1、Designation: D5712 10Standard Test Method forAnalysis of Aqueous Extractable Protein in Natural Rubberand Its Products Using the Modified Lowry Method1This standard is issued under the fixed designation D5712; the number immediately following the designation indicates the year oforiginal adoption or
2、, 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 determin-ing the amount of total
3、 aqueous extractable protein associatedwith natural rubber (NR) and its products. Water solubleproteins are extracted in a buffer solution and then precipitatedto concentrate them and also to separate them from watersoluble substances that may interfere with the determination.The extracted protein i
4、s redissolved and quantified colorimetri-cally by the modified Lowry method using a protein standard.1.2 For the purpose of this test method, the range of proteinmeasurement will be based on the limit of detection andquantitation and recorded in micrograms per dm2NR speci-men.1.3 The test method is
5、designed to be accurate and compat-ible with the industrial environment.1.4 Steps are included in this test method to minimize theeffects of interfering substances.1.5 It is recognized that other methods for the analysis ofleachable proteins exist and these may be used for routinequality control pur
6、poses provided they have been validated anda correlation established against the reference method specifiedby this test method.1.6 This test method has not been validated for use withlubricated products such as condoms. Condoms with differentlubricants as typically marketed, have not been tested in
7、anASTM ILS to determine if, and if so to what degree, thelubricant interferes with the assay.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health prac
8、tices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D3577 Specification for Rubber Surgical GlovesD3578 Specification for Rubber Examination GlovesD4483 Practice for Evaluating Precision for Test MethodStandards in the Rubber and C
9、arbon Black ManufacturingIndustries3. Terminology3.1 Definitions:3.1.1 backgroundthe absorbance measurement of theLowry assay in the absence of the protein analyte.3.1.2 calibrationthe standardization of an instrument set-ting.3.1.3 calibration solutionthe standard solution used toroutinely and repr
10、oducibly calibrate a measuring instrument.3.1.4 concentration rangethe recommended analyte con-centration range in g/mL that produces an absorbance mea-surement of 0.01 to 1.5 units at 600 to 750 nm.3.1.5 dilution factor (F)the ratio of the volume NaOH inmillilitres used to redissolve the test speci
11、men extract tovolume NaOH in millilitres used to redissolve the standardovalbumin proteins. For example, if protein in a 1-mL testextract is acid precipitated and redissolved in 0.25 mL, and theovalbumin protein standards are also redissolved in 0.25 mL,then the dilution factor ratio of the test ext
12、ract to that of thecalibration curve would equal one.3.1.6 extractantan aqueous buffer of pH 7.4 6 0.2 usedfor the extraction process.3.1.7 initial settingthe instrument setting to which thespectrophotometer is adjusted with the reference solution.3.1.8 interferentany substance that results in a fal
13、se posi-tive or negative measurement in the analytical test method.3.1.9 latex proteinaqueous extractable proteins andpolypeptides occurring in NR latex and its products.3.1.10 limit of detection (LOD)the lowest protein concen-tration that can be measured and be statistically different from1This tes
14、t method is under the jurisdiction of ASTM Committee D11 on Rubberand is the direct responsibility of Subcommittee D11.40 on Consumer RubberProducts.Current edition approved June 1, 2010. Published July 2010. Originally approvedin 1995. Last previous edition approved in 2005 as D5712 051. DOI: 10.15
15、20/D5712-10.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Dr
16、ive, PO Box C700, West Conshohocken, PA 19428-2959, United States.the blank. The LOD is expressed as 3.33 standard error of they-intercept of the calibration regression line divided by theslope of the calibration line.3.1.11 limit of quantitation (LOQ)the lowest protein con-centration that can be me
17、asured to produce quantitativelymeaningful results with acceptable precision and accuracy. TheLOQ is expressed as 103 standard error of the y-intercept ofthe calibration regression line divided by the slope of thecalibration line.3.1.12 linearitythe degree to which a graph of absorbanceversus concen
18、tration approximates a straight line.3.1.13 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 betweenindependent test results obtained within a single laboratory.3.1.15
19、reproducibilitythe variability or test error betweentest results obtained in different laboratories.3.1.16 spectrophotometric measurementthe unit of mea-surement of the instrument that is proportional to absorbance.3.1.17 standard solutionthe standard analyte to which thetest (unknown) sample being
20、measured is compared.3.1.18 water (dH2O)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 determina-tion of protein levels in NR and its products. This test methodinvolves the extracti
21、on of residual aqueous soluble proteinsfrom NR followed by the precipitation of these proteins toremove interfering, aqueous soluble substances. The proteincontent is then determined by the Lowry method of proteinanalysis using a protein standard for quantitation. Spectropho-tometric measurement is
22、performed at a fixed wavelength inthe range 600 to 750 Hz (nm). A wavelength of 750 nm isrecommended.5. Significance and Use5.1 This test method, for the determination of protein levelsin NR, is primarily intended to test NR materials for residualprotein content. It is assumed that all who use this
23、test methodwill be trained analysts capable of performing common labo-ratory procedures skillfully and safely. It is expected that workwill be performed in a properly equipped laboratory.6. Apparatus6.1 Spectrophotometer and cuvettes or microplate readerand 96-well microtiter plates.6.2 Pipettes, te
24、st tubes (for example, 1.5-mL polypropylenemicrocentrifuge (MC) tubes), test tube rack, vortex mixer, andcentrifuge for MC tubes.7. Reagents and Materials7.1 Whenever water is called for, distilled or deionizedwater should be used.All other reagents should be of analyticalquality.7.2 Extraction Buff
25、erAn aqueous buffer of pH 7.4 6 0.2NOTE 1The following buffer solutions could be used: phosphatebuffer; PBS, phosphate buffered saline; TES,N-trishydroxymethylmethyl-2-aminoethanesulfonic acid hemisodiumsalt buffer, or equivalent of sufficient buffering capacity (at least 25 mM)to maintain the extra
26、ct at pH 7.4 6 0.2.7.3 Modified Lowry Assay ReagentsA more detailed de-scription of the Lowry protein assay is discussed in Refs (1-7).37.3.1 Reagent AAlkaline tartrate solution prepared bydissolving 2.22 g sodium carbonate, 0.44 g sodium hydroxide,and 0.18 g sodium tartrate in water sufficient to m
27、ake 100 mL.Reagent A (alkaline tartrate):2.22 g sodium carbonate0.44 g sodium hydroxide0.18 g sodium tartrateq.s. 100 mL with distilled or deionized water (dH2O)7.3.2 Reagent BCopper sulfate solution prepared by dis-solving 7.0 g cupric sulfate pentahydrate in water sufficient tomake 100 mL.Reagent
28、B (copper sulfate):7.0 g cupric sulfate pentahydrateq.s. 100 mL with dH2O7.3.3 Reagent CAlkaline copper tartrate solution pre-pared by mixing 1 mL of Reagent B and 150 mL of ReagentA.Reagent C (alkaline copper tartrate):Mix reagents A use a vortex mixer orultrasonic water bath if needed. Ensure that
29、 the protein iscompletely redissolved to a clear solution. Should some proteinprecipitates remain, add a further measured quantity of thesodium hydroxide solution up to a total of 1 mL. Theredissolved protein solution may be stored prior to the deter-mination for not more than 24 h at 3 6 1C.NOTE 10
30、When storage of the extract for 24 h is necessary, it ispreferred to store the precipitated protein pellet rather than the redissolvedprecipitate. The precipitate can then be redissolved after storage.NOTE 11Lower centrifuge speeds may leave the protein insufficientlycompacted, which can lead to err
31、oneous results. The recommendedamount of sodium hydroxide solution (0.25 mL) used to redissolve theacid-precipitated sample concentrates the test extract 4-fold from theoriginal 1-mLvolume. When the volume used to redissolve the test extractis different from the volume used to redissolve the ovalbum
32、in proteinstandards, a dilution factor F is used in the calculation of extractableprotein to adjust the ratio of the two volumes. When the spectrophoto-metric absorbance measurement of the redissolved test extract is outsideof the limit of the calibration curve, the redissolved test extract may bedi
33、luted in 0.2 N NaOH so that the absorbance measurement of the dilutedsample is within the limits of the calibration standard curve. If anadditional quantity of sodium hydroxide solution is required, the degree ofconcentration will be different and must be allowed for in subsequentcalculations.9.4 Co
34、lor Development and Reading:9.4.1 Assay Procedure for 96-Well Microtiter Plate Modi-fied Lowry Method:9.4.1.1 Add 125 L of Reagent C.NOTE 12Optional Correction of InterferencesTo prepare the re-agent to correct for interferences, repeat all reagent additions but replaceReagent C with Reagent C8 (C p
35、rime, no copper sulfate present) andsubtract the absorbance in the absence of copper sulfate from the testsample absorbance containing copper sulfate (refer to 7.3.4 and 9.4.4).9.4.1.2 Add 60 L of redissolved specimen extracts (NRproteins), standard protein (ovalbumin), or reagent blank(minus protei
36、n analyte), mix well and let set for 15 min at roomtemperature (RT).9.4.1.3 Add 15 L of Reagent D, thoroughly mix immedi-ately, and let set for 30 min at RT.9.4.1.4 The absorbance of the final assay mixture in a96-well microtiter plate using a microplate reader (spectropho-tometer) is measured at a
37、wavelength of 750 nm (600 to 750nm optional) within1hofadding the Folin reagent. Alldeterminations are carried out from extractions of three indi-vidual NR specimens or products. Each of the three extracts isconcentrated by acid precipitation, and an average is calculatedfrom the three extracts.9.4.
38、2 Assay Procedure for Cuvette Modified Lowry Method:9.4.2.1 Add 2.5 mL of Reagent C.NOTE 13Optional Correction of InterferencesTo prepare the re-agent to correct for interferences, repeat all reagent additions but replaceReagent C with Reagent C8 (C prime, no copper sulfate present) andsubtract the
39、absorbance in the absence of copper sulfate from the testsample absorbance containing copper sulfate (refer to 7.3.4 and 9.4.4).9.4.2.2 Add 1.2 mL of redissolved specimen extracts, stan-dard protein, or reagent blank (minus protein analyte), mix welland let set for 15 min at RT.9.4.2.3 Add 0.3 mL of
40、 Reagent D, thoroughly mix imme-diately, and let set for 30 min at RT.9.4.2.4 Transfer 4 mL or less of the final assay mixture to acuvette and measure the absorbance in a spectrophotometer ata wavelength of 750 nm (600 to 750 nm optional) within 1 hof adding the Folin reagent. All determinations are
41、 carried outfrom sample extractions of three individual NR specimens orproducts. Each of the three extractions is concentrated by acidprecipitation, and an average is calculated from the threeextracts.9.4.3 Color DevelopmentFollowing the addition of diluteFolin reagent, color development reaches a m
42、aximum inapproximately 20 to 30 min at room temperature. There may bea gradual loss of signal of a few percent per hour.NOTE 14A standard calibration curve should be run at the sameapproximate time as the test samples for each Lowry assay. It is importantfor uniform results that in all subsequent de
43、terminations the time scales,equipment, and wavelength be consistent.9.4.4 Optional Correction of InterferencesUniversalmethods to eliminate interferences do not yet exist for thisassay. Aqueous extractable chemicals that are added to NR forcompounding and curing may interfere with the Lowry protein
44、assay. Interfering chemicals (for example, accelerators, syn-thetic polymers, and so forth) can cause a change in the colordevelopment; absorbance values are usually inflated. It isknown that the Lowry Folin phosphomolybdate/tungstate re-agent can form a color that absorbs in the 600 to 750 nm range
45、when reducing chemicals are present. The variation of theFolin reagent color can be a result of contamination fromchemicals external to the Lowry assay that affects the accuracyand reliability of low-level protein determinations. Since theprotein-induced color formation of the Lowry Folin reagentdep
46、ends less on the reducing potential of aromatic aminoacylresidues in proteins, and more on the reductant reaction of thecopper-polypeptide bond complexes in proteins (1-7),itispossible to correct for some interferences. A modification ofthe Lowry method, where the difference in color formationdeterm
47、ined by assaying protein extracts in the presence andabsence of copper, can be used to approximate the amount ofpeptide bonds in the protein extract. This correction method isincluded as an option in this test method to reduce the effectsof aqueous-soluble interfering chemicals in the assay. Thisapp
48、roach involves subtracting the test extract response pre-pared in the absence of copper sulfate from the proteinmeasurement in the presence of copper sulfate to produce aprotein signal, by difference. Equivalent care should be givento measuring the signal in the absence of copper as the testsample i
49、tself since any variability of the measurement cancontribute to the final measured value.NOTE 15The test method may not remove all substances that interferewith the Lowry colorimetric assay. Other methods of reducing the signalof interfering chemicals other than protein in the Lowry assay may beused. These include dialysis of the protein test extract in an aqueous bufferto remove the interfering chemicals, organic phase extraction of theprotein test extract to remove the interfering chemicals from the aqueousD5712 104phase, and double acid p
