ANALYTICAL

Analytical Biochemistry 322 (2003) 275–278

BIOCHEMISTRY

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Notes & Tips

Antibiotic interference in protein assays: comparative evaluationof six photometric methods

Thomas Marshall* and Katherine M. Williams

Analytical Biochemistry Group, Sunderland Pharmacy School, Fleming Building, The University of Sunderland, Sunderland SR1 3RG, UK

Received 15 May 2003

Methods commonly used for determination of total

protein [1] include the biuret assay [2], the Lowry assay[3], and the Coomassie Brilliant Blue (CBB)1 protein

dye-binding assay [4]. Recently, the bicinchoninic acid

(BCA) assay [5] has been introduced as an alternative to

the Lowry assay and the Pyrogallol Red-molybdate

(PRM) assay [6,7] recommended as an alternative to the

CBB assay. Other options include the benzethonium

chloride (BEC) assay [8], a turbidimetric method widely

used in hospital laboratories. An important consider-ation is the susceptibility of the assay to interference [1]

and a common source of interference is antibiotics [9].

The biuret assay is prone to interference from ampicillin

and vancomycin [10], penicillin interferes in the Lowry

assay [11–13], ampicillin and penicillin G interfere in the

BCA assay [14], and gentamicin and neomycin interfere

in the PRM assay [6,7]. The present study compares the

response of six protein assays to 24 antibiotics.

Materials and methods

Materials. Actinomycin D (A4262), amoxicillin

(A8523), ampicillin trihydrate (A6140), cephalothin so-

dium salt (C4520), chloramphenicol–water-soluble

powder (C3175), clindamycin hydrochloride (C5269),dihydrostreptomycin sequisulfate (D7253), geneticin di-

sulfate (G5013), gentamicin sulfate (G3632), hygromycin

B (H7772), kanamycin monosulfate (K4000), lincomycin

hydrochloride (L6004), mitomycin C (M0503), neomy-

cin trisulfate hydrate (N6386), paromomycin sulfate

* Corresponding author. Fax: +44-191-515-3747.

E-mail address: tom.marshall@sunderland.ac.uk (T. Marshall).1 Abbreviations used: CBB, Coomassie Brilliant Blue; BCA,

bicinchoninic acid; PRM, Pyrogallol Red-molybdate; BEC, benzetho-

nium chloride; BSA, bovine serum albumin; CSF, cerebrospinal fluid.

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2003.08.010

(P9297), polymyxin B sulfate (P1004), puromycin dihy-

drochloride (P8833), spectinomycin dihydrochloridehydrate (S9007), streptomycin sulfate (S6501), tetracy-

cline hydrochloride (T3383), tobramycin sulfate (T1783),

tylosin tartrate (T6134), and vancomycin hydrochloride

(V2002) were purchased from Sigma–Aldrich (Poole,

Dorset, UK). Penicillin G was supplied by Britannia

Pharmaceuticals (Redhill, Surrey, UK). The antibiotics

were solubilized at 10, 5, 1, 0.5, 0.1, 0.05, and 0.01 g/L in

0.1mol/L phosphate buffer, pH 7. Actinomycin D,amoxicillin, puromycin, and tetracycline proved less

soluble and were investigated at 1, 0.5, 0.1, 0.05, and

0.01 g/L. Bovine serum albumin (BSA; Sigma A7906)

was solubilized at 10 g/L in buffer, calibrated using the

Sigma biuret assay, and diluted to 5, 2, and 1 g/L (for

calibration of the protein assays).

Protein assays. For biuret assay [2], 0.5mL of anti-

biotic or 0.1–0.5mL of BSA protein calibrator (5 g/L), ina sample volume of 0.5mL adjusted with phosphate

buffer, was mixed with 0.5mL of biuret reagent (541-2;

Sigma–Aldrich). After 10min, absorbance was mea-

sured at 540 nm using a Jenway 6100 spectrophotometer

(Dunmow, Essex, UK) zeroed with a reagent blank. For

Lowry assay [3], 0.2mL of antibiotic or 0.02–0.1mL of

BSA protein calibrator (1 g/L), in a sample volume of

0.2mL, was mixed with 1mL of Lowry reagent C fol-lowed, after 15min, by 0.1mL of Lowry reagent E.

After 30min, absorbance was measured at 600 nm. For

BCA assay [5], 50 lL of antibiotic or 5–50 lL of BSA

protein calibrator (1 g/L), in a sample volume of 50 lL,was mixed with 1mL of BCA reagent (B9643; Sigma–

Aldrich) and incubated at 37 �C for 30min. Absorbance

was measured at 562 nm. For protein dye-binding assay

[4,6,7], 20 lL of antibiotic or 5–20 lL of BSA proteincalibrator (1–2 g/L), in a sample volume of 20 lL, wasmixed with 1mL of either CBB reagent (610-2; Sigma–

Aldrich) or PRM reagent (611-2L; Sigma–Aldrich).

276 Notes & Tips / Analytical Biochemistry 322 (2003) 275–278

After 10min, absorbance was measured at 595 nm (CBBassay) or 600 nm (PRM assay). For BEC assay [8], 20 lLof antibiotic or 5–20 lL of BSA protein calibrator (1 g/

L), in a sample volume of 20 lL, was mixed with 0.8mL

of 0.5mol/L sodium hydroxide containing 33mmol/L

EDTA. This was followed immediately by the addition

of 0.2mL of 2 g/L BEC. After 50min, absorbance was

measured at 600 nm.

The antibiotics were assayed at 10, 5, 1, 0.5, 0.1, 0.05,and 0.01 g/L and the absorbance/lg antibiotic calculated

from the sample dilution giving an absorbance reading

within the linear working range of the assay. This was

expressed as a percentage relative to the absorbance per

lg BSA.

The linearity of the response of the protein assays to

the antibiotics was subsequently investigated by mea-

suring absorbance (A) as a function of increasingamount of antibiotic in the absence of protein. The

additive effect of the interference was also investigated in

the presence of protein by comparing AANTIBIOTICþBSA

with AANTIBIOTIC +ABSA.

Results and discussion

Amoxicillin, ampicillin, cephalothin, actinomycin D,

polymyxin B, and tetracycline interfered strongly in the

biuret assay (Table 1). Polymyxin B gave a normal violet

Table 1

Antibiotic interference in protein assays expressed as a percentage relative t

Class Antibiotic B

Aminoglycoside Dihydrostreptomycin

Geneticin

Gentamicin

Hygromycin B

Kanamycin

Neomycin

Paromomycin

Streptomycin

Tobramycin

b-Lactam Amoxicillin 1

Ampicillin 1

Cephalothin 1

Penicillin G

Lincosamide Clindamycin

Lincomycin

Actinomycin D 1

Chloramphenicol

Mitomycin C

Polypeptide Polymyxin B

Aminonucleoside Puromycin

Aminocyclitol Spectinomycin

Tetracycline 1

Macrolide Tylosin tartrate

Glycopeptide Vancomycin

The results are the mean of four separate assays (CV <5%). The values ind

the absorbance/lg BSA (gradients of the BSA calibration curves: biuret¼protein concentration value of a sample will be increased by this percentage

provided that the interference is linear and additive and BSA is used as a p

color, the b-lactams (amoxicillin, ampicillin, and ceph-alothin) gave an abnormal yellow color; and actinomy-

cin D and tetracycline gave an abnormal lime green

color. Lower levels of interference were evident with the

aminoglycosides (bright blue except for hygromycin B

which produced turbidity), clindamycin, lincomycin

(bright blue), mitomycin C, puromycin, spectinomycin,

and vancomycin (Table 1). The Lowry assay responded

strongly to the b-lactam antibiotics, the lincosamides,polymyxin B, tetracycline, and vancomycin and less

strongly to the aminoglycosides, actinomycin D, puro-

mycin, and tylosin tartrate (Table 1). Dihydrostrepto-

mycin, neomycin, paromomycin, and streptomycin

produced turbidity in the Lowry assay when assayed at

10 g/L. The BCA assay showed intense interference with

the b-lactam antibiotics and tetracycline and lower levels

of interference with actinomycin D, polymyxin B, andvancomycin (Table 1). The PRM assay responded

strongly to polymyxin B and the aminoglycosides gen-

tamicin, neomycin, paromomycin, and tobramycin

(Table 1). The CBB assay did not respond significantly

to any of the antibiotics tested (interference <2% in all

cases). The BEC assay responded only to the polypep-

tide antibiotic polymyxin B (interference¼ 15%).

Linear and additive responses (AANTIBIOTIC +ABSA ¼AANTIBIOTICþBSA) were demonstrated for amoxicillin,

ampicillin, cephalothin, polymyxin B, and tetracycline

in the biuret assay (using 2 g/L BSA), for amoxicillin,

o the response of BSA

iuret Lowry BCA PRM

15 14 <1 3

14 32 <1 26

10 22 <1 304

49 8 1 1

10 2 2 21

12 36 <1 442

12 30 <1 135

15 35 6 7

8 <1 <1 259

50 307 448 1

50 95 508 <1

55 90 342 <1

4 101 437 <1

38 253 5 <1

44 293 7 <1

11 57 78 2

3 <1 0 <1

46 10 10 2

88 147 61 175

32 50 <1 7

29 8 1 <1

00 303 425 3

2 32 1 <1

26 137 30 <1

icate the absorbance/lg antibiotic expressed as a percentage relative to

0.00025, Lowry¼ 0.0072, BCA¼ 0.025, PRM¼ 0.013). The measured

when a sample contains protein and antibiotic at equal concentrations,

rotein calibrator.

Notes & Tips / Analytical Biochemistry 322 (2003) 275–278 277

ampicillin, lincomycin, polymyxin B, and tetracycline inthe Lowry assay (using 0.2 g/L BSA), and for gentamicin,

neomycin, paromomycin, polymyxin B, and tobramycin

in the PRMassay (using 1 g/L BSA). The BCA assay gave

a linear response with the b-lactam antibiotics and tetra-

cycline but the interference was less than additive with

BSA (0.4 g/L); i.e., AANTIBIOTICþBSA <AANTIBIOTIC

+ABSA. For those assays giving a linear and additive re-

sponse, Table 1 indicates the percentage increase in themeasured protein concentration value of a sample when

protein and antibiotic are present in equal amounts. An

increase in the ratio of protein to antibiotic in the sample

will proportionally reduce the percentage increase due to

interference and vice versa.

Antibiotics are widely used as therapeutic [15] and

antimicrobial agents (http://www.sigma-origins.co.uk/

pdfs/articles/1031848913.pdf) at levels (Table 2) whichwill interfere in protein assays. The PRM assay is rec-

ommended for clinical determination of urinary protein

[6,7] and the aminoglycosides gentamicin, kanamycin,

neomycin, streptomycin, and tobramycin will interfere

with this application (Tables 1 and 2). Modified versions

Table 2

Antibiotic levels (g/L) in human body fluids and tissue culture growth

medium

Antibiotic Blood

plasma

Urine Growth

medium

Dihydrostreptomycin NR NR 0.100

Geneticin NR NR 0.800

Gentamicin 0.004 >0.100 0.050

Hygromycin B NR NR 0.800

Kanamycin 0.030 1.000� 0.100

Neomycin 0.004 0.100� 0.050

Paromomycin NA NA 0.100

Streptomycin 0.050 0.900� 0.100

Tobramycin 0.012 0.600 NR

Amoxicillin 0.010 >0.300 NR

Ampicillin 0.014 1.000 0.100

Cephalothin 0.030 2.500 0.100

Penicillin G 0.012 0.540� 0.060

Clindamycin 0.010 0.030� NR

Lincomycin 0.018 NA 0.100

Actinomycin D NA NA 0.001

Chloramphenicol 0.018 0.100� 0.005

Mitomycin C NA NA 0.050

Polymyxin B NA 0.100� 0.050

Puromycin NR NR 0.100

Spectinomycin 0.160 2.000� 0.020

Tetracycline 0.005 0.300 0.010

Tylosin tartrate NR NR 0.008

Vancomycin 0.060 0.900� NR

The values for the body fluids are maximum levels following

therapeutic use [15]. Asterisks indicate estimates based upon the

maximum recommended dosage and the percentage of antibiotic ex-

creted unchanged within 24/48 h [15], assuming 1L urine/24 h. The

values for growth medium are those recommended by Sigma (http://

www.sigma-origins.co.uk/pdfs/articles/1031848913.pdf). In some cases,

the information is not available (NA) or the antibiotic not recom-

mended (NR).

of the biuret, Lowry, and BCA assays, incorporatingprotein precipitation, are also recommended for urinary

protein determination. Antibiotics bind strongly to

protein [15] and interference will persist if the antibiotics

are coprecipitated with the protein. Protein determina-

tion of blood plasma or cerebrospinal fluid (CSF) is

unlikely to be affected by antibiotic interference. Plasma

levels of antibiotics are low (Table 2) and antibiotics

diffuse only to a small extent into CSF unless the me-ninges are inflamed [15]. High levels of antibiotics are

routinely recommended as antimicrobial agents for tis-

sue culture (Table 2). Thus, interference will occur when

protein assays are applied to growth medium.

In summary, the present study evaluates the response

of six commonly used protein assays to 24 antibiotics.

The biuret and Lowry assays are sensitive to interference

from a wide range of antibiotics. The BCA assayis particularly prone to interference from b-lactamantibiotics (and tetracycline) and the PRM assay to in-

terference from the aminoglycosides (particularly gen-

tamicin, neomycin, paromomycin, and tobramycin). In

contrast, the CBB and BEC assays are essentially free

of antibiotic interference and should be used in prefer-

ence to other methods when this type of interference is

suspected.

References

[1] C.V. Sapan, R.L. Lundblad, N.C. Price, Colorimetric protein

assay techniques, Biotechnol. Appl. Biochem. 29 (1999) 99–108.

[2] A.G. Gornall, C.J. Bardawill, M.M. David, Determination of

serum proteins by means of the biuret reaction, J. Biol. Chem. 177

(1949) 751–766.

[3] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein

measurement with the Folin phenol reagent, J. Biol. Chem. 193

(1951) 265–275.

[4] M.M. Bradford, A rapid and sensitive method for the quantita-

tion of microgram quantities of protein utilizing the principle of

protein-dye binding, Anal. Biochem. 72 (1976) 248–254.

[5] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H.

Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J.

Olson, D.C. Klenk, Measurement of protein using bicinchoninic

acid, Anal. Biochem. 150 (1985) 76–85.

[6] Y. Fujita, I. Mori, S. Kitano, Color reaction between Pyrogallol

Red-Molybdenum (VI) complex and protein, Bunseki Kagaku 32

(1983) E379–E386.

[7] N. Watanabe, S. Kamel, A. Ohkubo, M. Yamanaka, S. Ohsawa,

K. Makino, K. Tokuda, Urinary protein as measured with a

Pyrogallol Red-Molybdate complex manually and in a Hitachi

726 automated analyzer, Clin. Chem. 32 (1986) 1551–1554.

[8] J. Iwata, O. Nishikaze, New micro-turbidimetric method for

determination of protein in cerebrospinal fluid and urine, Clin.

Chem. 25 (1979) 1317–1319.

[9] D.S. Young, Effects of Drugs on Clinical Laboratory Tests, fourth

ed., AACC Press, Washington, 1995.

[10] J.A. Lott, P. Warren, Estimation of reference intervals for total

protein in cerebrospinal fluid, Clin. Chem. 35 (1989) 1766–1770.

[11] H.A. Zondag, G.L. Van Boetzelaer, Determination of protein in

cerebrospinal fluid: sources of error in the Lowry method, Clin.

Chim. Acta 5 (1960) 155–156.

278 Notes & Tips / Analytical Biochemistry 322 (2003) 275–278

[12] D.J. Silverman, Interference with the Folin reaction by penicillin

and some of its semisynthetic derivatives, Anal. Biochem. 27

(1969) 189–191.

[13] F.W. Sunderman, Drug interference in clinical biochemistry, Crit.

Rev. Clin. Lab. Sci. 1 (1970) 427–449.

[14] T. Marshall, K.M. Williams, Drug interference in the Bradford

and 2,20-bicinchoninic acid protein assays, Anal. Biochem. 198

(1991) 352–354.

[15] J.E.F. Reynolds (Ed.), Martindale the Extra Pharmacopoeia, 31st

ed., Royal Pharmaceutical Society of Great Britain, 1996.