Immunoassays for Residue Analysis - ACS Publications - American

Chapter 7

Molecular Modeling Studies of Ceftiofur A Tool for Hapten Design and Monoclonal Antibody Production

Downloaded by UNIV OF IOWA on September 28, 2014 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch007

Beate G. Rose, Sandra A. Buckley, Carol Kamps-Holtzapple, Ross C. Beier, and Larry H. Stanker Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 2881 F & Β Road, College Station, TX 77845-9594

Competitive indirect enzyme-linked immunosorbent assays (ciELISA's) are described for the detection of ceftiofur using different strategies for hapten design, use of immunogens, and plate-coating antigens. Molecular modeling data are presented to demonstrate investigation of hapten design. Mice were immunized using an extended immunization protocol to develop the required immune response. Serum antibody levels were evaluated for each conjugation method using both homologous and heterologous conjugates as plate -coatingantigens. A heterologous assay system proved to be more sensitive than a homologous system. Thus a heterologous assay system was employed for the production of monoclonal antibodies for the detection of ceftiofur. On the basis of preliminary experiments, desfuroylceftiofur proved to be the most advantageous hapten. Desfuroylceftiofur was prepared in situ and linked to maleimide -activated carrier proteins, i.e., BSA and KLH. Three antibodies were isolatedfrommice immunized with the KLH conjugate. The limit of detection by the antibodies Cef-36, Cef-68, and Cef-116 for ceftiofur was in the range of 0.33-32.33 ppb. Results from antibody characterization demonstrate that certain structural features are necessary for antibody binding. Cross-reactivity studies with structurally related cephalosporins, as well as penicillins were performed using the monoclonal antibodies Cef-68 and Cef-116. Ceftiofur (Figure 1) is an F D A approved veterinary cephalosporin for the treatment of respiratory diseases in cattle, horses, and swine (1-3). In a recent paper we discussed immunization strategies focusing on different aspects of hapten-design and the advantage of using heterologous plate assay systems (4). Current publications on the detection of ceftiofur mainly include HPLC and microbiological assays as illustrated in Table I. However, detection methods for ceftiofur which are specific, deliver faster results and do not require the use of expensive sophisticated equipment have not yet This chapter not subject to U.S. copyright Published 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


ROSE ET AL.

II N 2

S

Molecular Modeling Studies of Ceftiofur

Ο

ΚΑ N0CH3

Η ,

o

^ Y ^ ^ s 4 - 4 j Ο

COOH

COOH Β

Figure 1. Structures of ceftiofur (A) and desfuroyl ceftiofur (B).

In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

IMMUNOASSAYS FOR RESIDUE ANALYSIS

Table I: Methods of Detection for Ceftiofur.


Method HPLC HPLC HPLC HPLC HPLC Charm Test II MA Delvotest-P HPLC HPLC HPLC LC/EMS LC/EMS HPLC HPLC HPLC Agar gel MA MA LC, MS Colorimetry Agar gel , MICS Agar gel a

b

c

d

d

e

f

f

e

e

Medium

Reference

body fluids, tissues body fluids, tissues plasma plasma, urine milk milk milk milk milk, mammary tissue blood, plasma bovine serum, milk milk milk serum blood serum, urine serum, urine, milk milk serum, milk blood blood mice blood

(5)

(1000 350 >1000

560 160 72 3

>1000 e —

e —

a

Values are an average of 2-5 determinations. Antisera were diluted to represent approximately 50% maximum activity in a titration ELISA. These dilutions were used in the ciELISA experiments and tested on the corresponding plate coating antigens. Immunogen prepared in Freund's adjuvant. Immunogen prepared in Ribi adjuvant. Experiment not done due to insufficient material. — Indicates that no inhibition was observed at a concentration of 10 μg/mL in the ciELISA. b

c

d

e

areas represent an increase in negativity from white to gray to dark gray). The negative charge introduced by the furan ring fragment in the ceftiofur molecule is not significant since this ring is hydrolyzed once in the animal tissue, thus desfuroylceftiofur should be considered as the actual analyte (the free thiol is denoted by the arrow in Figure 4B). Notice that addition of s-SMCC in hapten ΙΠ does not alter the electronic profile of the desfuroylceftiofur portion (Figure 4C). Ceftiofur, desfuroylceftiofur and hapten III are virtually identical (Figure 4) with respect to three-dimensional structure and to surface charge, particularly in that portion of the molecule containing the thiazolyl ring fragment (denoted by the arrow in Figure 4A). Hapten I and hapten II differ in this region since the cross-linkers introduce additional electronegative charges due to their functional groups (Figure 5). Also, the threedimensional shape is significantly altered by the folding of the cross-linkers parallel to the cephem nucleus and the rotation of the thiazolyl ring fragment towards the furan ring fragment. However, as we found in the blood sera study, these differences do not rule out an immune response, but the models explain why the sensitivity is affected when comparing homologous and heterologous assay systems. We make the presumption that, like ceftiofur, hapten I and hapten II lose the furan ring as a result of thioesterase hydrolysis. Additionally, haptens I and Π were modeled without the furan ring to represent the immunizing hapten more closely. Figure 6 shows the three-dimensional isosurfaces for haptens I and II without the furan ring (compare to structure of desfuroylceftiofur in Figure 4). Replacement of the fiiran ring by a thiol group projects an overall positive charge on the cephem nucleus and the thiazolyl ring fragment contrasting the negatively charged portion of




I

I

I

I

i1

ο

Molecular Modeling Studies off Ceftiofur

91


7. ROSE ET AL.

Figure 4. Three-dimensional stereoscopic views of electron density diagrams shaded with electrostatic potential isosurfaces for plate A : ceftiofur, plate B: desfuroyl ceftiofur, and plate C: hapten III.




92

Figure 5. Stereoscopic views of electron density diagrams shaded with electrostatic potential isosurfaces for plate A : hapten I and plate B: hapten II.



93


7. ROSE ET AL.

Figure 6. Stereoscopic views of electron density diagrams shaded with electrostatic potential isosurfaces for plate A : hapten I without furan ring and plate B: hapten II without furan ring.


94



the cross-linker. The thiazolyl ring fragment is twisted even further towards the cephem nucleus than it is in desfuroylceftiofur. From these calculations it appears that the electrostatic interactions required for antibody binding may be altered enough to affect the immune response when bulky functional groups are substituted at the site of binding. These structural and electronic differences explain why differences in sensitivity can be observed when analyzing homologous and heterologous assay systems. In the heterologous assay the antibody preferentially binds the free analyte, presumably since the bound antigen is electronically and sterically different to the antibody binding pocket. Such differences in structure and electronics have been reported in recent publications using molecular modeling as a tool for hapten design and antibody characterization (33-37; chapter by Beier et al., this volume). ciELISA of Monoclonal Antibodies. Both the blood sera study and the molecular modeling results showed that using the hydrolyzed form of ceftiofur as the immunogen would probably provide the most sensitive hapten for the production of monoclonal antibodies. The procedure was followed as described in (21). Out of ten clones, three active antibodies were isolated. The three antibodies Cef-36, Cef-68, and Cef-116 were further subcloned. Using the ciELISA method, the anti-ceftiofur antibodies were tested against unconjugated ceftiofur. The results are shown in Table IV with antibody Cef-116 having the highest affinity for free ceftiofur. Table V illustrates the cross-reactivity between ceftiofur and related cephalosporins, including penicillins for the two antibodies Cef-68 and Cef-116. Three of the cephalosporins tested are similar to ceftiofur in that they contain the 2-(2-arninothiazol-4-yl)-2methoxy-iminoacetamide fragment (thiazolyl ring fragment) at the C-7 position of the cephem nucleus. The anti-ceftiofur antibodies recognized all cephalosporins containing the thiazolyl ring fragment. Only the structurally related cephalosporins, ceftriaxone, cefotaxime, and cefteram showed cross-reactivity. Ceftazidime contains the thiazol ring, but not the acetamide oxime group and could not be detected. Cefuroxime containing a furan ring instead of the thiazol ring could be detected by Cef-116, albeit to a lesser extent. Chemical structures of ceftiofur and the related cephalosporins are represented in Figure 7. None of the penicillins were detected by either Cef-68 or Cef-116. It appears that both the thiazol ring and the acetamide oxime portions are important for antibody binding and the cephem nucleus plays a minor role. This fact is corroborated by the molecular modeling studies. Conclusion The model which best fits the conjugation pattern necessary to detect ceftiofur in biological fluids should be one in which immunogens and plate coating antigens are varied. More sensitive results can be obtained when using a heterologous assay system. Ideally, one would like the antibodies to recognize antibiotics such as ceftiofur in meat and dairy products. We now have a sensitive antibody which detects ceftiofur and certain related cephalosporins. Useful information about hapten design and the characteristics necessary for antibody binding was gained by molecular modeling studies. The three-dimensional structural and electronic calculations showed which functionalities of the ceftiofur molecule are recognized by the antibody and thus provide a reasonable comparison to actual experimental results.


ROSE ET AL.


Tabic IV. Competitive inhibition results for antibodies Ccf-36, Cef-68, and Cef-116. Inhibition at 50% ( ppb) for Antibodies

Ceftiofur

3

Cef-36

Cef-68

Cef-116

15.00

32.33

0.33


Values are an average of six determinations. Table V . Cross-reactivity of ceftiofur, related cephalosporins, and penicillins for antibodies Cef-68 and Cef-116. % Cross-Reactivity Compound Ceftiofur Ceftriaxone Cefotaxime Cefteram Ceftazidime Cefuroxime Cephalothin Cefoxitin Cefazolin Cefadroxil Cefamandole Cephradine Cephapirin Cefaclor Cefsulodin Cefoperazone Ampicillin Amoxicillin Cloxacillin Penicillin G

a

Cef-68

Cef-116

100 84 41 16 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

100 98 69 9 0

Immunoassays for Residue Analysis - ACS Publications - American

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