ). Mark each glove at a point on the cuff 20 cm from the tip of the middle finger. Take one glove and
2
insert it inside the other so that they fit together (this can be done conveniently using rods to insert the thumb
into the thumb, etc.; however, the method of doing this is not critical as long as the gloves are exposed to
minimum handling). Repeat the process with two further pairs of gloves of the same size.
3
7.2.3.2 Pour sufficient dye solution (6.1) into the inner glove to fill all of the fingers. Introduce 25 cm of
extractant (6.2) between the inner and outer glove. Manipulate gently to remove any air bubbles and seal the
gloves with a clamp (5.9) at the 20 cm mark.
7.2.3.3 Fix the gloves to a shaker and shake for 120 min ± 5 min at 25 °C ± 5 °C. If small droplets of
liquid are noted on the outer surface, suggesting the presence of pinholes in the outer glove, discard the
samples and repeat the extraction with a fresh pair of gloves.
7.2.3.4 Remove the clamp and separate the gloves carefully, taking care not to contaminate the extract
with the dye solution in the inner glove.
7.2.3.5 Decant the extract from the outer glove into a centrifuge tube (5.3). If it is coloured blue, it is
indicative of a pin-hole or cross-contamination. In such cases, discard the solution and repeat the extraction
2
with a fresh pair of gloves. Clarify the extract by centrifugation at not less than 20 000 m/s (2 000 × g) for
15 min. Store the extract at a temperature of not more than 7 °C and carry out the determination within 48 h.
Alternatively, frozen aliquots of the extract may be stored at −10 °C or lower for up to 15 days.
7.2.3.6 Cut both gloves at the 20 cm mark to remove the cuffs. Remove surplus liquid from the cuffs by
blotting and allow to dry at room temperature. Determine the mass of the cuffs (m ) to an accuracy of not less
c
than 0,001 g. Calculate the average mass (m ) of the extracted part of the gloves: m = (m + m − m )/2 where
s s 1 2 c
m and m are the masses of the original gloves and m is the combined mass of the un-extracted cuffs.
1 2 c
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ISO 12243:2003(E)
7.3 Preparation of standard protein solutions
Prepare standard solutions of protein by dilution of the protein stock solution (6.4) with extractant solution (6.2),
3 3 3 3 3
to make solutions with concentrations of e.g. 40 µg/cm , 20 µg/cm , 10 µg/cm , 5 µg/cm , and 2,5 µg/cm .
Use the extractant (6.2) as a blank. The solutions are stable for 2 days refrigerated (see the Note).
NOTE The lower concentrations can readily be prepared by two-fold serial dilution of the appropriate more
concentrated solution. The standard solutions should cover a wide range of concentrations the precise values of which are
known since the exact concentration of the stock solution has been determined (see 6.4). These solutions are also
required for the verification procedure described in Annex A.
7.4 Precipitation and concentration of protein
7.4.1 General
Carry out single determinations at 25 °C ± 5 °C.
3
7.4.2 Accurately transfer 4 cm each of extractant (6.2) (as a blank), the standard protein solutions
3 3
(see 7.3) and the three glove extracts to 10 cm centrifuge tubes (5.3). Add 0,4 cm of DOC (6.3.6), mix and
3 3
allow to stand for 10 min, then add 0,4 cm of TCA (6.3.7) and mix. Add 0,4 cm of PTA (6.3.8), mix (see
the Note) and allow to stand for a further 30 min.
NOTE The amount used is to ensure a sufficient quantity for analysis using a cuvette. If a micro-plate reader is used,
the quantities may be reduced proportionately. If a large number of samples is involved, it is particularly important to
ensure that the centrifuge tubes are clearly identified.
2
7.4.3 Centrifuge at not less than 60 000 m/s (6 000 × g) for 30 min. It is important that the protein is
properly compacted. If necessary, extend the time of centrifugation. Decant the supernatant liquid and drain
3
by inverting each centrifuge tube on an absorbent paper towel. Add 0,8 cm of 0,2 mol/l sodium hydroxide
solution (6.3.5) to each tube, including the blank, to redissolve the precipitated protein. Use a vortex mixer or
ultrasonic water bath (5.7) if needed.
Ensure that the protein has completely redissolved to give a clear solution. Should some protein precipitate
3 3
remain, add a further measured quantity of sodium hydroxide solution up to 3,2 cm (i.e. a total of 4,0 cm ).
The same amount of sodium hydroxide shall be used for each of the solutions. The recommended amount of
3
sodium hydroxide solution (0,8 cm ) gives a concentration factor F of 5. If the same amount of sodium
hydroxide is not used for each sample, then F will vary from one sample to another:
Volume of extract before precipitation
F=
Volume of NaOH used to redissolve the protein
The redissolved-protein solution should preferably be used the same day. If the determination cannot be
carried out at once, the pellet may be stored for not more than 24 h at a temperature not exceeding 7 °C.
3
In cases where complete dissolution in not achieved after addition of 4,0 cm of NaOH, centrifuge at
2
60 000 m/s (6 000 × g) for 15 min to give a clear protein solution.
7.5 Colour development
7.5.1 Switch on the spectrophotometer and zero it in accordance with the manufacturer’s instructions.
3 3
7.5.2 To 0,8 cm of the redissolved-protein solutions, including the blank from 7.4.2, add 0,3 cm of
3
reagent A (6.3.1) and mix well. Add 0,1 cm of reagent B (6.3.2), mix, and allow to stand for at least 15 min
but no longer than 1 h before measuring the absorbance.
3
NOTE Only 0,8 cm of the redissolved-protein solution is used for the colour reaction, regardless of the final volume
of the redissolved-protein solution.
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ISO 12243:2003(E)
If precipitation occurs on standing due to the presence of certain interferants, centrifuge to give a clear
solution prior to colour measurement.
7.5.3 Spectrophotometric measurement
Transfer the solutions prepared in 7.5.2 to cuvettes and measure the absorbance versus the blank at 750 nm
(preferred) or a specific wavelength in the range 600 nm to 750 nm within 1 h of adding reagent B. For uniform
results, the time scales, equipment and chosen wavelength must remain consistent. Determine the protein
content, in micrograms per gram of glove, as described in 8.3.
Or
7.5.4 Measurement using a micro-plate reader
3
Transfer 0,49 cm of the solutions prepared in 7.5.2 to a flat-bottom microtitre plate (see 5.10.2) and measure
the absorbance versus the blank at a specific wavelength in the range 600 nm to 750 nm within 1 h of adding
reagent B. Determine the protein content, in micrograms per gram of glove, as described in 8.3.
8 Calculation of results
8.1 Calibration curve
Prepare a calibration curve by plotting the concentration of the original protein solutions (see 7.3) against their
absorbance after undergoing precipitation and being redissolved (see 7.5.3 or 7.5.4).
NOTE Some protein is lost during the concentration process. The method assumes that the same percentage of
protein is lost from the standards as from the test samples during concentration.
8.2 Calculation of concentrations
Determine the concentration c of each of the three extracted samples, in micrograms per cubic centimetre of
extract, by using their absorbance to read them directly from the curve. Report the median value.
NOTE In the event that the calibration curve is non-linear, the value can be calculated by polynomial regression. It is
suggested that commercial computer software for curve fitting and calculation of unknown concentrations is more practical.
8.3 Calculation of extractable-protein content
8.3.1 Procedure A — Cut-glove procedure
Calculate the extractable-protein content E, in micrograms per gram of glove, from the equation:
Vc×× 5
E=
F× m
where
V is the volume of extractant used, in cubic centimetres;
c is the protein concentration in the redissolved-protein solution, in micrograms per cubic centimetre;
F is the concentration factor;
m is the mass, in grams, of the whole glove.
NOTE The value of 5/F will be 1 unless it has been necessary to use other than the recommended amount of sodium
hydroxide — see comment in 7.4.3.
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ISO 12243:2003(E)
The extractable protein per glove, in micrograms, is obtained by multiplying the result obtained above by m:
Extractable protein per glove = E× m
8.3.2 Procedure B — Glove-in-glove procedure
Calculate the extractable-protein content E, in micrograms per gram of glove, from the equation:
Vc×× 5
E=
F× m
s
where
V is the volume of extractant used, in cubic centimetres;
c is the protein concentration in the redissolved-protein solution, in micrograms per cubic centimetre;
F is the concentration factor;
m is the mass, in grams, of the glove sample extracted (see 7.2.3.6).
s
NOTE The value of 5/F will be 1 unless it has been necessary to use other than the recommended amount of sodium
hydroxide — see comment in 7.4.3.
The extractable protein per glove, in micrograms, is obtained by multiplying the result obtained above by m:
Extractable protein per glove = E× m
where
m is the mass, in grams, of a whole glove =+()mm/2;
12
m and m are the respective masses of the original pair of gloves.
1 2
8.3.3 Conversion to mass per unit surface area
Regulatory authorities may require the results to be expressed in terms of surface area, e.g. micrograms per
unit area. Conversion to these values is as follows:
Vc×× 5
2
Extractable protein in µg/dm =
F× A
where A is the total surface area of the glove (see 7.2.2.3), in square decimetres.
9 Precision
9.1 Background
An interlaboratory test programme (ITP) to evaluate the precision of the method was conducted in 2002 using
the precision procedures and guidelines described in ISO 9272 (in preparation). The existing ISO/TR 9272
may be consulted for other details and terminology.
Both extraction procedures were evaluated: the cut-glove procedure and the glove-in-glove procedure. The
ITP was conducted with four materials with increasing measurement levels. Seven laboratories participated in
the ITP, and a Type 1 precision was evaluated. A test result is the mean of three replicates on each of two
separate test days, and precision is given in terms of test results, i.e. a mean value for each of two test days.
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ISO 12243:2003(E)
The precision results as determined by this ITP shall not be applied to accep
...
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