Hapten modification approach for switching immunoassay specificity from selective to generic

Journal of Immunological Methods 388 (2013) 60–67

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Research paper

Hapten modification approach for switching immunoassay specificity from selective to generic Maksim A. Burkin ⁎, Inna A. Galvidis Department of Hybridomas, Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Moscow 105064, Russia

a r t i c l e

i n f o

Article history: Received 1 October 2012 Received in revised form 29 October 2012 Accepted 4 December 2012 Available online 9 December 2012 Keywords: ELISA specificity Low molecular weight analyte Cross-reactivity profile Hapten conjugate design Macrolides Glycopeptide antibiotics

a b s t r a c t The cross-reactivity profile of polyclonal antibodies against a low molecular weight analyte is strongly influenced by design of the coating or enzyme-linked hapten. The hapten modification effect on immunoassay specificity was studied. Heterology in hapten type and linking method were applied. The influence of these factors on analyses of two groups of antibiotics, 16-membered macrolides and glycopeptides was studied. This approach was used to convert the selective ELISAs to tylosin and eremomycin for group determination of tylosin\tilmicosin, tylosin\spiramycin and eremomycin\vancomycin. It was shown that the analytical spectrum of the developed polyclonal antibody-based immunoassays could be expanded and depended mainly on the type of coating hapten but not on the linking method. Modification of the hapten type in coating conjugates applied in present study served as a mechanism for switching specificity of the ELISA between selective and group. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Numerous low molecular weight compounds of natural and anthropogenic origin are physiologically active. Therefore they are not indifferent for human, animals, plants and ecosphere as a whole. The following groups of compounds: anti-infective agents, hormones and endocrine disruptors, growth promoters, abused drugs, other pharmaceuticals, pesticides, pollutants and toxins are the target analytes in areas of food control, environmental and drug monitoring, forensic medical examination, veterinary and sanitary inspection, research activities and others. Thus the development of analytical methods should be a match to the growing number of analytes. Although technologies are being intensively developed based on antibody-like properties of artificial biomimetic receptors, such as molecular-imprinted polymers (Baggiani et al., 2006) and aptamers (Jayasena, 1999; Giovannoli et al., 2008; Fodey et al., 2011), which are not time-consuming and require no animal involvement the traditional immunoassay remains popular and widely used. The immunoassay is well-known ⁎ Corresponding author. Tel./fax: +7 495 9170393. E-mail address: [email protected] (M.A. Burkin). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2012.12.002

to be specific, sensitive and inexpensive tool capable for high throughput screening in the mentioned areas of activities (Rebe Raz and Haasnoot, 2011; Kumar et al., 2004; Galve et al., 2002; Schwenzer et al., 1983; Tsai and Lin, 2005). Here we demonstrate an approach permitting versatile usage of immune sera or already developed polyclonal antibodiesbased immunoassays. The principle of this approach is to change assay specificity converting cross-reactivity profile of related analogs, metabolites or derivatives. Owing to structurally modified hapten conjugates used as coating antigens in competitive ELISA only a part of an antibody population of antiserum binds whereas the remaining part appears to be nonreactive. A part of antibodies express some different epitope specificity from initial one. The modified (heterologous) antigen may differ from the homologous antigen and immunogen in hapten type, its spatial orientation on the carrier, in presence/ absence and length of the spacer arm, in method of conjugation that determines the chemical structure of the linkage. In present paper the attempts to extend analytical spectrum for existing methods using coating antigen modification are described. The effect of modifications in hapten type and linking method on analysis was studied. The object under study was the ELISA developed for determination of veterinary

M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

macrolide antibiotics tylosin and tilmicosin. Spiramycin (SPIR) is another 16-membered macrolide used to combat bacterial and mycoplasmal infections in cattle, pigs and poultry. The residues of SPIR are therefore also to be estimated in tissues of edible animals (Council Regulation (EU), 2010; Codex Alimentarius Commission, 2011). To convert the assay for SPIR identification the same antibodies against tylosin were used. One more model was selective ELISA for determination of glycopeptide eremomycin, a human antibiotic candidate. The possibility of recognition in the other members of glycopeptide family, vancomycin, teicoplanin and ristomycin A using this assay was also assessed.

2. Methods 2.1. Chemicals Tylosin base (TYL) and desmycosin (DMN) were the gift from Prof. G.A. Korshunova (Belozersky Research Institute of Physicochemical Biology, Moscow State University). Eremomycin (ERM) and ristomycin A were the gift from Prof. G.S. Katrukha (Gause Institute of New Antibiotics). Tilmicosin (TMN), spiramycin (SPIR), vancomycin (VCM), teicoplanin, glucose oxidase (GO), bovine serum albumin (BSA), gelatine (Gel), N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc), Freund's complete adjuvant, о-phenylenediamine, and glutaraldehyde (ga) were purchased from Chimmed (Moscow, Russia). Antisera against BSA–TYL and GO–ERM(ga) were developed in recent works (Burkin and Galvidis, 2012; Burkin and Burkin, 2009).

2.2. Synthesis of conjugated antigens based on 16-membered macrolides 2.2.1. Gel–SPIR The solutions containing 4mg of Gel (0.025 μmol) in 1 mL of 0.05 M carbonate buffer pH 9.6 (CB) were combined with the solutions of SPIR (10 mg/mL) corresponding 10-, 30- and 100-fold molar excess of antibiotic over carrier (0.21, 0.63, 2.11 mg, respectively) and stirred for 3 h at room temperature. Then each mixture was supplemented with 0.1 mL of sodium borohydride solution (2 mg/mL) and stirred for another 1 h. The resulting conjugates were dialyzed exhaustively against 0.15 M phosphate buffered saline, pH 7.0 (PBS).

2.2.2. Gel–TYL, Gel–DMN and BSA–TYL The syntheses of coating antigens Gel–TYL × 10, Gel– DMN × 175 and immunogen, BSA–TYL × 100 with corresponding hapten load by synthesis did not differ from the preceding description and detailed in Burkin and Galvidis (2012).

2.3. Synthesis of conjugated antigens based on glycopeptide antibiotics Several functionally active groups in glycopeptide molecule allowed conjugating of hapten in different sites.

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2.3.1. Conjugating using hapten NH2 group 2.3.1.1. BSA–ERM(ga), BSA–VCM(ga) and GO–ERM(ga). Two solutions containing 4 mg of BSA (0.06 μmol) in 1 mL of distilled water were supplemented with 25-fold molar excess of ERM and VCM (234 μL and 217 μL, 10 mg/mL solutions, respectively) and 30 μL of freshly prepared 2.5% glutaraldehyde. After incubation at room temperature for 2 h under stirring, 100 μL of 2 mg/mL sodium borohydride was added to each reaction mixture and stirred for another 2 h. Thus obtained conjugates BSA–ERM×25(ga) and BSA–VCM×25(ga) were dialyzed for 2 days against PBS (3×5 L). The immunogen GO–ERM × 50(ga) was prepared before using the same procedure. 2.3.1.2. Gel(pi)–ERM, Gel(pi)–VCM. Crystal sodium periodate (2.4 mg, 114 nmol) was added to 18 mg of Gel (2 × 57 nmol) in 2 mL of 0.01 M acetate buffer (pH 5.0) and mixed for 15 min with magnet stirrer. Oxidized glycoprotein was dialyzed against 2 × 5 L of 0.01 M acetate buffer (pH 5.0) during the night at 4 °C. The resulting volume of dialyzate was divided into two equal portions, which were supplemented with 2 mL-solution of ERM (2.22 mg) and of VCM (2.06 mg) in CB (pH 9.6). These mixtures of protein and hapten taken in molar ratio 1/25 were stirred for 2 h and then for another 2 h after addition of sodium borohydride solution (0.1 mL, 2 mg/mL). The resultant conjugates were dialyzed exhaustively against PBS. 2.3.2. Conjugating using hapten COOH group 2.3.2.1. BSA–ERM(edc), BSA–VCM(edc). Two portions of VCM 1.3 mg and 4.35 mg (0.9 and 3 μmol, respectively) in 0.5 mL of water were supplemented with 15 mg of EDC and stirred for 30 min. The 1 mL water solutions of BSA (4 mg, 0.06 μmol) were added to activated VCM and mixed during the night. The resultant conjugates BSA–VCM×15(edc) and BSA– VCM×50(edc) were purified from the unreacted ingredients using dialysis. BSA–ERM× 50(edc) was prepared using the same procedure detailed in Burkin and Burkin (2009). 2.3.2.2. BSA–ERM(ae) and BSA–VCM(ae). Two solutions containing 4.67 mg of ERM (3 μmol) in 1 mL of DMSO and 4.35 mg of VCM (3 μmol) in 1 mL of DMF were supplemented with 52 μL of N-hydroxysuccinimide (4.5 μmol) and 87 μL of EDC (4.5 μmol) each one from 10 mg/mL solutions in DMF and mixed using magnet stirrer for 1.5 h. The solutions of activated glycopeptides 860 μl (2.25 μmol) were added to BSA (3 mg, 45 nmol) in 0.5 mL of CB (pH 9.6). Thus, the mixtures BSA/hapten prepared with molar ratio 1/50 were incubated overnight under stirring and then dialyzed exhaustively. 2.3.3. Conjugating using hapten phenyl and resorcyl groups 2.3.3.1. BSA–ERM(f), BSA–VCM(f). The synthesis of VCM conjugate using formaldehyde condensation method was made according to the procedure described for BSA–ERM(f) (Burkin and Burkin, 2009). The mixture of BSA (4 mg, 0.06 μmol), VCM (2.34 mg, 3 μmol) and formaldehyde (0.3 mL of 37% solution, 3690 μmol) was incubated overnight at room temperature under stirring and then dialyzed.

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M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

O O

O HO

O O

HO O

O

O O

OH

HO

N O

O

OH O O

tylosin (TYL)

O

O HO

O O

O

O

O O

OH

N OH

O

N

O HO

HO O

O

O

HO O

O

O O

OH

desmycosin (DMN)

N OH

N

O

O tilmicosin (TMN)

O O HO O

O O O

OH

HO

N O

O mycaminose

OH O mycarose

spiramycin (SPIR) Fig. 1. Structures of 16-membered macrolides. The sites of coupling between macrolides (aldehydes at C20 position) and carriers are indicated by arrows.

2.3.4. Conjugating using hydroxyls in carbohydrate moieties of hapten 2.3.4.1. Gel–ERM(pi), Gel–VCM(pi). Two water solutions of ERM (740 μg, 475 nmol) and two solutions of VCM (688 μg, 475 nmol) in 0.5 mL were supplemented with equimolar quantity and threefold molar excess of sodium periodate (103 and 309 μg, 1 mg/mL solution) and mixed for 20 min. The portions of Gel solutions (3 mg, 19 nmol) in 0.5 mL of CB (pH 9.6) were added to solutions of oxidized antibiotics and mixed for 2 h. Then 100 μL of sodium borohydride solution (2 mg/mL) was added to each reaction mixture, stirred for another 2 h and dialyzed against PBS (3 × 5 L). All the dialyzates were stored at − 20 °C as solutions with concentration of 1 mg/mL and 50% content of glycerol. 2.4. ELISA procedure To optimize the ratios of immunoreagents an indirect immunoassay check board titration was carried out. Interaction between antisera in serial dilutions and whole spectrum of synthesized coating antigens in concentration range (0.05– 1.5 μg/mL) that provided absorbance of reaction between 0.8

and 1.2 were determined and then used in competitive indirect ELISA. For this purpose 96-well plates (Costar) were filled with 0.2 mL of conjugate solutions in optimal concentration in CB and incubated at 4 °C for 16 h. The wells were washed 3–5 times with PBS containing 0.05% Tween 20 (PBS-t). Then 0.1 mL of antiserum in optimal dilution in PBS-t with 1% BSA and 0.1 mL of antibiotic standard solutions (1000–0.01 ng/mL and 0 ng/mL) were added to each well. The plates were incubated in chamber of thermoshaker at 25 °C for 1 h and then washed again; the wells were filled with 0.2 mL solution of anti-rabbit IgG antibodies conjugated with horseradish peroxidase. After 1-h incubation and washing, the wells were filled with 0.2 mL of the substrate solution containing 0.4 mg/mL o-phenylenediamine and 0.005% hydrogen peroxide in 0.15 M citrate–phosphate buffer, pH 5.0. The enzymatic reaction was stopped after 45 min with 50 μL of 4 M sulphuric acid per well. Absorbance was measured at 490 nm using Dynatech MR 5000 reader (Germany). The absorbance in wells with null analyte concentration (B0) was taken as 100% antibody binding level. The antibody binding rate (%) for each concentration of antibiotic (B) (n=4) was calculated as ratio B/B0 ×100. Standard curves were plotted using OriginPro 8.0 software. Among the coating antigens with

M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

A

TYL (100%) TMN ( - - %) SPIR (0.4%)

80 60 0.4

110

40

3. Results and discussion

20 0 0,01

0,1

1

10

100

ng/mL

B

Gel-DMN õ 175

100 TYL (100%) TMN (100%) SPIR ( - -%)

B/B0

80 60 0.9 0.9

40 20 0 0,01

0,1

1

10

100

ng/mL

C

Gel-SPIR õ 10

100

TYL (100%) TMN (0.4%) SPIR (50%)

80

B/B0

The competitive tandem ELISA based on antiserum against BSA–TYL ×100 and two coating antigens Gel–TYL×10 and Gel– DMN × 175 have been developed previously and allowed simultaneously selective determination of TYL and equal recognition of TYL and TMN, respectively (Burkin and Galvidis, 2012). To convert the assay specificity toward another veterinary macrolide, SPIR we needed to isolate a portion of antibodies focused on common epitopes for TYL and SPIR. The SPIR-based coating conjugate is just the affinity sorbent that restricted specificity spectrum of whole antiserum against BSA–TYL and bound only antibodies required for SPIR recognition. SPIR was conjugated with Gel using the same manner as TYL and DMN in reaction between aldehyde at C20 position in lactone ring of macrolide and protein amines (shown by arrows, Fig. 1). The formation of SPIR-conjugates was assessed on their immunochemical properties as coating antigens. Among the conjugates with various hapten-loads Gel–SPIR × 10 was chosen for the following examinations on assay specificity. Gel–SPIR × 10-ELISA showed the best assay sensitivity. The cross-reactivity of SPIR was examined in three ELISA variants with Gel–TYL × 10, Gel–DMN × 175 and Gel–SPIR× 10 immobilized on plates (Fig. 2). Homologous assay format (Gel–TYL × 10) remained TYL-selective. Although SPIR could be detected, this detection was 275 times weaker (0.36%, IC50 = 110 ng/mL). DMN as a coating hapten lacking mycarose (the only common moiety for TYL and SPIR) could not involve in the reaction of the part of anti-BSA–TYL antibodies which were specific to this common fragment of molecule. Thus, SPIR expressed no inhibitory activity at 100 ng/mL level in Gel– DMN-ELISA. And vice-versa, this part of antibodies against mycarose moiety that could distinguish TYL and TMN was not detectable because it could not bind with immobilized DMNantigen. So TYL and TMN were indistinguishable, they were recognized equally. The replacement of hapten in the coating antigen by SPIR resulted in conversion of specificity quite the contrary. Owing SPIR coated on the plate only antibodies to mycaminose–mycarose (common moiety) could be bound. When this part of antibodies were involved in reaction the recognition of SPIR became much better and reached 50% (IC50 = 0.6 ng/mL). TMN lacking a mycarose fragment was a poor competitor (0.39%, IC50 = 77 ng/mL). Thus, introduction of heterologous hapten determinant in coating antigen helped the better detection of corresponding analyte and modified cross-reactivity profile. The immunoassay developed using the available antibodies against TYL had limit of SPIR detection of 0.1 ng/mL that was better if compared with analogical assays described before (Albrecht et al., 1996; Situ and Elliott, 2005). The ELISA system based on antibodies raised against glycopeptide antibiotic ERM conjugated with GO using glutaraldehyde as a crosslinking agent was another object of study

Gel-TYL õ 10

100

B/B0

different hapten loads those ones which ensured the highest sensitivity of analyte determination were selected for the further studies. The cross-reactions of antibodies with macrolide or glycopeptide derivates were assessed as the ratio of the main analyte (TYL or ERM) concentration that induced half-inhibition of antibody binding (IC50) with coating antigen to the corresponding concentration of analog expressed in percents.

63

60 77

0.3 0.6

40 20 0 0,01

0,1

1

10

100

ng/mL Fig. 2. Coating antigen influence on competitive ELISA characteristics for determination of 16-membered macrolides tylosin, tilmicosin and spiramycin. Standard curves are shown for assay formats using antibodies against BSA–TYL and coating conjugates Gel–TYL × 10 (A), Gel–DMN × 175 (B) and Gel–SPIR × 10 (C). The IC50 (ng/mL) and cross-reactivity (%) values are indicated in frames and in parentheses, respectively.

(Burkin and Burkin, 2009). Selectivity of ERM determination was confirmed in several assay formats using coating antigens homologous in hapten but different in conjugating procedures. To direct assay specificity towards the other glycopeptides vancomycin, teicoplanin and ristomycin A the conjugates of corresponding haptens were synthesized and tested as solid-phase antigens. Complex structure of glycopeptide

64

M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

OH

NH2 HO

HO

OH

OH O

O

NH2

HO

HO

O

HN

NH

NH

O

O

OH O

O

NH

NH

O

NH

NH2

NH

OH O

O

O

OH

NH2 HO

NH

O

O

Cl NH

O

O

O OH

ristomycin A

NH

OH

OH O NH

NH

HO O

NH R

O

HO HO

NH2

OH O

O

O

HO

O

OH OH OH

Cl

O O

O

O O

O

vancomycin (VCM)

NH

Cl

NH

O HO

OH

OH

O

O

OH

Cl

O

HN

O

HO

O O

O

NH2

NH

HO

HO

OH

O

O

O HO

HO

NH

NH

O

eremomycin (ERM)

O

O

O

NH

NH

HN

OH OH

OH

O

O

O

NH2

HO

O

O

O

OH

O

HO

HO

HO

OH OH

O

O O

O O

OH OH

O

O

OH

O

OH

Cl

O O

O

OH

O

NH

NH

O

HN

O NH

O NH

NH

NH2

O

HO HO

O O

HO HO

O OH

OH O OH

HO OH

teicoplanin A2 R = - (CH2)2- CH = CH - (CH2)4- CH3 Fig. 3. Structures of glycopeptide antibiotics.

molecules allowed several approaches to be used for preparing conjugated antigens (Fig. 3). The various procedures involved different reactive groups of antibiotics in linking. They influenced on orientation of hapten on the carrier and were applied to reveal their role in assay specificity. Coupling glycopeptides with protein carrier by their amines were realized using two procedures. As a result the haptens were linked with the carrier distantly through glutaraldehyde spacer (C5) or zero-length spacer when hapten amines reacted with periodate-oxidized glycoprotein. There are several amines available in glycopeptide antibiotics so the resultant conjugates might have nonuniform structure. The strictly defined

Table 1 Conjugates glycopeptide-carrier prepared using different coupling methods. Reactive groups of hapten

Amines Carboxyl Phenyl, resorcyl Carbohydrate hydroxyls

Conjugates based on haptens ERM

VCM

BSA–ERM × 25(ga) Gel(pi)–ERM × 25 BSA–ERM × 50(edc) BSA–ERM × 50(ae) BSA–ERM × 50(f) Gel–ERM × 25(pi)

BSA–VCM × 25(ga) Gel(pi)–VCM × 25 BSA–VCM × 50(edc) BSA–VCM × 50(ae) BSA–VCM × 50(f) Gel–VCM × 25(pi)

Coupling methods are indicated in parentheses (ga) — glutaraldehyde; (pi) — periodate oxidation; (edc) — carbodiimide coupling method; (ae) — active ester method; and (f) — formaldehyde condensation;

M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

orientation hapten on the carrier was achieved when single carboxyl was involved in linking. The different procedures, carbodiimide coupling and active ester methods forming the same bonding between hapten and carrier were compared.

A 100

Despite preactivation of hapten carboxyl with EDC the possibility of reaction between protein carboxyl and hapten amines could never be totally eliminated. The conditions of synthesis using active ester method ruled out this possibility.

100

BSA-ERMx25ga ERM VCM (1.7%)

80

65

BSA-VCMx25ga ERM VCM (100%)

80

60 4.2

B/B0

B/B0

60 247

6.0 6.0

40

40

20

20

0

0 0,1

1

10

100

1000

0,1

1

ng/mL

B 100

10

100

1000

ng/mL 100

Gel(pi)-ERMx25 ERM VCM (0.19%)

80

Gel(pi)-VCMx25 ERM VCM (52%)

80

0.9

B/B0

60

B/B0

60 470

2.6 5.0

40

40

20

20

0

0 0,1

1

10

100

1000

0,1

1

ng/mL

C 100

10

100

1000

ng/mL 100

BSA-ERMx50edc ERM VCM (<1%)

80

BSA-VCMx50edc ERM VCM (70.8%)

80

>1000

10.1

B/B0

60

B/B0

60

6.8 9.6

40

40

20

20

0

0 0,1

1

10

ng/mL

100

1000

0,1

1

10

100

1000

ng/mL

Fig. 4. The influence of coating hapten and coupling method on competitive ELISA characteristics for determination of eremomycin and vancomycin. Standard curves are shown for assay formats using antibodies against GO–ERM(ga) and coating conjugates based on ERM (left column) and VCM (right column). Conjugates were synthesized using coupling methods: glutaraldehyde (ga) (A) and periodate oxidation of the carrier (pi) (B) involved hapten amines; carbodiimide coupling (edc) (C) and active esters method (ae) (D) involved hapten carboxyl; formaldehyde condensation (f) (E) involved hapten phenyl and resorcyl groups; and periodate oxidation of hapten carbohydrate hydroxyls (pi) (F). The IC50 (ng/mL) and cross-reactivity (%) values are indicated in frames and in parentheses, respectively.

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M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

D 100

100

BSA-ERMx50ae ERM VCM (0.31%)

80

BSA-VCMx50ae ERM VCM (60.9%)

80

60 2.2

711

B/B0

B/B0

60

5.6 9.2

40

40

20

20

0

0 0,1

1

10

100

1000

0,1

1

ng/mL

E 100

100

BSA-ERMx50f ERM VCM (1.7%)

80

100

1000

BSA-VCMx50f ERM VCM (98.6%)

80

4.3

B/B0

60

B/B0

60 254

7.1 7.2

40

40

20

20

0

0 0,1

1

10

100

1000

0,1

1

ng/mL

F

10

ng/mL

100

10

100

1000

ng/mL 100

Gel-ERMx25pi ERM VCM (0.68%)

80

Gel-VCMx25pi ERM VCM (92.6%)

80

4.9

B/B0

60

B/B0

60 721

5.0 5.4

40

40

20

20

0

0 0,1

1

10

100

1000

0,1

ng/mL

1

10

100

1000

ng/mL Fig. 4 (continued).

Formaldehyde condensation served as an approach to couple hapten's phenyl or resorcyl groups to protein amines similar to that described earlier (Burkin et al., 2002; Burkin and Galvidis, 2009). Carbohydrate moieties of glycopeptides were also regarded as the sites of conjugating with carrier. As a result of the periodate oxidation of carbohydrate hydroxyls to aldehydes, the last ones were able to react with protein amines.

To avoid excessive cleavage of the carbohydrate fragment of the molecule, 1:1 and 3:1 ratios between sodium periodate and hapten were chosen and compared. The formation of conjugates was confirmed before using UV spectrometry (Burkin and Burkin, 2009) and was also assessed on binding characteristics in ELISA. The structures of glycopeptides teicoplanin and ristomycin A (Lomakina, et al., 1982)

M.A. Burkin, I.A. Galvidis / Journal of Immunological Methods 388 (2013) 60–67

differed significantly from ERM and VCM, so they were not active as competitors even when they were used as coating haptens (data not shown). The ERM- and VCM-conjugates variously loaded with hapten were examined and those which appeared to ensure the best assay sensitivity were selected. Both groups of antigens in Table 1 were taken for the following examination of specificity as the models of various hapten design. Six different coupling methods ensured five variants of chemical bond between the hapten and the carrier (edc = ae) and at least four sites of linking according to types of reactive groups. Such variety of conjugate design could evoke changes in VCM cross-reactivity rate. Though variety in linking resulted in nine-fold differences in VCR cross-reactivity (from 1.7% for BSA–ERM(ga) and BSA–ERM(f) to 0.19% for Gel(pi)–ERM) all of the coating ERM-conjugates ensured selectivity of assay (Fig. 4). An application of VCM-coating antigens changed the situation dramatically. The recognition of VCM increased significantly and ranged from 52% in Gel(pi)–VCM-ELISA to 100% when BSA– VCM(ga) was coated. VCM being used as a coating hapten ensured group determination for ERM and VCM (Fig. 4). Because of equivalence between ERM and VCM the limit of detection for these analytes calculated as IC20 value was about 1 ng/mL for all group variants of ELISA. The assay of VCM appeared to be significantly more sensitive than therapeutic drug monitoring tests based on bioassays, chromatographic techniques and different immunoassay formats developed before (Ackerman, et al., 1983; Schwenzer, et al., 1983; Pfaller, et al., 1984; Kitahashi and Furuta, 2001; Favetta et al., 2001) and comparable with the assay described by Lam, and Le Chris (2002). Thus, this case also demonstrated that assay specificity to these two glycopeptides depended mainly on type of coating hapten. Conjugating procedure and hapten orientation on the carrier in this case did not make much of a difference. The possible reason of this fact was multi-variant hapten linking in immunogen and inducing of antibodies against differently oriented hapten.

4. Conclusions The present paper deals with cross-reactivity profile conversion of polyclonal antibody-based immunoassay for low molecular weight analytes. The cross-reactivity of related analogs, metabolites or derivatives is an important characteristic of assay that should be taken into consideration in the analysis. Sometimes its influence on assay performance may be undesired. But high cross-reactivity may be useful for group determination of related compounds. Modification of hapten design in coating conjugates applied in present study served as a mechanism for switching specificity of ELISA between selective and group. This approach proved to be simple and useful for correction specificity of assay depending on the purposes of application.

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