ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 2, pp. 210–214. © Pleiades Publishing, Inc., 2009.Original Russian Text © M.A. Burkin, A.A. Burkin, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 2, pp. 232–236.

210

INTRODUCTION

Eremomycin is a polycyclic glycopeptide antibioticisolated from

Nocardia

orientalis

[1, 2]. It is severaltimes more active and less toxic than its structural ana-logues vancomycin, teicoplanin, and ristamycin. Simi-larly to other glycopeptides, eremomycin is effectivewith respect to Gram-positive aerobic and anaerobicmicroorganisms, such as coagulase-positive and coagu-lase-negative staphylococci, streptococci (including themultiple-drug-resistant strains), enterococci, coryne-bacteria, and clostridia. The binding of the antibiotic tothe terminal dipeptide D

-

Ala

-

D

-

Ala, in the precursor ofPeptidoglycan, blocks the synthesis of the latter. Inaddition, eremomycin disturbs the structure and func-tion of the cytoplasmic membrane of the bacterial celland the synthesis of RNA at the ribosomal level. Ere-momycin exerts a bacteriostatic effect on enterococci,coagulase-negative effect on staphylococci, and some

α

-hemolytic streptococci and a bactericidal effect onother microorganisms [3].

Pharmacokinetic studies and, in particular, drugconcentration monitoring in biological fluids (bloodplasma, serum, and urea) imply the use of an adequateanalytical method. Biological tests based on the sup-pression of the growth of antibiotic-sensitive microor-ganisms are time-consuming and nonspecific and, as aresult, are inapplicable when drug combinations areprescribed. Chromatographic analysis of these com-pounds is labor-consuming and expensive. The simplic-

ity, specificity, and sensitivity of immunochemicaltechniques make them more attractive compared to thefirst group of methods.

Various methods for the immunological determi-nation of vancomycin and teicoplanin described inthe literature include the polarization fluorescenceimmunoassay [4–6], radioimmune assay [5, 7],immunofluorescence immunoassay [7], as well asthe receptor–antibody sandwich assay for teicopla-nin, in which the albumin-

ε

–aminocaproyl–D

-

Ala

-

D

-

Ala conjugate is used as a receptor for selectiveanalyte adsorption [8].

The procedure of obtaining antibodies against ere-momycin (1558 Da) implies its covalent binding to aprotein carrier. Its structure allows several approachesto be used for obtaining conjugated antigens. Forinstance, the amino groups of the glycosidic moiety ofthe molecule can be bound to the amino groups of theprotein using a dialdehyde. The carboxy group can bethen activated by carbodiimide, followed by interactionwith free amino groups in protein macromolecules.Formaldehyde condensation makes it possible to per-form synthesis with the use of resorcyl or phenyl frag-ments of the eremomycin molecule, similarly to theprocedure for the estrogen-like mycotoxin zearalenone,which was described in our earlier paper [9].

Enzyme Immunoassay for the Determination of the Glycopeptide Antibiotic Eremomycin

M. A. Burkin and A. A. Burkin

Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, RAMS, Moscow, 105064 Russia e-mail: burma68@yandex.ru

Received October 22, 2007

Abstract

—An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using rabbitpolyclonal antibodies against the eremomycin–glucose oxidase conjugated antigen. This technique allows theglycopeptide antibiotic eremomycin to be determined both in aqueous solutions (with a sensitivity as high as0.1 ng/ml) and in blood plasma. The cross-reactivity of the antibodies with vancomycin was 0.4% of that foreremomycin, while teicoplanin was almost not recognized. Experiments with blood plasma samples diluted1 : 10 showed that the assay was linear over the concentration range 1–30 ng/ml and that the variation coeffi-cient did not exceed 10%. The high sensitivity and selectivity of this test make it suitable for pharmacokineticstudies and drug monitoring analysis.

DOI:

10.1134/S0003683809020161

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ENZYME IMMUNOASSAY FOR THE DETERMINATION OF THE GLYCOPEPTIDE 211

The goal of this study was to obtain antibodiesagainst eremomycin and develop an enzyme immu-noassay based on these antibodies for eremomycinquantification in biological fluids.

MATERIALS AND METHODS

Synthesis of conjugated antigens.

Three solutionscontaining 8 mg of gelatin (0.05

µ

mol) and 10 mg ofglucose oxidase (GO, EC 1.1.3.4; 0.05

µ

mol; Sigma,United States) in 1.5 ml of distilled water were supple-mented with 41, 83, 208, and 415

µ

l of 10 mg/ml ere-momycin (0.25, 0.5, 1.25, and 2.5

µ

mol, which corre-sponded to 5-, 15-, 25-, and 50-fold molar excess,respectively) and 30

µ

l of freshly prepared 2.5% glut-araldehyde. After incubation at room temperature for2 h under stirring, each reaction mixture was supple-mented with 100

µ

l of 2 mg/ml sodium borohydrideand incubated under stirring for another 2 h. Thusobtained conjugates, gelatin–eremomycin(5)a, gelatin–eremomycin(10)a, gelatin–eremomycin(25)a, and GO–eremomycin(50)a, were placed in dialysis bags and dia-lyzed for 2 days against 0.5% NaCl (three portions5 liters each).

Three samples containing 580, 1740, and 5800

µ

gof eremomycin (0.35, 1.05, and 3.5

µ

mol) in 0.6 ml ofdistilled water were supplemented with 15 mg of 1-ety-hyl-3–(3-dimethyl-aminopropyl) carbodiimide (78

µ

mol;Sigma, United States). After incubation at

30°ë

for30 min under stirring, 5 mg of BSA (0.07

µ

mol; ICNBiomedicals, United States) dissolved in 1 ml of0.05 M carbonate–bicarbonate buffer (pH 9.0) wasadded to the mixtures. The reaction mixtures were incu-bated at room temperature for 16 h and then dialyzedagainst 0.5% NaCl. Thus synthesized antigens weredesignated as BSA–eremomycin(5)c, BSA–eremomy-cin(10)c, and BSA–eremomycin(50)c.

To prepare BSA–eremomycin(50)f, 5 mg of BSA(0.07 mol) dissolved in 2 ml of distilled water wasmixed with 5800

µ

g of eremomycin (3.5

µ

mol) in0.6 ml of water and 0.3 ml of 37% formaldehyde(3690

µ

mol; Fluka, Germany). The mixture was incu-bated at

37°ë

overnight and then dialyzed. During dial-ysis, a precipitate was formed that was then dissolvedby the addition of one drop of 1 N NaOH.

Thus obtained conjugates were mixed with an equalvolume of glycerol and stored at –10 to

–15°ë

as solu-tions with a concentration of 1 mg/ml (with respect toprotein).

Changes in the UV spectra of the conjugates relativeto the spectrum of the carrier and/or the interaction ofobtained conjugates with antibodies detected in theELISA served as criteria of the binding of the hapten tothe carrier. UV spectra were recorded with a Hitachi-557 spectrophotometer (Japan). ELISA was performedin microplates (Costar, United States) and analyzedwith a Dynatech MR5000 photometer (Germany).

Obtaining antiserum.

Dark-colored rabbits weighing2.5–3 kg were subcutaneously injected at 10–15 points ontheir backs with GO–eremomycin(50)a emulsified inFreund’s complete adjuvant at a dose of 100

µ

g.Repeated immunization was performed with the samedoses of GO–eremomycin(50)a in aqueous solutions atone-month intervals. The animals were bled from themarginal ear vein seven days after each injection. Bloodserum was separated, mixed with an equal volume ofglycerol, and stored at –10 to

–15°ë

until use.

The development of immune response in the ani-mals was monitored by analyzing the interaction ofsequentially taken sera with each of the conjugates syn-thesized in the concentration range 0.05–0.5

µ

g/ml.Thus found optimum ratios of immune reagents werethen used in competitive analysis.

OHOH

HN

HO

O

HO

NH

ONH

O

NH

O

NH2

NH

O

NH

O

H3C

CH3

NH

CH3OH

Cl

OO

OO

NH2H3CHO

H3C

O

O

HO

OO

OH

OH

NH2H3CHO

H3C

O

O

212

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M. A. BURKIN, A. A. BURKIN

For this purpose, microplate wells were filled with0.2 ml of conjugated antigens in a 0.05 M carbonate–bicarbonate buffer (pH 9.5) and incubated at

4°ë

for16 h. After incubation, the wells were washed four orfive times with 0.15 M NaCl supplemented with 0.05%Tween 20 and then filled with 0.1 ml of eremomycinsolutions (1000–0.1 and 0 ng/ml) and 0.1 ml of antise-rum in 0.01 M phosphate-buffered saline (pH 7.5) sup-plemented with Tween 20 and 1% BSA (PBS-t). Afterincubation for 1 h, the microplate wells were washedagain and filled with 0.2 ml of a solution of antispeciesantibodies conjugated with horseradish peroxidase(Sigma, United States). After incubation for 1 h andsubsequent washing, wells were filled with 0.2 ml of asubstrate solution containing 0.4 mg/ml

o

-phenylenediamine (Sigma, United States) and 0.005% H

2

O

2

in a0.15 M citrate-phosphate buffer (pH 5.0). After incuba-tion for 45 min, the enzymatic reaction was stopped byadding 50

µ

l of 4 M sulfuric acid containing 0.1 MNa

2

SO

3

, and the resultant solution was assayed colori-metrically at 492 nm.

The level of antibody binding in wells with a zeroeremomycin concentration was taken as a control(100%) and used to calculate the percent of antibodybinding for each concentration of the relevant com-pound. The concentration-dependence curves werethen constructed.

The cross-reactivity of antibodies was calculated asthe ratio of the concentration of eremomycin thatcaused a 50% inhibition of antibody binding to the

solid phase (IC

50

) to the respective concentration ofstructurally similar antibiotics (vancomycin and teico-planin) and expressed in percent. The calibration curveswere constructed, IC

50

values were calculated, and theresults were statistically processed using MicrosoftExcel 2002 software.

RESULTS AND DISCUSSION

The spectrograms shown in Figs. 1a, 1b, and 1cdepict the changes in the spectra of proteins whichoccurred as a result of eremomycin binding and conju-gate formation. The intensity of peaks at 280 nm, whichcorresponds to the maximum absorption of eremomy-cin, increased in the following order: BSA–eremomy-cin(5)c < BSA–eremomycin(10)c < BSA–eremomy-cin(50)c and gelatin–eremomycin(5)a < gelatin–ere-momycin(10)a < gelatin–eremomycin(25)a (Fig. 1),with a simultaneous increase in the hapten–proteinratio in the reaction. The immunochemical activity ofthe conjugates (i.e., the ability to induce generation ofantieremomycin antibodies in animals) as well as the abil-ity of the solid-phase antigen to bind these antibodies andthe inhibition of this binding by free eremomycin addi-tionally corroborated the efficiency of synthesis.

Even in the first antiserum obtained in response toimmunization with GO–eremomycin(50)a, eremomy-cin could be detected by ELISA with a sensitivity ofapproximately 1.0 ng/ml. The serum obtained after thenext immunization allowed the detection limit of the

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

220 280 340nm

D

220 280 340 220 280 340

1

2

3

4

5

6

7

89

10

Fig. 1.

UV spectra of eremomycin-based conjugated antigens (0.1 mg/ml). Designations:

1

, glucose oxidase (GO), 0.1 mg/ml;

2

, GO–eremomycin(50)a;

3

, eremomycin, 50

µ

g/ml, H

2

O;

4

, BSA–eremomycin(5)c;

5

, BSA–eremomycin(10)c;

6

, BSA–eremo-mycin(50)c;

7

, gelatin–eremomycin(5)a;

8

gelatin–eremomycin(10)a;

9

, gelatin–eremomycin(25)a; and

10

, BSA–eremomy-cin(50)f.

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ENZYME IMMUNOASSAY FOR THE DETERMINATION OF THE GLYCOPEPTIDE 213

assay to be reduced by more than an order of magni-tude. This serum was used in further experiments.

All synthesized conjugates were immunochemi-cally active. After the selection of optimal conditions ofanalysis, the working concentrations of the serum andantigens were determined. Figure 2 shows the results oferemomycin quantitation using three different solid-phase antigens (BSA–eremomycin(50)c, BSA–eremo-mycin(50)f, and gelatin–eremomycin(25)a). In parallelexperiments, eremomycin was quantitated in normalhuman plasma, a biological fluid that is more difficultto analyze than blood serum or urea. As can be seenfrom the diagram, the patterns of the calibration curvesobtained as a result of ELISA in PBS-t and in the buffercontaining 10% of normal human plasma did not differsignificantly. Thus, the analysis variants can be used forthe determination of eremomycin concentration not onlyin aqueous solutions but also in plasma. Furthermore, tak-ing into account the expected level of eremomycin con-centration in plasma (on the order of 1–100

µ

g/ml) andthe working range of assay (~

0.1–10

ng/ml), it will benecessary to significantly dilute the test sample; as aresult, plasma interference will be negligible.

Although the sensitivity of all considered variants ofanalysis was close, in further analysis we used the mostsensitive variant; gelatin–eremomycin(25)a was usedas a solid-phase antigen (Fig.2). The study of the spec-ificity of the antibodies performed with the use of somestructural analogues of eremomycin demonstrated thestrongly specific nature of eremomycin recognition.For example, the cross-reactivity of antibodies againsteremomycin and vancomycin was 100 and 0.4%,respectively, and teicoplanin was almost not recognizedregardless of the immobilized conjugate type.

Thus, this was the first study to obtain high-specific-ity antibodies as a result of the immunization of rabbits

with eremomycin conjugated to glucose oxidase. Wedeveloped an indirect competitive enzyme immunoas-say that makes it possible to determine the content oferemomycin both in aqueous solutions (with a sensitiv-ity as high as 0.1 ng/ml) and in plasma (in the concen-tration range 1–30 ng/ml, with an allowance for tenfoldsample dilution) (Fig.3). The variation coefficient didnot exceed 10%. The high sensitivity of the assay,which is greater than the sensitivity of similar immu-nochemical methods used to monitor the therapeuticconcentrations of vancomycin and teicoplanin by one[6], two [8], and three [4, 5, 7] orders or magnitude,makes it possible to quantify eremomycin, eliminatingthe possible interferences of the carrier and drugs bysimply diluting the test sample.

ACKNOWLEDGMENTS

This study was supported by Bryntsalov-A ZAO.

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100

80

60

40

20

00.03 0.1 0.3 1 3 10

Binding, %

c

,

ng/ml

Fig. 2.

Determination of eremomycin content in (

1, 3, 5

)aqueous solutions and (

2, 4, 6

) 10% human plasma usingthe basis of interaction of anti-GO–eremomycin(50)a serumwith different solid-phase antigens: (

1, 2

) BSA–eremomy-cin(50)c, (

3, 4

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5, 6

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100

80

60

40

20

00.1 0.3 1 3

Binding, %

c

,

ng/ml

y

= 21.3

x

+ 6.5

R

2

= 0.9955

Fig. 3.

Standard curve for the determination of eremomycincontent in human plasma using ELISA, constructed on thebasis of the linear regression equation with the approxima-tion significance level specified.

214

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