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

Wis.; borough, with

and albumin.

Fisons England) solution. lauryl

Scientific perform

Apparatus similarly

Ltd., in their

Loughreaction

PI.MP

SAMPLER 60/I, II
1mm)

It

r-(mu

Brij-35 oxyethylene Mo.)

Dissolve 25 g of Brij-35 (polyether; Sigma Chemical Co., St. Louis, water, with warming, and dilute to 100

016
0.16

Air
Somple

Sen
inset

suc
!2.........Workirig
14 timrns H31 Air

in distilled

BCP

Stock acetic acetic acid (AR

acid solution. Dilute grade) to 1 liter with

150 ml distilled

of glacial water.

0 60 L2..._Workint

Diln.d

.w.nnl.

BCP
reogent Air

INSET

Working BCP reagent, 40 j.tmol of BCP per liter. Dissolve 10 g of sodium acetate trihydrate (AR grade) in about 800 ml of distilled water. Add 10 ml of stock acetic of stock acid BCP solution, solution, 1 ml of Brij-35 and and acid dilute solution, to 1 liter with and 1 ml distilled
COLORIMETER
15mm flow cell

.60

2 50

From

Ibm

cell

-p

water. Check the pH 0.03 with stock acetic dium least hydroxide a week chloride solution. at room

if necessary adjust to 5.2 solution or 0.5 mol,liter soThe reagent Dissolve is stable for at

RECORDER

600nm

temperature. solution. 9 g of sodium albumin Hospital [cat. no. Supply

Fig.
Inset

1. Flow
shows

diagram

for the BCP method

system

Sodium

air re-injection

chloride in 1 liter of distilled water. Standards. For this we used human B5158; Dade Biochemicals, American (U.K.)

were known

measured, serum and

a standard albumin

curve

was

plotted, were

and determined. Hoechst

un-

concentrations

Ltd.,

Didcot,

England]

specified

as 100% albumin assayed by the the contents chloand concen-

by moving-boundary macro-Kjeldahl

electrophoresis and procedure. Reconstitute

Equipment Pharmaceuticals

reagents were obtained from Ltd., Hounslow, England.

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.

BCP
exhibits

and The

Albumin
working BCP maxima reagent at 436 absorbance

dilute with sodium chloride solution trations up to 60 g/liter. Equipment. AutoAnalyzer I modules: Mkl Pump, Colorimeter (with 15-mm cell), and Basingstoke, The dilution injected used Recorder (Technicon England). system 200-fold. to minimize BCG Particular pattern, of about with air in a modified bubble The

Mk2 Sampler, N tubular flow Co. Ltd.,

and 590 nm, tively. With increases, Brij-35 noted causes in the trum Addition but causes with

with absorbances increasing pH that at 436 the opposite (2). to

of 0.91 and the absorbance

0.11, respecat 590 nm of also

Instruments (Figure 1) gives

nm decreases. Addition effect, a phenomenon the working BCP

continuous-flow

a sample is (as

BCG

resampled stream sample interaction E. C. Albutt, is necessary peaks will personal to achieve result.

of albumin

reagent a shift specan abon ab-

method; care or noisy

an increase peak from of albumin

in absorbance at 590 nm with 590 to 603 nm. The difference plus reagent vs. reagent shows at 603 nm. effect of reagent

communication). a regular BCG We with

sorbance maximum We examined the

conditions

Method used the the standards method of Northam described above, and and Widdowson measured (1), ab-

sorbance at 636 used in the U.K., and buffer 1 ml

nm. In this method, a reagent with 40 mol solution pH 3.8) (250 is used.

which is widely of BCG per liter g!liter) in citrate

sorbance of the albumin!BCP complex at 603 nm by adding 0.1 ml of a 40 g!liter solution of albumin to 20 ml of BCP reagent, using a reagent blank. Conditions other than those under examination were kept as in the original gradually method over he (above). The absorbance pH interval 5.1 to 5.6, from increased 0.275 to

of a Brij-35

(50 mmol/liter,

0.324. Similarly, increasing the BCP concentration from 40 to 100 i.tmol!liter caused an increase in absorbance from 0.292 to 0.313. Addition of Brij-35 solution up to
0.5 mL/liter increased the absorbance, but from 0.5 to

Electroimmunoassay Laurell rocket technique standards (as above) were diluted chloride solution in diameter, cut contained human albumin mmol,liter, pH strength under stained The was used. Serum and 250-fold with sodium

1.5 ml,liter the absorbance gradually at higher Brij-35 Reagent conditions were

was constant, concentrations. chosen (a)

declining to achieve the

and 5 sl was applied to a well 2.5 mm in a gel layer 1.5 mm thick. The gel per liter, monovalent antibuffer (50 at a field antiserum, 8.6). 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, and (b) to be such that small inaccuracies in the routine preparation of the BCP reagent (i.e., buffer, minimum od.
CLINICAL CHEMISTRY, Vol. 24, No. 1. 1978 81

of 10 V!cm for filter paper layers, with Coomassie

3 h, the plates were pressed dried with a hair-dryer, and Brillant Blue R. Peak heights

Brij-35, effect

and BCP upon the

concentration) reproducibility

would of the

have meth-

0.50

0.40

V
//

LU 0

z
0

0.30

cr1

cr1 4

0.20

0.10

//
Fig. 2. Standard
ious sources B, freeze-dried

20
ALBUMIN

30

40
g/liter

50

60
Fig. 3. Recorder trace of analysis of freeze-dried human

curves A, purified
human

prepared
human serum

with use of albumin

albumin (Dade

from varTravenol

serum 0 Pak
water 1, 1 in

Biochemicals);

I unassayed (Travenol Laboratories Ltd.) was reconstituted with distilled to 57.5 g of albumin per liter. This was diluted in sodium chloride solution:

(0 Pak I unassayed,

5;

2,

2 in

5; 3,

3 in

5; 4, 4

in 5;

5, undiluted.

Chart

speed,

50.8 cm/h

C, purified human albumin (Hoechst Pharmaceuticals Ltd.); D, purified human albumin (Sigma Chemical Co.). In A-D, absorbance was measured at 603 nm by the BCP method. E, purified human albumin (Hoechst Pharmaceuticals
Laboratories Ltd.);
Ltd.); absorbance measured at 636 nm by the BCG method

Sample groups ceded

interaction. of three specimens

To

evaluate of low

this,

we analyzed pre-

concentration,

with
Standards. rations described (specified inpurities); albumin albumin solution correct method, related (Figure highest were above; Three obtained: from purified that Hoechst human from Dade albumin prepaLtd. Biochemicals

by three of high concentration. At 60 samples!h, a sample!wash ration of 1!1, serum containing 11.5 per liter assayed was increased to an apparent next after serum containing Interaction Approach 12.1 57.5

g of albumin g/liter when

Pharmaceuticals

as >99% albumin, and from Sigma containing preparations to give for moisture concentration up 2). to an The Dade with

with no detectable protein Chemical Co. (crystallized per 100 g). These in sodium chloride We did not With were the BCP linearly gave light the path

g of albumin per liter. specimens was negligible.

from low to high to the continuous

1-3 g of globulins were dissolved or other and albumin albumin BCP impurities. absorbance concentration

aspiration plateau was within two transmission lines (i.e., 91% of steady state in terms of absorbance units, 97% in terms of transmission units) at a concentration
of

a concentration

of 60 g!liter.

57.5 g
Precision.

of

albumin distributing 13.0 g/liter

per

liter

(Figure precision

3). was diluted both measured with sodium 35.9 precision concentraby conof albumin

Within-batch (serum between serum

randomly centration chloride g!liter

20 specimens pool 20 pool). assess pools For

of 60 g!liter in a 1-cm

preparation (0.45

solution) (undiluted

of concentration concentrations

absorbance

at 603 nm). The lower absorbances, impurities because and, only the

Hoechst and Sigma possibly reflecting in particular, Dade albumin the was

preparations gave the presence of of moisture, in vacTravenol was than the conwith provided and Dade abalbusupplied

the CV was we prepared tions sera

0.43%. To two serum

between-batch with albumin

presence

of 23.1 g/liter with low and

and 39.6 g!liter normal albumin

(by pooling patients concentrations, re-

uum-sealed ampoules. A freeze-dried serum Laboratories reconstituted recommended centration, use by Hoechst which of a commercial Ltd., 10 ml, was

spectively),

(Q Pak
to achieve then serum

I unassayed; Norfolk, water the high standard Linearity by the rather

Aliquots

divided into aliquots, and stored at 4 #{176}C. of each serum pool were analyzed, in 20 batches weeks. Each batch We used the same acetic acid solution, from bro-

Thetford,

England) albumin

in 6 ml of distilled

each, on different days during four included eight to 15 serum specimens. manifold, stock BCP solution, stock and

electroimmunoassayed Ltd.

assayed

Pharmaceuticals to that 2).

Brij-35 solution throughout this period. Specificity. 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). 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).
Vol. 24, No.

cals, Pharmacia (Great Britain) Ltd., London] eluate from the gel columns was examined presence of albumin by immunoelectrophoresis

CLINICAL

CHEMISTRY,

1, 1978

Table 1. Apparent Albumin Concentration of Albumin-Free Serum Eluates Prepared by Affinity Chromatography
Nature of

Table

2. Reaction

of BCG (a)

with Pure Human

with Pure Human of Pure Human

Transferrin Transferrin

Alone, and (b) in the Presence

specImen

Total proteIn g/liter

AIbumIn BCG method

concn, BCP method

g/liter RID8

Albumin
(a)

Apparent
Transferrln concn 9/lIter albumin concn

%
transferrin

of

Crohns

disease

36.0 31.0 18.0 17.0

11.0 7.0 6.5 5.5

0.5

0.7

Diffuse band on electrophoresis Nephrotic syndrome

Glomerular
a

0
0 0

0
0

assayed
albumin

as

5.0
10.0 0

4.3
6.5

86 65 47 45 41

nephritis
immunodiffusion.

20.0
30.0 40.0

9.5
13.5 16.5
(b)

Radial

monovalent antihuman Comparison precipitin disappeared Eluates tients method, from were and

antihuman-albumin serum arc (Hoechst with the original corresponding the analyzed the gel columns by the

serum

and

polyvalent Ltd.). the had paBCG

Transferrin 6.7 g/Iiter + pure human albumin g/llter

Pharmaceuticals serum showed that to serum albumin proteins from method,

Apparent

% of
transferrln

albumin concn.

assayed
albumIn

as

and all other

detectable BCP radial

remained. four the 8.5

17.0 26.5

of serum

12.0

52
30

19.0 28.0
35.0 43.5

method
apparent method, concentration by either methods, detected Human

(Hoechst

Mancini Pharmaceuticals

immunodiffusion Ltd.) (Table 1). The assayed of the was by the BCG total protein not detected also Ltd.), impurities, to give conTime
after mixing

33.5
42.5

albumin concentrations, were between 23 and of the the except eluate. BCP or the in specimen (Hoechst

22 22 15

32%

Albumin radial 1, where

immunodiffusion albumin was

by immunoelectrophoresis. transferrin Pharmaceuticals with no detectable chloride solution

Table 3. Rate of Color Development of the Reaction of BCG with Albumin, Serum, 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

globulin prepn

in the range 5 to 40 g/liter. arc by immunoelectrophoresis antiserum. BCG Each methods. solution Transferrin

We detected only against a was analyzed had a signifiby

A
lOs 30 s 1 mm 5 mm

.310 .300
.300

.330 .321 .320 .321

.320

.281

.221 .221 .223 .231 .249

.282
.280

.060 .080
.108

.041
.050

.053 .061 .094 .095

timed from mixing

cant reaction with BCG (Table 2) even in the presence of albumin but showed no reaction with BCP. Color development of the BCP reagent with purified human lOs action preparations ately or after reaction with human stantaneous. ment 3). We estimated animal-based bumin for quality-control the serum albumin specimens, 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. No re-

.301
.305

.280 .282 .282

.150
.190

30 mm
60 mm
a Absorbance

.307 .321 .250 at 636 nm vs. reagent at intervals 20 cl of protein solution with 4 ml of BCG reagent.

.202
shown,

solutions,

1 h. Some serum samples BCG while with other solutions transferrin continued color over solutions,

albumin method,

concentration 23.9 g/liter

of 39.0 by the BCP

g/liter method.

by

the

BCG

development a period

Effect of Interfering Method

Blank correction. 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. sera reg of were 1

were analyzed bcp itself had absorbance.

Icteric

calibration. sera

Two freeze-dried (Wellcome Reagents

bovine-based Ltd., Beck-

concentrations corrections per grossly albumin

up to 500 izmollliter of between 0.5 and 1.5 liter. Similar sera at lOg corrections (approximately of hemoglobin the greatest in one extreme milk.

enham,
of 31.0 12.6 Pure Ltd.) the the and

England) and 36.0 12.9

had apparent g/liter by the respectively,

albumin concentrations BCG method, but only by the BCP method. of by by an

albumin

g/liter,

hemolyzed per liter

per
corexand to with
83

bovine serum albumin (Hoechst Pharmaceuticals in sodium chloride solution at a concentration gave method method. an apparent albumin concentration 15.0 g/liter serum had BCG BCP of 43.0 g/liter, An unassayed but only equine

liter). Grossly lipemic sera required rections, 3.5 g/liter being necessary ample where the serum looked salicylate, standards,
CHEMISTRY,

40 g/liter

like

Salicylate. pure human

Sodium albumin
CLINICAL

added to serum did not interfere

Vol. 24, No. 1, 1978

so BCP

//

so BCG

40

40

30 /

30

C 20
4

C 20 .4 .7.
7.

4
./

u #/
/

/ / / /

/ /

/ /

10

.,,

/,4
/,

/
Albumin 9/liter

/
/ / /
/

/ /

EIA
30 40 50

/
10

Albumi,, 20

g/liter 30 40

E IA
50

10

20

Fig.

4. Comparison methods

of results

by the

BCP

and

electroimmu-

Fig. 5. 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.,

mg/liter. Bilirubin.

5), the regression (n= 160,r=O.986). We further used above) the human albumin standardization. 4).

equation compared BCG and (Hoechst Similar

being (with

y(BCG) different

l.Olx sera with

+ 6.77 to those

specified as essentially serum and to purified a concentration of 500 was noted at concentrations The apparent albumin lution was 39.4 g/liter 250 zmol, and tively. Sera with be underestimated bilirubin were by the

was added to standards up to

BCP methods, Pharmaceuticals results were

purified Ltd.) for (Table Diag-

umol/liter. Minor interference exceeding 170 /Lmol/liter. of a 40 g/liter in the presence per liter, respectend to sera with liter soof

obtained

concentration and 38.3 g/liter of bilirubin

A
nostics

freeze-dried Ltd.) was

serum (Validate; General standardized by electroimmunoassay

500 zmol

high bilirubin by the BCP between

concentrations method. Five 113 and

with use maceuticals calibration BCG as good methods and

of purified human Ltd.) Although gave as that (Table closer between 4). agreement electroimmunoassay

albumin (Hoechst Pharthe freeze-dried serum for between methods, results it was and by the still not BCP

concentrations

192 mol/

estimated by the noassay. Concentrations 0.2 range g to for 4.6 g/liter However, mol/liter), ference 17 sera

BCP method by the BCP (mean with bilirubin

and electroimmumethod were lower 2.4 g/liter). in concentrations

electroimmunoassay

difference,

Discussion
The BCP BCG methods. 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. method (1),

25 to 79 jzmol/liter analyzed by both in albumin concentration

(mean concentration, 52 methods, the mean difwas only 0.1 g/liter.

Comparison of the BCP with Electroimmunoassay

Sera with albumin 45 g/ liter were assayed and by electroimmunoassay. formed rioration. (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, an effect that is greatest at the isoelectric point (pH 4.3) of the complex (9). Precipitation is inhibited, but not eliminated, by the surfactant Brij-35. Neither precipitation nor the associated with BCP, negative even in the baseline absence pH effect (1) were observed due BCG of Brij-35, possibly

working purified were

day, to avoid sample detehuman albumin standards used for all three methods. range Sera hemolysis, methequation n = conof

Biochemicals) selected bilirubin,

Sera were albumin, showing presence Results ods (Figure

to provide the maximum and globulin concentration. also (lipemia, selected.

to the higher reagent Several comparisons

used in the BCP method. have been made between

other abnormalities of drugs, etc.) were

and specific methods (11-15). BCG methods overestimate

Although all agree that serum albumin concen-

by the BCP and electroimmunoassay 4) agreed well, with the regression

=

tration, the magnitude of the inaccuracy varies. This may reflect several factors: reagent composition (10), calibration material, time allowed for color development, and the source of specimens. agreement with electroimmunoassay in the normal range when freeze-dried In our study, was much better serum was used

being y(BCP) 160, r = 0.986). siderably

84 CLINICAL

0.95x Results than

+ 1.72 by the

(in g albumin/liter, BCG method were

higher

by electroimmunoassay
Vol. 24. No. 1, 1978

(Figure

CHEMISTRY,

Table 4. Regression Equations Describing Comparisons between BCP, BCG, and

Electroimmunoassay (EIA) Methods, with Various

concentration rarely by more regression has been underestimates line suggested

were than of BCP

sometimes two standard against that sera. the (12)

underestimated, deviations electroimmunoassay. 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,

bilirubin is undesirable but other molecules, particularly lites, in the line. The could degree high also interfere of scatter specificity reaction of the restriction and

it is acceptable. drugs and their this is probably about for human albumin, could binding and the

Clearly, metaboreflected regression

y x

BCP
EIA 160 27.7 27.9

BCG
EIA 160 27.7 34.8

BCG
BCP 160 27.9 34.8

BCG
BCP 120 29.4 35.8 1.02

BCG
EIA 80 31.0 32.5 0.85

BCP
EIA 80 31.0 30.4 0.95

of results of BCP with by bilirubin of BCP

No. specimens (n)

albumin, its susceptiwell be manito relatively

its

Mean x
(9/liter)

instantaneous bility

to interference

Mean

festations

(g/Iiter)

Slope Intercept Correl. coeff.

0.95 1.72 0.986

1.01 6.77 0.986

1.06 5.27 0.989

few high-affinity binds to numerous

binding sites on serum fragments of bovine

albumin. BCG serum albumin,

5.81 0.977 1.82

6.22 0.988 1.00

1.21 0.988 1.13

although it also has a high affinity for the fragment that is the site for bilirubin binding (22). Human serum albumin is known anionic at the to have molecules high-affinity and hydrophobic which are electrostatic. site two general classes of binding
(23).

SD about regression

1.37

1.48

1.30

site for Binding electrostatic

other site

than fatty acids is by a combination forces, while binding but can numerous BCG albumin

of at in to but vary bind

line (Si)

the low-affinity sites, number, 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). Possibly in the all that is required zation on a cationic celles, of BCG used prompt action electrostatic reduced Investigation improvement specifically a BCP method for to improve measurement However, (25). serum gel, or protein. with binding in number of BCG

on serum

(24)

for the spectral change is immobilisupport (9) such as detergent miWe hypothesize sites, the pH that proteins which of the the was slow due could reagent. further of dyes the offers BCG reto be interfering to cationic by increasing

these proteins in test sera. 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, 25, 26).

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. stage

might permit or the design However, than of development

determination. respects of existing

developed color slowly, but still had reaction within 10 s (Table 3). Consequently, would not seem to be a complete solution and places system that advantage transferrin (Table considerable constraint can be used. of BCP is its specificity by the or with 1), which original (21). solutions great lack of reaction the albumin-free contained most concentration, The apparent estimated to explain

to the problem the analytical The principal albumin, pure human preparations serum sessed

on for with serum other

better performance methods. 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. The require a supply of high-titer

with large methods antisera, and (15). The BCP with

proteins in their immunochemically of these sufficiently

as asalbumin

are subject
method,

to several however,

technical
shows

excellent

agreement

concentration method was between method. In the min are control of human

by the BCG the difference and

serum

electroimmunoassay, capacity of existing inexpensive mens.

yet retains dye-binding

the simplicity and methods, permitting of many speci-

results

by electroimmunoassay equine for use reactivity However, and bovine in calibration is much nonhuman

the

BCG

same-day-day

analysis

BCP method, not suitable because albumin. their

albuor quality than that is mis-

less

References
1. Northam, albumin by B. E., and AutoAnalyzer Widdowson, G. M., using bromocresol No. 11, 1 (1967). Determination green. of serum Assoc. Gun. stanwith

material

clearly unsuitable for use with specific immunochemical methods, and its use with BCG methods can be leading (10). Dye-binding by competing by salicylate. specimens methods molecules. Purified containing

Biochem.,
2. Doumas, dards and bromocresol 3. McPherson, Modification 117(1972). 4. Lolekha,

Tech.

Bull.

are susceptible
The BCP bilirubin endogenous

to interference

B. T., Watson, W. A., and Biggs, H. G., Albumin the measurement of serum albumin concentration green. Clin. Chins. Act.s 31,87 (1971). I. G., and Everard, of the bromocresol P. H., and Charoenpol, D. W., Serum green method. W., Improved albumin

method is unaffected has little effect but bilirubin at high

estimation:

Clin.

Chim.

Ac/a
method

37,

automated

CLINICAL

CHEMISTRY,

Vol.

24, No.

1, 1978

85

for determining 20, 617 5. Beng,

determination

serum

albumin

with

bromcresol

green.

Clin.
method

Chem.
for

(1974). C. G., and Lim, K. L., An improved automated of serum albumin using bromcresol green. 59, 14 (1973). Von
von

termination the bromcresol 15. Slater, bumin in the 33 (1975).

and

estimation of acute green reaction. Clin. P. M., and Ann. of patients. Federation p5.

phase

Chem.

reactants 22,616 J. R.,

by the (1976). 12, Tech. Newsletter

use

of

L., Carter,
sera

Hobbs, Clin.

Measurement Bull.

of al34, No.

Am. J. Clin.

Biochem. Chemistry,

Pat ho!. 6.
zur

Schirardin,
Bestimmung

H., and
Serum

Ney, J., Albumin

Eine vereinfachte
mit Hilfe von (1972).

Micromethode
Bromkresolgrun. of serum dye-binding al-

16. International 13, March 1976,

of Clinical

J. Clin.

Chem.

Clin.

Biochem.

10,338

7. Westgard, J. 0., and Poquette, M. A., Determination bumin with the SMA 12/60 by a bromcresol green method. Clin. Chem. 18,647 (1972). 8. Leonard, P. J., Persaud, plasma albumin by BCG analyzer and a comparison Clin. Chim. Acta 35, 409 J., dye

17. Webster, D., A study isolated serum globulin 18. Pinnell, dye binding gland, 1976, 19.

of the interaction fractions. Clin.

of bromocresol Chim. Acta 53,

green with 109 (1974). by En-

and Motwani, R., The estimation of binding on the Technicon SMA 12/60 with the HABA dye binding technique. (1971).

A. E., Investigation methods. M.Sc. p 111. U., and red/protein

of serum albumin determination Thesis, University of Birmingham,

salts
295,

Kragh-Hansen, on the phenol 438 (1973).

M#{216}ller,J. V., Effect of pH and inorganic interaction. Biochim. Biophys. Ac/a human serum albumin: sulphonphthalein. of albumin Acta in 49, from 49

9. Beng, C. G., Rasanayagam, L., Lim, K. L., and Lau, K. S., Solubility and absorption spectra of complexes resulting from interaction among human albumin, bromocresol green and detergents. Clin. Chim. Acta 52, 257 (1974). ID. Spencer, K., and Price, C. P., Influence of reagent quality and reaction conditions on the determination of serum albumin by the bromocresol green dye binding method. Ann. Clin. Biochem. 14, 105 (1977). II. Webster, D., Bignell, A. H. C., and Attwood E. C., An assessment of serum of the suitability albumin. Clin. of bromocresol green for the determination 101 (1974). of bromocresol determination

20. Carter, P., Ultramicroestimation of Binding of the cationic dye 5,5-dibromo-o-cresol Microchem. J. 15, 531 (1970). 21. Travis, by J., and Pannell, R., Selective plasma (1973). 22. Peters, of affinity T., chromatography.

removal

Clin.
Recent biosynthesis.

Chim.
progress Clin.

standing
(1977). 23.

its

Jr., Serum structure

albumin:
and acid

the under23, 5

Chem.

Chim.

Acta

53,

Res.
green of serum

Spector, 16, 165

A. A., Fatty (1975).

binding

to plasma green (1964).

albumin. by human

J. Lipid.
serum green 23, 663 by

12. Ferreira, P., and Price, and immunoprecipitation albumin. Clin. Chim. Acta

C. P., A comparison methods for the 55, 259 (1974).

24. Rodkey, F. L., Binding of bromocresol albumin. Arch. Biochem. Biophys. 108, 25. Webster, and serum (1977).

510

13. Laurell, C-B., The use of electroimmunoassay specific proteins as a supplement to agarose

gel

for determining electrophoresis. albumin

D., The immediate reaction between bromcresol as a measure of albumin content. Clin. Chem. and Durran, S. M., Albumin green method. Clin. Chem.

J.
de-

Clin.
14.

Pat ho!.
Gustafsson,

28, Suppl.

(Assoc.

Gun.

Pat hoE.)

6,22(1975). of serum

J. E. C., Improved

specificity

26. Corcoran, R. M., a modified bromcresol

determination 23,765 (1977).

88

CLINICAL

CHEMISTRY,

Vol. 24, No. 1, 1978