1、Designation: D5712 10D5712 15Standard Test Method forAnalysis of Aqueous Extractable Protein in Latex, NaturalRubber, and ItsElastomeric Products Using the ModifiedLowry Method1This standard is issued under the fixed designation D5712; the number immediately following the designation indicates the y
2、ear oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers an analytical test for determ
3、ining the amount of total aqueous extractable protein associated withnatural rubber (NR) and itsNR, latex, and elastomeric products. Water soluble proteins are extracted in a buffer solution and thenprecipitated to concentrate them and also to separate them from water soluble substances that may int
4、erfere with the determination.The extracted protein is redissolved and quantified colorimetrically by the modified Lowry method using a protein standard.1.2 For the purpose of this test method, the range of protein measurement will be based on the limit of detection and quantitationand recorded in m
5、icrograms per dm2 NRtest specimen.1.3 The test method is designed to be accurate and compatible with the industrial environment.1.4 Steps are included in this test method to minimize the effects of interfering substances.1.5 It is recognized that other methods for the analysis of leachable proteins
6、exist and these may be used for routine qualitycontrol purposes provided they have been validated and a correlation established against the reference method specified by this testmethod.1.6 This test method has not been validated for use with lubricated products such as condoms. Condoms with differe
7、nt lubricantsas typically marketed, have not been tested in anASTM ILS to determine if, and if so to what degree, the lubricant interferes withthe assay.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of t
8、his standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D3577 Specification for Rubber Surgical GlovesD3578 Specification for Rubber Examination GlovesD4483 Practice for Evaluat
9、ing Precision for Test Method Standards in the Rubber and Carbon Black Manufacturing Industries3. Terminology3.1 Definitions:3.1.1 backgroundthe absorbance measurement of the Lowry assay in the absence of the protein analyte.3.1.2 calibrationthe standardization of an instrument setting.3.1.3 calibra
10、tion solutionthe standard solution used to routinely and reproducibly calibrate a measuring instrument.3.1.4 concentration rangethe recommended analyte concentration range in g/mL that produces an absorbance measurementof 0.01 to 1.5 units at 600 to 750 nm.1 This test method is under the jurisdictio
11、n of ASTM Committee D11 on Rubber and is the direct responsibility of Subcommittee D11.40 on Consumer Rubber Products.Current edition approved June 1, 2010July 1, 2015. Published July 2010September 2015. Originally approved in 1995. Last previous edition approved in 20052010 asD5712 05D5712 10.1. DO
12、I: 10.1520/D5712-10.10.1520/D5712-15.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an
13、ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In
14、 all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.5 dilution factor (F)the ratio of the volume NaOH in millilitres use
15、d to redissolve the test specimen extract to volumeNaOH in millilitres used to redissolve the standard ovalbumin proteins. For example, if protein in a 1-mL test extract is acidprecipitated and redissolved in 0.25 mL, and the ovalbumin protein standards are also redissolved in 0.25 mL, then the dilu
16、tionfactor ratio of the test extract to that of the calibration curve would equal one.3.1.6 extractantan aqueous buffer of pH 7.4 6 0.2 used for the extraction process.3.1.7 initial settingthe instrument setting to which the spectrophotometer is adjusted with the reference solution.3.1.8 interferent
17、any substance that results in a false positive or negative measurement in the analytical test method.3.1.9 latex proteinaqueous extractable proteins and polypeptides occurring in NR latex and its products.3.1.10 limit of detection (LOD)the lowest protein concentration that can be measured and be sta
18、tistically different from theblank. The LOD is expressed as 3.3 standard error of the y-intercept of the calibration regression line divided by the slope of thecalibration line.3.1.11 limit of quantitation (LOQ)the lowest protein concentration that can be measured to produce quantitatively meaningfu
19、lresults with acceptable precision and accuracy. The LOQ is expressed as 10 standard error of the y-intercept of the calibrationregression line divided by the slope of the calibration line.3.1.12 linearitythe degree to which a graph of absorbance versus concentration approximates a straight line.3.1
20、.13 Lowryfor the purpose of this test method, the word “Lowry” is used to represent any modified form of the originalLowry assay method.3.1.14 repeatabilitythe variability or test error between independent test results obtained within a single laboratory.3.1.15 reproducibilitythe variability or test
21、 error between test results obtained in different laboratories.3.1.16 spectrophotometric measurementthe unit of measurement of the instrument that is proportional to absorbance.3.1.17 standard solutionthe standard analyte to which the test (unknown) sample being measured is compared.3.1.18 water (dH
22、2O)a liquid (H2O) purified by distillation (distilled water) or deionization (deionized water).4. Summary of Test Method4.1 This colorimetric test method is used for the determination of protein levels in NR NR, latex, and itselastomeric products.This test method involves the extraction of residual
23、aqueous soluble proteins from NR NR, latex, and elastomeric productsfollowed by the precipitation of these proteins to remove interfering, aqueous soluble substances. The protein content is thendetermined by the Lowry method of protein analysis using a protein standard for quantitation.quantificatio
24、n. Spectrophotometricmeasurement is performed at a fixed wavelength in the range 600 to 750 Hz (nm). A wavelength of 750 nm is recommended.5. Significance and Use5.1 This test method, for the determination of protein levels in NR,latex, is primarily intended to test NR NR, latex, andelastomeric mate
25、rials for residual protein content. It is assumed that all who use this test method will be trained analysts capableof performing common laboratory procedures skillfully and safely. It is expected that work will be performed in a properlyequipped laboratory.6. Apparatus6.1 Spectrophotometer and cuve
26、ttes or microplate reader and 96-well microtiter plates.6.2 Pipettes, test tubes (for example, 1.5-mL polypropylene microcentrifuge (MC) tubes), test tube rack, vortex mixer, andcentrifuge for MC tubes.7. Reagents and Materials7.1 Whenever water is called for, distilled or deionized water should be
27、used.All other reagents should be of analytical quality.7.2 Extraction BufferAn aqueous buffer of pH 7.4 6 0.2NOTE 1The following buffer solutions could be used: phosphate buffer; PBS, phosphate buffered saline; TES, N-trishydroxymethylmethyl-2-aminoethanesulfonic acid hemisodium salt buffer, or equ
28、ivalent of sufficient buffering capacity (at least 25 mM) to maintain the extract at pH 7.4 6 0.2.7.3 Modified Lowry Assay ReagentsA more detailed description of the Lowry protein assay is discussed in Refs (1-7).37.3.1 Reagent AAlkaline tartrate solution prepared by dissolving 2.22 g sodium carbona
29、te, 0.44 g sodium hydroxide, and 0.18g sodium tartrate in water sufficient to make 100 mL.3 The boldface numbers given in parentheses refer to a list of references at the end of the text.D5712 152Reagent A (alkaline tartrate):2.22 g sodium carbonate0.44 g sodium hydroxide0.18 g sodium tartrateq.s. 1
30、00 mL with distilled or deionized water (dH2O)7.3.2 Reagent BCopper sulfate solution prepared by dissolving 7.0 g cupric sulfate pentahydrate in water sufficient to make100 mL.Reagent B (copper sulfate):7.0 g cupric sulfate pentahydrateq.s. 100 mL with dH2O7.3.3 Reagent CAlkaline copper tartrate sol
31、ution prepared by mixing 1 mL of Reagent B and 150 mL of Reagent A.Reagent C (alkaline copper tartrate):Mix reagents A use a vortex mixer or ultrasonicwater bath if needed. Ensure that the protein is completely redissolved to a clear solution. Should some protein precipitates remain,add a further me
32、asured quantity of the sodium hydroxide solution up to a total of 1 mL. The redissolved protein solution may bestored prior to the determination for not more than 24 h at 3 6 1C.1 to 8C.NOTE 10When storage of the extract for 24 h is necessary, it is preferred to store the precipitated protein pellet
33、 rather than the redissolved precipitate.The precipitate can then be redissolved after storage.NOTE 11Lower centrifuge speeds may leave the protein insufficiently compacted, which can lead to erroneous results. The recommended amount ofsodium hydroxide solution (0.25 mL) used to redissolve the acid-
34、precipitated sample concentrates the test extract 4-fold from the original 1-mL volume.When the volume used to redissolve the test extract is different from the volume used to redissolve the ovalbumin protein standards, a dilution factor Fis used in the calculation of extractable protein to adjust t
35、he ratio of the two volumes. When the spectrophotometric absorbance measurement of theD5712 154redissolved test extract is outside of the limit of the calibration curve, the redissolved test extract may be diluted in 0.2 N NaOH so that the absorbancemeasurement of the diluted sample is within the li
36、mits of the calibration standard curve. If an additional quantity of sodium hydroxide solution is required,the degree of concentration will be different and must be allowed for in subsequent calculations.9.4 Color Development and Reading:9.4.1 Assay Procedure for 96-Well Microtiter Plate Modified Lo
37、wry Method:9.4.1.1 Add 125 L of Reagent C.NOTE 12Optional Correction of InterferencesTo prepare the reagent to correct for interferences, repeat all reagent additions but replace ReagentC with Reagent C (C prime, no copper sulfate present) and subtract the absorbance in the absence of copper sulfate
38、 from the test sample absorbancecontaining copper sulfate (refer to 7.3.4 and 9.4.4).9.4.1.2 Add 60 L of redissolved specimen extracts (NR proteins), test specimen extracts, standard protein (ovalbumin), orreagent blank (minus protein analyte), mix well and let set for 15 min at room temperature (RT
39、).9.4.1.3 Add 15 L of Reagent D, thoroughly mix immediately, and let set for 30 min at RT.9.4.1.4 The absorbance of the final assay mixture in a 96-well microtiter plate using a microplate reader (spectrophotometer)is measured at a wavelength of 750 nm (600 to 750 nm optional) within 1 h of adding t
40、he Folin reagent. All determinations arecarried out from extractions of three individual NRtest specimens or products. Each of the three extracts is concentrated by acidprecipitation, and an average is calculated from the three extracts.9.4.2 Assay Procedure for Cuvette Modified Lowry Method:9.4.2.1
41、 Add 2.5 mL of Reagent C.NOTE 13Optional Correction of InterferencesTo prepare the reagent to correct for interferences, repeat all reagent additions but replace ReagentC with Reagent C (C prime, no copper sulfate present) and subtract the absorbance in the absence of copper sulfate from the test sa
42、mple absorbancecontaining copper sulfate (refer to 7.3.4 and 9.4.4).9.4.2.2 Add 1.2 mL of redissolved specimen extracts, standard protein, or reagent blank (minus protein analyte), mix well andlet set for 15 min at RT.9.4.2.3 Add 0.3 mL of Reagent D, thoroughly mix immediately, and let set for 30 mi
43、n at RT.9.4.2.4 Transfer 4 mL or less of the final assay mixture to a cuvette and measure the absorbance in a spectrophotometer at awavelength of 750 nm (600 to 750 nm optional) within 1 h of adding the Folin reagent. All determinations are carried out fromsample extractions of three individual NRte
44、st specimens or products. Each of the three extractions is concentrated by acidprecipitation, and an average is calculated from the three extracts.9.4.3 Color DevelopmentFollowing the addition of dilute Folin reagent, color development reaches a maximum inapproximately 20 to 30 min at room temperatu
45、re. There may be a gradual loss of signal of a few percent per hour.NOTE 14Astandard calibration curve should be run at the same approximate time as the test samples for each Lowry assay. It is important for uniformresults that in all subsequent determinations the time scales, equipment, and wavelen
46、gth be consistent.9.4.4 Optional Correction of InterferencesUniversal methods to eliminate interferences do not yet exist for this assay.Aqueous extractable chemicals that are added to NR NR, latex, and elastomeric products for compounding and curing mayinterfere with the Lowry protein assay. Interf
47、ering chemicals (for example, accelerators, synthetic polymers, and so forth) cancause a change in the color development; absorbance values are usually inflated. It is known that the Lowry Folinphosphomolybdate/tungstate reagent can form a color that absorbs in the 600 to 750 nm range when reducing
48、chemicals arepresent. The variation of the Folin reagent color can be a result of contamination from chemicals external to the Lowry assay thataffects the accuracy and reliability of low-level protein determinations. Since the protein-induced color formation of the LowryFolin reagent depends less on
49、 the reducing potential of aromatic aminoacyl residues in proteins, and more on the reductant reactionof the copper-polypeptide bond complexes in proteins (1-7), it is possible to correct for some interferences. A modification of theLowry method, where the difference in color formation determined by assaying protein extracts in the presence and absence ofcopper, can be used to approximate the amount of peptide bonds in the protein extract. This correction method is included as anoption in this test method to reduce the effects of a
