CLIN. CHEM.

24/1,

80-86

(1978)

New Automated Determination
Andrew E. Pinnell’

Dye-Binding with Bromcresol

Method for Serum Albumin Purple

and Barry

E. Northam

We describe a new automated dye-binding serum albumin determination with bromcresol that has several advantages over an existing green (BCG) method. The continuous-flow sensitive, linear, and precise, with negligible

method for purple (BCP) bromcresol method is
sample in-

because proteins. 3-globulin

of

a nonspecific proteins and fractions

reaction are found the include

with in the

some
ai-, az-,

serum and reacBCG (2, reaction

These

acute-phase

teraction at an analytical rate of 60 samples per hour. Unlike BCG, BCP did not react with an albumin-free serum globulin preparation or pure human transferrin solutions. Reaction with serum was instantaneous; in contrast, BCG exhibits a slow nonspecific reaction with some specimens.
The specificity of BCP was demonstrated by good agreement with results of “rocket” immunoelectrophoresis (EIA) where y(BCP) = 0.95x (EIA) + 1.72. The BCG method overestimated serum albumin concentration where j4BCG) = 1.Olx(EIA) + 6.77. Precipitation, which affects the BCG method, was not observed with BCP. Blank corrections

tants (14, 17). y-Globulins do not react with 17). The nonspecific reaction is slow but the with albumin is immediate, method depends in part velopment in the analytical (18) number with It was hypothesized would reduce the protein nonspecific However, stricted sorbance values. purple sons. interactions so that upon the system the error duration (14).

of the BCG of color depH dye! in the red (19). are rereagent ab-

that increased reagent of weak electrostatic reduction case with phenol BCG methods of the high

a consequent

reaction, as is the direct colorimetric to a pH near 4 because and decreasing Consequently, (BCP) was changes dye

were negligible, salicylate did not interfere, and bilirubin affected the method only if present in very high concentration. The method offers a solution to the poor accuracy
of existing desirable BCG methods features. while retaining many of their

effect of albumin at higher pH we sought a new dye. Bromcresol in detail from yellow for three reain the color to purple

investigated

This

Additional
green

Keyphrases:
compared

electroimmunoassay,
.

bromcresol

methods

specificity

pH interval 5.2-6.8, permitting use of a reagent at about pH 5. BCP is structurally similar to BCG, so we hoped that the desirable properties of the latter would be retained. Louderback et al. have reported2 that BCP reacts only with albumin, and a manual method for this dye has been described (20). We have devised an automated method and compare BCG method

Dye-binding nation are Bromcresol methods ceptible benzoic advantages. precipitate effect” pends

methods for serum albumin simple, rapid, economical, and (BCG) is widely described (1-8). used BCG

determiprecise.

green have been

and many is less susdis-

here its performance with that of an established method and a specific electroimmunoassay (ETA), with encouraging results.

to interference acid (1), and The causing (1, 3, 9). on the source

than 2-(4’-hydroxyazobenzene) is sensitive, but has several and albumin

BCG/albumin turbidity The of the

complex may partly the “negative baseline of the (10), Most by BCG complex so the serious methods dechoice is the and

Materials
BCP Method

and Methods

absorptivity is critical. results

Stock absolute

BCP ethanol.

solution, Dissolve

40

mmol 0.54

of BCP g of BCP

per (pH

liter Indicator

of

of calibration poor agreement specific Expert methods (16). BCG

material between

methods (11-15). Panel on Proteins should be used overestimates

Consequently, has recommended only for screening serum albumin

the IFCC that BCG purposes

Grade; B.D.H. Chemicals, Poole, England) in 15 ml of absolute ethanol. When a clear orange solution is obtained, dilute to 25 ml with absolute ethanol. The reagent is stable at 4 #{176}C for at least three months. Preliminary examination indicates that BCP from other manufacturers brook, England; (Koch-Light Aldrich Laboratories Chemical Co. Inc., Ltd., ColnMilwaukee,

concentration

Department of Clinical Chemistry, B4 6NH, U.K. Current address: Department Hospital, Received Dudley, Aug. West Midlands, 15, 1977; accepted

General of Clinical

Hospital, Chemistry,

Birmingham, The Guest
2

Louderback,

A., using Chem.

Mealy, bromcresol 14, 793

E.

H., purple (1968).

and

Taylor,

N.

A.,

A new

dye

DY1 4SE, U.K. Oct. 14, 1977.

binding technic in serum. Clin.

for determination Abstract.

of albumin

80

CLINICAL

CHEMISTRY,

Vol. 24, No. 1, 1978

0. and dilute to 100 I 016 0.’liter soThe reagent Dissolve is stable for at RECORDER 600nm temperature.5 to Electroimmunoassay Laurell “rocket” technique standards (as above) were diluted chloride solution in diameter.) were known measured.. 1978 81 of 10 V!cm for filter paper layers. a phenomenon the working BCP continuous-flow a sample is (as BCG resampled stream sample interaction E. Dilute grade) to 1 liter with 150 ml distilled of glacial water. respecat 590 nm of also Instruments (Figure 1) gives nm decreases.tmol!liter caused an increase in absorbance from 0.K. Standards.K. serum and a standard albumin curve was plotted. tively.d .’liter the absorbance gradually at higher Brij-35 Reagent conditions were was constant. 9 g of sodium albumin Hospital [cat. with warming. of about with air in a modified bubble The Mk2 Sampler. Dade Biochemicals. Addition of Brij-35 solution up to 0..MP SAMPLER 60/I. The gel per liter. and 590 nm. Brij-35 noted causes in the trum Addition but causes with with absorbances increasing pH that at 436 the opposite (2).91 and the absorbance 0. Supply Fig.. cut contained human albumin mmol. chosen (a) declining to achieve the and 5 sl was applied to a well 2. Dissolve 10 g of sodium acetate trihydrate (AR grade) in about 800 ml of distilled water...60 2 50 From Ibm cell -p water. and and Widdowson measured (1).e..324.16 Air Somple Sen inset suc !±2.’liter.5 mm in a gel layer 1. ab- sorbance at 636 used in the U. borough. Results Reaction Optimum (without Brij-35) of the vacuum-sealed ampoule ride solution as recommended in 3 ml of sodium by the manufacturer to give between conditions. Vol. Sigma Chemical Co. The difference plus reagent vs. reagent shows at 603 nm. Serum and 250-fold with sodium 1. American (U.Workirig 14 timrns H31 Air in distilled BCP Stock acetic acetic acid (AR acid solution. N tubular flow Co.1 to 5.2 ± solution or 0. solution. Hounslow. with Coomassie 3 h. The absorbance pH interval 5. England. BCP exhibits and The Albumin working BCP maxima reagent at 436 absorbance dilute with sodium chloride solution trations up to 60 g/liter. AutoAnalyzer I modules: Mkl Pump. Reconstitute Equipment Pharmaceuticals reagents were obtained from Ltd. increasing the BCP concentration from 40 to 100 i.. 24. Conditions other than those under examination were kept as in the original gradually method over he (above). using a reagent blank. With increases. no. of albumin reagent a shift specan abon ab- method. but from 0.) Dissolve 25 g of “Brij-35” (polyether.5 mol. Ltd. Addition effect.nnl. care or noisy an increase peak from of albumin in absorbance at 590 nm with 590 to 603 nm. BCP reogent Air INSET Working BCP reagent. Fisons England) solution.5 mL/liter increased the absorbance.1 ml of a 40 g!liter solution of albumin to 20 ml of BCP reagent.Wis. For this we used human B5158.. lauryl Scientific perform Apparatus similarly Ltd. St.. buffer. No.8) (250 is used. and (b) to be such that small inaccuracies in the routine preparation of the BCP reagent (i. CLINICAL CHEMISTRY.. to minimize BCG Particular pattern. In this method. Check the pH 0. the plates were pressed dried with a hair-dryer. C. and Basingstoke.tmol of BCP per liter.. is necessary peaks will personal to achieve result. with and albumin. and Brillant Blue R. The dilution injected used Recorder (Technicon England).5 ml. minimum od. effect of reagent communication). Albutt. Flow shows diagram for the BCP method system Sodium air re-injection chloride in 1 liter of distilled water.275 to of a Brij-35 (50 mmol/liter. Didcot.03 with stock acetic dium least hydroxide a week chloride solution. II 1mm) It r-’(mu Brij-35 oxyethylene Mo. 1 ml of Brij-35 and and acid dilute solution. from increased 0.313. pH strength under stained The was used. effect and BCP upon the concentration) reproducibility would of the have meth- ... which is widely of BCG per liter g!liter) in citrate sorbance of the albumin!BCP complex at 603 nm by adding 0. and buffer 1 ml nm.6). After and barbital electrophoresis 15 g of agarose best compromise between maximum absorbance of the BCP!albumin complex and minimum reagent absorbance at 603 nm. solution.. concentrations.11. a regular BCG We with sorbance maximum We examined the conditions Method used the the standards method of Northam described above._Workint Diln. to 1 liter with and 1 ml distilled COLORIMETER 15mm flow cell . England] specified as 100% albumin assayed by the the contents chloand concen- by moving-boundary macro-Kjeldahl electrophoresis and procedure. monovalent antibuffer (50 at a field antiserum. a reagent with 40 mol solution pH 3.. water. at room if necessary adjust to 5. were and determined. Similarly.6. 8.w. 1. to of 0. Peak heights Brij-35. Inset 1. system 200-fold. Hoechst un- concentrations Ltd. Colorimeter (with 15-mm cell). 40 j. Louis..5 mm thick. in their Loughreaction PI..292 to 0. Add 10 ml of stock acetic of stock acid BCP solution.. 0 60 L±2. Equipment.

A freeze-dried serum Laboratories reconstituted recommended centration.). 57. specimens was negligible. To two serum between-batch with albumin presence of 23.e. London] eluate from the gel columns was examined presence of albumin by immunoelectrophoresis CLINICAL CHEMISTRY. use by Hoechst which of a commercial Ltd.). in vacTravenol was than the conwith provided and Dade abalbusupplied the CV was we prepared tions sera 0. 24. Brij-35 solution throughout this period. related (Figure highest were above. 97% in terms of transmission units) at a concentration of a concentration of 60 g!liter. absorbance was measured at 603 nm by the BCP method. 4 in 5. to an The Dade with with no detectable protein Chemical Co.1 57. I unassayed (Travenol Laboratories Ltd. and from Sigma containing preparations to give for moisture concentration up 2). albumin albumin solution correct method. from low to high to the continuous 1-3 g of globulins were dissolved or other and albumin albumin BCP impurities. cals. purified human albumin (Sigma Chemical Co.5 g Precision. The for the against The linear range for the BCG than in the BCP method and the was 82 method maximum was narrower absorbance less (Figure 2). absorbance measured at 636 nm by the BCG method Sample groups ceded interaction. re- uum-sealed ampoules. E. 3.).45 solution) (undiluted of concentration concentrations absorbance at 603 nm). absorbance concentration aspiration plateau was within two transmission lines (i. rations described (specified inpurities).30 cr1 cr1 4 0.43%. purified human albumin (Hoechst Pharmaceuticals Laboratories Ltd. 1. manifold.). Pharmacia (Great Britain) Ltd.5 g of albumin g/liter when Pharmaceuticals as >99% albumin. 2. This was diluted in sodium chloride solution: (0 Pak I unassayed. from bro- Thetford. Each batch We used the same acetic acid solution. Specificity. 4. assayed Pharmaceuticals to that 2). Norfolk. Biochemicals by three of high concentration.5 g of albumin per liter. impurities because and. 3. a sample!wash ration of 1!1. 50. in 20 batches weeks.0. water the high “standard” Linearity by the rather Aliquots divided into aliquots.0 g/liter per liter (Figure precision 3). Ltd. purified human prepared human serum with use of albumin albumin (Dade from varTravenol serum 0 Pak water 1. At 60 samples!h. of albumin distributing 13.20 0... assess pools For of 60 g!liter in a 1-cm preparation (0. 2 in 5. 91% of steady state in terms of absorbance units. These in sodium chloride We did not With were the BCP linearly gave light the path g of albumin per liter. Three obtained: from purified that Hoechst human from Dade albumin prepaLtd.1 g/liter with low and and 39. 5. Standard ious sources B. In A-D. with Standards. and stored at 4 #{176}C. stock BCP solution. of each serum pool were analyzed. stock and electroimmunoassayed Ltd. 10 ml. D. 1 in Biochemicals). serum containing 11. we analyzed pre- concentration. Albumin was selectively removed serum by affinity chromatography on cyanogen mide-activated linked to Blue agarose Dextran (Sepharose) 2000 [Pharmacia gel sorptivity were similar min standard (Figure given covalently Fine Chemi(21). Recorder trace of analysis of freeze-dried human curves A. 5.50 A 0. Dade albumin the was preparations gave the presence of of moisture.) was reconstituted with distilled to 57. 1978 . was spectively). The lower absorbances. freeze-dried 0 20 ALBUMIN 30 40 g/liter 50 60 Fig.5 per liter assayed was increased to an apparent next after serum containing Interaction Approach 12. 2.40 V // LU 0 z 0 0.9 precision concentraby conof albumin Within-batch (serum between serum randomly centration chloride g!liter 20 specimens pool 20 pool). (“Q Pak to achieve then serum I unassayed”. on different days during four included eight to 15 serum specimens. only the Hoechst and Sigma possibly reflecting in particular. Vol.10 // Fig.8 cm/h C. (crystallized per 100 g). England) albumin in 6 ml of distilled each. was diluted both measured with sodium 35. No. of three specimens To evaluate of low this.. purified human albumin (Hoechst Pharmaceuticals Ltd. 3 in 5.6 g!liter normal albumin (by pooling patients’ concentrations. Chart speed. undiluted.

5 g/liter being necessary ample where the serum looked salicylate.320 . concentration 23. We estimated animal-based bumin for quality-control the serum albumin specimens.0 10.5 86 65 47 45 41 nephritis immunodiffusion.5 16.280 . enham. from were and antihuman-albumin serum arc (Hoechst with the original corresponding the analyzed the gel columns by the serum and polyvalent Ltd.330 .231 . but only by the BCP method.041 . where immunodiffusion albumin was by immunoelectrophoresis.5 method apparent method..202 shown. to give conTime after mixing 33.050 . 3.0 18.282 . Apparent % of transferrln albumin concn.5 and 1. 20.223 . were between 23 and of the the except eluate. of by by an albumin g/liter.5 42.080 .Table 1.5 (b) Radial monovalent antihuman Comparison precipitin disappeared Eluates tients method. Apparent Albumin Concentration of Albumin-Free Serum Eluates Prepared by Affinity Chromatography Nature of Table 2. BCP or the in specimen (Hoechst 22 22 15 32% Albumin radial 1. CHEMISTRY.7 g/Iiter + pure human albumin g/llter Pharmaceuticals serum showed that to serum albumin proteins from method.0 g/liter.).0 30.094 .221 . Color development of the BCP reagent with purified human lOs action preparations ately or after reaction with human stantaneous.060 . BCG Each methods.0 6. 1978 .6 Pure Ltd. 24.321 . An unassayed but only equine liter).3 6. Reaction of BCG (a) with Pure Human with Pure Human of Pure Human Transferrin Transferrin Alone.5 13.0 26. Rate of Color Development of the Reaction of BCG with Albumin. Icteric calibration. of 31. solution Transferrin We detected only against a was analyzed had a signifiby A lOs 30 s 1 mm 5 mm . BCP method g/liter RID8 Albumin (a) Apparent Transferrln concn 9/lIter albumin concn % transferrin of Crohns disease ‘‘ 36. 1.0 28.0 0 4.5 5.5 17. sera Two freeze-dried (Wellcome Reagents bovine-based Ltd. solutions.9 had apparent g/liter by the respectively. Serum. standards.282 . an apparent albumin concentration 15.108 .0 11. The assayed of the was by the BCG total protein not detected also Ltd.) (Table 1). hemolyzed per liter per corexand to with 83 bovine serum albumin (Hoechst Pharmaceuticals in sodium chloride solution at a concentration gave method method.0 12.0 7.7 Diffuse band on electrophoresis Nephrotic syndrome Glomerular a 0 0 0 0 0 assayed albumin as 5. albumin concentrations BCG method.250 at 636 nm vs.5 0. and Protein Preparations a AlbuminHuman albumin Serum 2 Human transferrin free specified as >99% pure was dissolved in sodium centrations one precipitin polyvalent the BCP and 1 3 globulin prepn in the range 5 to 40 g/liter. 40 g/liter like Salicylate. No.0 31. Grossly lipemic sera required rections.307 . impurities.053 .300 .301 .5 albumin concentrations. four the 8. concentration using Dade of various human alwith albumin For BCG after albumin mixing) was observed or transferrin or serum and was with was stable the instantaneous for at least albumin-free either exhibited sera and color (less than serum immedia slow purified was developof 1 h (Table in1 h.320 .095 timed from mixing cant reaction with BCG (Table 2) even in the presence of albumin but showed no reaction with BCP.) the the and England) and 36. pure human Sodium albumin CLINICAL added to serum did not interfere Vol.061 .221 .190 30 mm 60 mm a Absorbance .305 . Similar sera at lOg corrections (approximately of hemoglobin the greatest in one extreme milk.9 g/liter of 39.281 .0 40.300 .0 9. sera reg of were 1 were analyzed bcp itself had absorbance.5 liter. No re- . assayed albumIn as and all other detectable BCP radial remained.150 .249 .280 .0 by the BCP g/liter method.0 35.0 43. the had paBCG Transferrin 6. ment 3). arc by immunoelectrophoresis antiserum.0 12.282 .0 17.5 0. concentration by either methods. transferrin Pharmaceuticals with no detectable chloride solution Table 3.321 . and (b) in the Presence specImen Total proteIn g/liter ‘AIbumIn” BCG method concn. albumin method.5 of serum 12. . 1 h. by the BCG development a period Effect of Interfering Method Blank correction. Some serum samples BCG while with other solutions transferrin continued color over solutions.0 g/liter serum had BCG BCP of 43.321 . BCP reagent from Normal sera had with bilirubin quired blank apparent required for g of apparent Substances Sera which a negligible on the BCP with been use of a omitted.).0 52 30 19. reagent at intervals 20 cl of protein solution with 4 ml of BCG reagent. detected Human (Hoechst Mancini Pharmaceuticals immunodiffusion Ltd.310 . Beck- concentrations corrections per grossly albumin up to 500 izmollliter of between 0.

showing presence Results ods (Figure to provide the maximum and globulin concentration. siderably 84 CLINICAL 0. formed rioration. / Albumin 9/liter / / / / / / / / EIA 30 40 50 / 10 Albumi.986).r=O. 1. in concentrations electroimmunoassay difference. Five 113 and with use maceuticals calibration BCG as good methods and of purified human Ltd. This may reflect several factors: reagent composition (10). General standardized by electroimmunoassay 500 zmol high bilirubin by the BCP between concentrations method. “negative even in the baseline absence pH effect” (1) were observed due BCG of Brij-35. Pharmaceuticals results were purified Ltd. with the regression = tration.. the mean difwas only 0. (Dade in the The same same Method and BCG in the Method range 13 to BCG tends to form a green concentrations by the two dye-binding Each analysis methods was per- fibrillar precipitate with albumin.4 g/liter 250 zmol. We further used above) the human albumin standardization.) was serum (“Validate”. Precipitation is inhibited. allows a high little sample BCG In the method provides an alternative to existing The continuous-flow system described rate of analysis with good precision and interaction. 7. . 4.) were and specific methods (11-15).2 range g to for 4. Sera with be underestimated bilirubin were by the was added to standards up to BCP methods. Discussion The BCP BCG methods. 5. equation compared BCG and (Hoechst Similar being (with y(BCG) different = l.986). No. to the higher reagent Several comparisons used in the BCP method. method (1).4 g/liter).4 . Comparison of the BCP with Electroimmunoassay Sera with albumin 45 g/ liter were assayed and by electroimmunoassay. also (lipemia. etc. by the surfactant Brij-35. but not eliminated.72 by the (in g albumin/liter. time allowed for color development. agreement with electroimmunoassay in the normal range when freeze-dried In our study. 25 to 79 jzmol/liter analyzed by both in albumin concentration (mean concentration. the regression (n= 160./ u #/ “/ / / / / / / / / 10’ .7. mol/liter). selected. 52 methods. /.) for (Table Diag- umol/liter. 24.. Comparison methods of results by the BCP and electroimmu- Fig.95x Results than + 1. Bilirubin. and tively. Neither precipitation nor the associated with BCP. 4).so BCP // so BCG 40 40 - S 30 / 30 C 20 4 C 20 . Concentrations 0. methequation n = conof Biochemicals) selected bilirubin... ference 17 sera BCP method by the BCP (mean with bilirubin and electroimmumethod were lower 2. to avoid sample detehuman albumin standards used for all three methods. agreement electroimmunoassay albumin (Hoechst Pharthe freeze-dried serum for between methods. calibration material.Olx sera with + 6. respectend to sera with liter soof obtained concentration and 38. an effect that is greatest at the isoelectric point (pH 4. 1978 (Figure CHEMISTRY.1 g/liter. BCG methods overestimate Although all agree that serum albumin concen- by the BCP and electroimmunoassay 4) agreed well. Sera were albumin.6 g/liter However.3 g/liter of bilirubin A nostics freeze-dried Ltd. and the source of specimens.4 /‘. 4 . have been made between other abnormalities of drugs. BCG method were higher by electroimmunoassay Vol.77 to those specified as essentially serum and to purified a concentration of 500 was noted at concentrations The apparent albumin lution was 39.) Although gave as that (Table closer between 4). mg/liter. range Sera hemolysis. . 20 g/liter 30 40 E IA 50 10 20 Fig. possibly working purified were day. of a 40 g/liter in the presence per liter. 5). the magnitude of the inaccuracy varies. Minor interference exceeding 170 /Lmol/liter. r = 0.3) of the complex (9). results it was and by the still not BCP concentrations 192 mol/ estimated by the noassay. was much better serum was used being y(BCP) 160. Comparison of results by the BCG method and electro- noassay immunoassay at concentrations bilirubin 100% human (Sigma bilirubin) albumin up to at least Chemical methods the BCP method Purified 300 Co.

yet retains dye-binding the simplicity and methods. with binding in number of BCG on serum (24) for the spectral change is immobilisupport (9) such as detergent miWe hypothesize sites.4 35.9 34.8 1. particularly lites.986 1. Dye-binding by competing by salicylate. 1 (1967).02 BCG EIA 80 31.. Modification 117(1972). serum gel.9 BCG EIA 160 27. in the line.988 1.8 BCG BCP 120 29. of serum Assoc.. stanwith material clearly unsuitable for use with specific immunochemical methods. an error of 3 g of albumin Pure human transferrin preparations significant this method was still present albumin-free of this hypothesis of the BCP method albumin at its present in most limitations (17) that method. 1. W. W.989 few high-affinity binds to numerous binding sites on serum fragments of bovine albumin. G.13 although it also has a high affinity for the fragment that is the site for bilirubin binding (22). 25. No. permitting of many speci- results by electroimmunoassay equine for use reactivity However. are susceptible The BCP bilirubin endogenous to interference B.977 1. Regression Equations Describing Comparisons between BCP.81 0.7 34. which original (21). and Biggs. Doumas.72 0. I..0 32.06 5. and its use with BCG methods can be leading (10). The apparent estimated to explain to the problem the analytical The principal albumin. the (12) underestimated.. but still had reaction within 10 s (Table 3).s 31. metaboreflected regression y x BCP EIA 160 27. technical shows excellent agreement concentration method was between method. Purified containing Biochem. bilirubin is undesirable but other molecules. G. their albuor quality than that is mis- less References 1.8 BCG BCP 160 27. and (15). 24.. Chins. than of development determination. of BCG used prompt action electrostatic reduced Investigation improvement specifically a BCP method for to improve measurement However. number. stage might permit or the design However.988 1. H. 2. and Electroimmunoassay (EIA) Methods. and Everard.7 27. further of dyes the offers BCG reto be interfering to cationic by increasing these proteins in test sera. Clin. with Various concentration rarely by more regression has been underestimates line suggested were than of BCP sometimes two standard against that sera. solutions great lack of reaction the albumin-free contained most concentration.85 BCP EIA 80 31.0 30. using bromocresol No. Ac/a method 37. Tech. D.5 0. Determination green. 0. Chim. its susceptiwell be manito relatively its Mean x (9/liter) instantaneous bility to interference Mean y festations (g/Iiter) Slope Intercept Correl. is principally rather (Table dried than material purified partly human interfering compensate albumin proteins for the for calibration freezeof inthe presence with two classes of binding 4). T.27 0. would not seem to be a complete solution and places system that advantage transferrin (Table considerable constraint can be used. the pH that proteins which of the the was slow due could reagent. Human serum albumin is known anionic at the to have molecules high-affinity and hydrophobic which are electrostatic. Northam. Bull. respects of existing developed color slowly.. The could degree high also interfere of scatter specificity reaction of the restriction and it is acceptable. and Charoenpol. not suitable because albumin. Consequently. drugs and their this is probably about for human albumin. specimens methods molecules. coeff.21 0.95 of results of BCP with by bilirubin of BCP No. Because of the do the BCG methods should be are usu- it has been suggested analyzed by a specific abnormal sera Such methods for dealing immunochemical monovalent problems as demonstrated ally slow and impracticable numbers of specimens.87 (1971). The BCP with proteins in their immunochemically of these sufficiently as asalbumin are subject method.22 0. A.. BCG method the from similarly effect but the It of Albumin Preparations Used for Standardization Pure albumin Dade human stds from Hoechst Freeze-dried human serum icteric Consequently.986 1. SD about regression 1. The slow rate of color development terfering accuracy after mixing proteins of BCG serum (14) methods with per has been by reagent liter and (14.01 6. BCG.30 site for Binding electrostatic other site than fatty acids is by a combination forces. and AutoAnalyzer Widdowson. pure human preparations serum sessed on for with serum other better performance methods.. Watson. capacity of existing inexpensive mens. to several however. deviations electroimmunoassay.4 0. of BCP is its specificity by the or with 1).. Improved albumin method is unaffected has little effect but bilirubin at high estimation: Clin.82 6.95 1.37 1. automated CLINICAL CHEMISTRY. BCG serum albumin.77 0. G. W. albumin by B. Serum green method. E. 11. 4. while binding but can numerous BCG albumin of at in to but vary bind line (Si) the low-affinity sites. In the min are control of human by the BCG the difference and serum electroimmunoassay. (25). Albumin the measurement of serum albumin concentration green. H. Possibly in the all that is required zation on a cationic celles. Act. McPherson. Lolekha. Vol. site two general classes of binding (23). 26). The require a supply of high-titer with large methods antisera. 1978 85 . could binding and the Clearly.. or protein. dards and bromocresol 3. 5.00 1. M. Gun.48 1. of the bromocresol P. and bovine in calibration is much nonhuman the BCG “same-day-day” analysis BCP method. specimens (n) albumin.Table 4.

Rasanayagam. Clin. Effect of pH and inorganic interaction. Clin. P. Acta 53. 15. S.. Recent biosynthesis. and Price. A study isolated serum globulin 18. A. F. 108. An improved automated of serum albumin using bromcresol green. C... albumin. K. The immediate reaction between bromcresol as a measure of albumin content. Clin. plasma albumin by BCG analyzer and a comparison Clin. R.. C-B. Biochem. J. J. Acta 52. Newsletter use of L. 8. 1978 .. Leonard. Chem. on the phenol 438 (1973). of serum J. by En- and Motwani.. Tech. 18. 22. 24. Influence of reagent quality and reaction conditions on the determination of serum albumin by the bromocresol green dye binding method.. G. Biochem. and Durran. reactants” 22. Peters. P. Clin. Selective plasma (1973).. Spencer. 12. 105 (1977).J. and red/protein of serum albumin determination Thesis. Webster. 19... by J. Fatty (1975). 409 J.. of the interaction fractions. The use of electroimmunoassay specific proteins as a supplement to agarose gel for determining electrophoresis.. 1.. a modified bromcresol determination 23. of patients. from 49 9. U. Webster. 14. and immunoprecipitation albumin. 5 Chem.. International 13. C.338 7. its Jr. dye 17. 257 (1974). Am. R.. Clin. serum green 23. and Poquette. K. Clin. M. of albumin Acta in 49. L. Chim. Clin. J. p 111. S. 165 A. 510 13. K. Persaud. of bromocresol green for the determination 101 (1974). and Serum Ney. Bestimmung H.647 (1972).. Biochem. R. C.22(1975). M. A. Biochim. Improved specificity 26.. A. 24..5’-dibromo-o-cresol Microchem. An assessment of serum of the suitability albumin. Chem. Pat hoE. L. and estimation of “acute green reaction. Ultramicroestimation of Binding of the cationic dye 5. D. J. No. removal Clin. albumin D. of serum dye-binding al- 16. Chemistry. Federation p5. 23.. Ferreira. Clin. of affinity T.. 6. (Assoc.. P. 59. salts 295. zur Schirardin. by human J. Chem. II. Carter... Biophys. green with 109 (1974). J. Laurell. method Chem. Clin... K. Acta 53. 25. The estimation of binding on the Technicon SMA 12/60 with the HABA dye binding technique.Sc. for (1974). and Ann. M#{216}ller.. Von von termination the bromcresol 15.. Suppl. Pat ho!.for determining 20. Clin. and Lim. A comparison methods for the 55. Res. Pat ho!. of al34. and Pannell. Carter.. 1976. chromatography. L. Rodkey. 0. Chim. progress Clin. Serum structure albumin: and acid the under23. bromocresol green and detergents. No. and Attwood E. green of serum Spector. Albumin Eine vereinfachte mit Hilfe von (1972). A. Investigation methods. Chim.. 10. 617 5.. P. V.. March 1976. 14 (1973). 14. P. P. E. Beng.. H. M.. (1971). sera Hobbs. C. Acta C.. 21. Biochem. C.. Acta 35. 16. Corcoran. University of Birmingham. L. bumin in the 33 (1975). Webster. Clin. M. Arch. and serum (1977). of bromocresol Chim. Albumin green method. standing (1977). M. G. of bromocresol determination 20. Binding of bromocresol albumin. Chim. 663 by 12. Travis. 28. Measurement Bull.616 J. and Lau. Kragh-Hansen. Chim. ID. Bignell. C. Determination bumin with the SMA 12/60 by a bromcresol green method. J. Pinnell. D. Beng. Chem. phase Chem.. Ac/a human serum albumin: sulphonphthalein. 259 (1974). Vol.. determination serum albumin with bromcresol green. Micromethode Bromkresolgrun. by the (1976). Clin. Westgard.765 (1977). Solubility and absorption spectra of complexes resulting from interaction among human albumin. Gun. Lim. dye binding gland. Ann. binding to plasma green (1964). Gustafsson. Biophys. Lipid. Clin.. R. and Price. of Clinical J.) 6. 531 (1970). de- Clin. Clin. Slater.. E. 88 CLINICAL CHEMISTRY.

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