Production, characterization, and cross-reactivity studies of monoclonal antibodies against the coccidiostat nicarbazin

Article abstract of DOI:10.1021/jf010208j

A cELISA was developed for the coccidiostat nicarbazin. On the basis of previous computer-assisted molecular modeling studies, p-nitrosuccinanilic acid (PNA-S) was selected as a hapten to produce antibodies to 4,4′-dinitrocarbanilide (DNC), the active com

Full text of DOI:10.1021/jf010208j

4542
J. Agric. Food Chem. 2001, 49, 4542−4552
P r od u ction , Ch a r a cter iza tion , a n d Cr oss-Rea ctivity Stu d ies of
Mon oclon a l An tibod ies a ga in st th e Coccid iosta t Nica r ba zin
†
†
†
‡
Ross C. Beier, Laura H. Ripley, Colin R. Young, and Colleen M. Kaiser
Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of
Agriculture, 2881 F & B Road, College Station, Texas 77845-4988, and Biology Department, Texas A&M
University, College Station, Texas 77843
A cELISA was developed for the coccidiostat nicarbazin. On the basis of previous computer-assisted
molecular modeling studies, p-nitrosuccinanilic acid (PNA-S) was selected as a hapten to produce
antibodies to 4,4′-dinitrocarbanilide (DNC), the active component of the coccidiostat nicarbazin.
Synthesis is described for the hapten [p-nitro-cis-1,2-cyclohexanedicarboxanilic acid (PNA-C)] used
in a BSA conjugate as a plate coating antigen. Monoclonal antibodies (Mabs) were isolated that
compete with nicarbazin, having IgMκ isotype. Because of the lack of water solubility of nicarbazin,
N,N-dimethylformamide (DMF) (3%, v/v) and acetonitrile (ACN) (10%, v/v) were added to the assay
buffer to achieve solubility of nicarbazin and related compounds. The Nic 6 Mabs had an IC35 value
for nicarbazin of 0.92 nmol/mL, with a limit of detection of 0.33 nmol/mL. Nic 6 exhibited high
cross-reactivity for PNA-S and PNA-C, and 3-nitrophenol, 4-nitrophenol, and 1-(4-chlorophenyl)-
3
-(4-nitrophenyl) urea. However, Nic 6 had little or no cross-reactivity with 15 other related
compounds.
Keyw or d s: Broilers; coccidiostat; coccidiosis; DNC; 4,4′-dinitrocarbanilide; ELISA; hapten; im-
munoassay; monoclonal antibodies; nicarbazin; p-nitro-cis-1,2-cyclohexanedicarboxanilic acid; p-
nitrosuccinanilic acid
INTRODUCTION
Coccidiosis causes significant economic losses to the
poultry industry (1). The parasites multiply in the
intestinal tract and cause tissue damage. This invasion
results in the interruption of feeding, digestive pro-
cesses, and nutrient absorption, dehydration, blood loss,
and increased susceptibility to other disease agents (2).
Nicarbazin was the first agent found to give satisfac-
tory control of coccidiosis in broiler production (3, 4). It
is the oldest anticoccidial in use today (5). Nicarbazin
is used worldwide as a feed additive to prevent out-
breaks of cecal and intestinal coccidiosis in poultry (6,
F igu r e 1. Nicarbazin is an equimolar complex of 4,4′-
dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimi-
dine (HDP).
7
), and it is used to increase the rate of weight gain.
Nicarbazin is composed of an equimolar complex of 4,4′-
dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimeth-
ylpyrimidine (HDP) (Figure 1). It was demonstrated in
number of the other coccidiostats tested (11). However,
antimicrobial resistance continues to be a problem with
respect to animal drug use. Ten field isolates of Eimeria
sp. obtained from North Germany were studied for
sensitivity to various anticoccidials. Many of the isolates
1
967 that nicarbazin inhibited egg production from
female adult worms of the parasite Schistosoma man-
soni in infected mice (8). Nicarbazin acts by inhibiting
the full development of second-generation meronts, thus
reducing the pathogenic effect and numbers of oocysts
excreted (9). Nicarbazin was one of six out of the 71
compounds screened that completely inhibited cryptospo-
ridial growth at a concentration of 1 µM using a
screening method that assessed anticryptosporidial and
cytotoxic effects of putative chemotherapeutic com-
pounds (10).
(9 out of 10) showed varying resistance to anticoccidials,
including nicarbazin (12). Battery trials conducted
during commercial broiler production in Lower Saxony,
Germany, found some isolates that were partially
resistant to nicarbazin and other anticoccidials (13).
The complex DNC‚HDP is 10 times more potent in
the control of Eimeria tenella, the primary coccidia cecal
pathogen, than is DNC by itself. When HDP was used
alone it was observed to have no anticoccidial activity
Following drug withdrawal, there is no evidence of
residual anticoccidial activity with nicarbazin or a
(
3). It was proposed that ultrafine crystals that were
*
To whom correspondence should be addressed: Ross C.
obtained as a result of complex formation between DNC
and HDP allow for better dissolution of DNC resulting
in improved anticoccidial activity (14). Chickens also
excrete DNC more slowly than they do HDP (15). The
U.S. Food and Drug Administration has established a
Beier, USDA, ARS, SPARC, 2881 F & B Road, College Station,
TX 77845-4988. Phone: 979/260-9411, Fax: 979/260-9332,
E-Mail: RCBeier@ffsru.tamu.edu.
†
‡
U.S. Department of Agriculture.
Texas A&M University.
1
0.1021/jf010208j CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/21/2001

Monoclonal Antibodies to Nicarbazin
J. Agric. Food Chem., Vol. 49, No. 10, 2001 4543
nicarbazin withdrawal period of 4 days with a tolerance
of 4 ppm (16).
The Food Safety and Inspection Service (FSIS) uses
high performance liquid chromatography (HPLC) with
UV detection to analyze for DNC (17). The FSIS method,
as well as other published methods, uses a variety of
organic solvents (e.g., ethyl acetate, acetonitrile, hexane,
dimethylformamide, and methanol) for various steps
during the analyses of nicarbazin. A number of analyti-
cal techniques have been used to quantify nicarbazin.
Pulse polarographic determinations were used to quan-
tify nicarbazin in chicken tissues (18, 19). Liquid
chromatographic methods have been used to quantify
nicarbazin in feeds (20, 21) and chicken tissues (22-
2
5). Near-infrared reflectance spectroscopy has been
used to quantify granulated nicarbazin (26), and super-
critical fluid extraction followed by HPLC analysis was
used for nicarbazin determination in poultry feed, eggs,
and muscle tissue (27). Liquid chromatography-ther-
mospray mass spectrometry (MS) (25), liquid chroma-
tography-atmospheric pressure chemical ionization MS
F igu r e 2. Synthesis and structures of haptens for conjugation
to carrier proteins: (A) PNA-S and (B) PNA-C.
symposium. Unfortunately, those early hybridomas did
not continue to produce viable antibodies (39). Our
recent fusions and isolated hybridomas are the subject
of this paper.
(
28), and liquid chromatography-electrospray MS (6)
were used to quantify nicarbazin levels in chicken
tissues, eggs and feeds, respectively. All of these meth-
ods are time-consuming and result in production of
substantial organic solvent wastes.
Our laboratory has had a long-term interest in
developing monoclonal antibodies and immunochemical
methods for the determination of coccidiostats and
antibiotics used in animal production. Detection meth-
ods based on immunoassays can greatly increase the
rate of sample throughput, allow screening of increased
numbers of samples without increasing the cost of
analysis, and eliminate or greatly reduce the amount
of solvent waste generated during analysis procedures.
In this paper, we describe four hybridoma cell lines
two of which (Nic 6, ATCC PTA-2647; and Nic 7, ATCC
[
PTA-2648) have been deposited with the American Type
Culture Collection (ATCC)]. These hybridoma cell lines
produce Mabs that may be used for the detection of the
coccidiostat nicarbazin. Details of production and char-
acterization of these antibodies are reported in this
paper. The effect of pH and the use of solvents while
carrying out the competitive enzyme-linked immun-
osorbent assay (cELISA) are discussed.
MATERIALS AND METHODS
Ch em ica ls a n d Ma t er ia ls. Acetonitrile (ACN), N,N-di-
methylformamide (DMF), ethyl acetate (EtOAc), EM Science
methanol (MeOH), and Kieselgel 60 F254 HPTLC plates (EM
Science No. 13728) were obtained from VWR Scientific Prod-
ucts Corp. (Suwanee, GA); bovine serum albumin (BSA, No.
A-7030), keyhole limpets (Megathura crenulata) hemocyanin
(KLH, No. H-2133), polyoxyethylene-sorbitan monolaurate
(Tween 20), sodium carbonate, sodium bicarbonate, magne-
We have used computer-assisted molecular modeling
as a tool to understand antibody-ligand interactions
29-33), to demonstrate that different energy state
conformations of a hapten can exist and to show that
different hapten energy states can influence the outcome
of the immune response (34), to predict hapten design
(
(
35, 36), and to understand physical behavior of mol-
ecules (37). Some of these molecular modeling applica-
tions are discussed in a review on the use of immunoas-
say for detection of antibiotics in foods and feeds (38).
sium chloride, NaCl, Na
MA base, 8-azaguanine (Sigma A-5284 Hybri-Max), pristane
2,6,10,14-tetramethylpentadecane, Sigma P-1403) 95%, HAT
2 4
HPO , TRIZMA hydrochloride, TRIZ-
(
media supplement (hypoxanthine, aminopterin and thymidine,
Sigma H-0262), HT media supplement (hypoxanthine and
thymidine, Sigma H-0137), and goat anti-mouse IgG (whole
molecule) peroxidase conjugate (Sigma A-5278) were obtained
from Sigma Chemical Co. (St. Louis, MO). Ammonium acetate,
1-benzyl-3-(4-nitrophenyl)urea, 1,3-bis(4-nitrophenyl)urea, 1-(3-
chlorophenyl)-3-(2-methoxy-5-nitrophenyl)urea, 1-(3-chlorophe-
nyl)-3-(4-methoxy-3-nitrophenyl)urea, 1-(4-chlorophenyl)-3-(4-
nitrophenyl)urea, cis-1,2-cyclohexanedicarboxylic anhydride,
Our original approach to the production of monoclonal
antibodies (Mabs) to DNC was to make a hapten from
the drug DNC and use it for immunization. This
methodology failed to produce Mabs that could compete
with “free” DNC, as was discussed by Beier and Stanker
(35). Computer-assisted molecular modeling was used
to study this conjugate as well as to help determine an
alternative hapten to use as an antigen (35). Figure 2
shows the chemical structure of p-nitrosuccinanilic acid
1
-(2-fluorophenyl)-3-(2-methoxy-4-nitrophenyl)urea, 2,6-luti-
dine, 1-(3-methoxyphenyl)-3-(3-nitrophenyl)urea, 2-nitroa-
niline, 3-nitroaniline, 4-nitroaniline, 2-nitrobenzyl alcohol,
(hapten-1). This DNC-mimic was shown by molecular
modeling to be a good candidate as a hapten. It was
thought that a DNC-mimic-protein conjugate could be
used as an immunizing agent for production of antibod-
ies to DNC. A comparison of the computer-assisted
electrostatic potential isosurfaces of the molecular
models of DNC and p-nitrosuccinanilic acid were com-
pared (35). A strikingly close similarity was seen in the
electrostatic potential isosurfaces of these two com-
pounds.
3
-nitrobenzyl alcohol, 4-nitrobenzyl alcohol, 4-nitrophenethyl
alcohol, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol and suc-
cinic anhydride were obtained from Aldrich Chemical Co., Inc.
(Milwaukee, WI). N-hydroxysulfosuccinimide (Sulfo-NHS; No.
2451) and N,N′-dicyclohexylcarbodiimide (DCC; No. 20320)
were obtained from Pierce Chemical Co. (Rockford, IL). Su-
pelcosil LC-ABZ chromatography column was obtained from
Supelco (Bellefonte, PA). BALB/c mice and ICR mice were
obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN).
Spectra/Por 4 dialysis membrane tubing, molecular weight
cutoff (MWCO) 12 000-14 000 was obtained from Spectrum
Medical Industries, Inc. (Los Angeles, CA). Ribi-Corixa adju-
vant (No. R700, MPL + TDM emulsion in 2% oil-Tween-80)
A preliminary report was published (39) on the
development of Mabs to nicarbazin where preliminary
data from our “fusion 2” products were discussed in a

4544 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Beier et al.
was obtained from CORIXA Corp. (Hamilton, MT). Nonfat dry
milk (NFDM) J anet Lee instant nonfat dry milk fortified with
vitamins A & D contained 35.5% protein (Albertson’s Inc.,
3
NaO
5
) was obtained in a 56% yield. To convert the product to
the acid form, the material was extracted from pH 3.00
biphthalate buffer (Micro Essential Laboratory Inc., Brooklyn,
NY) with dichloromethane and dried with sodium sulfate.
High-resolution mass measurement was obtained by electro-
spray MS on a PE-Sciex QSTAR Pulsar mass spectrometer of
Boise, ID, and was obtained from a local grocery store). Iscoves
Modified Dulbecco’s Medium (Gibco BRL no. 12200-069),
penicillin/streptomycin (Gibco BRL No. 15140-122), and L-
glutamine (Gibco BRL No. 25030-081) were obtained from
Gibco/Life Technologies (Grand Island, NY). Fetal bovine
serum was obtained from J RH Biosciences (Lenexa, KS).
Sample dilutions were made with a portable pipet-aid, electri-
cally actuated (Drummond Scientific Co., Broomall, PA). A
multichannel pipettor was used to conduct the cELISA [Finnpi-
pet, digital multichannel 50-300 µL pipet (LSI North America,
Needham Heights, MA)]. Costar 96w microtiter plates (No. 07-
-
the M-H ion and was observed at m/z 291.0978 (calculated
for C14
-
H
15
N
2
O
5
, 291.098097).
P r ep a r a tion of Im m u n ogen a n d P la te Coa tin g An ti-
gen s. The immunogen and plate coating conjugate were
synthesized using an N-hydroxysuccinimide (NHS)-enhanced,
carbodiimide-mediated coupling reaction described by Staros
et al. (40). These conjugations were conducted in a manner
similar to the method used for the coccidiostat salinomycin
(41).
2
00-90) and Costar 24w plates (No. 3524) were obtained from
CoStar Corp. (Cambridge, MA). Nylon cloth sieve w/100 µm
pore size (No. 34-1800-04) and Nunc-Immuno plates F96
MaxiSorp and Nunc-lids (Nunc # 439454) used for immunoas-
says were obtained from PGC Scientifics Corp. (Frederick,
MD). K-Blue substrate was obtained from Elisa Technologies
KLH Conjugate. The immunogen was produced by dissolv-
ing Sulfo-NHS (17 mg, 0.078 mmol) in dry DMF (1 mL),
followed by addition of a solution of PNA-S (20.1 mg, 0.077
mmol) in dry DMF (0.25 mL). To this solution was added DCC
(15.2 mg, 0.074 mmol) and dry DMF for a final volume of 2.5
mL DMF. After the sample was stirred for 2 h at room
temperature, the reaction mixture was added to a solution of
(Lexington, KY). Antibody isotype was determined using SBA
Clonotyping System/AP (No. 5300-04, Southern Biotechnology
Associates, Inc., Birmingham, AL). The miniPERM bioreactor
2
KLH (42 mg) in H O (6 mL, pH 8.5). During the addition,
(
IV-76001059) was obtained from Sartorius (New York, NY).
NaOH (0.1 N) was used to keep the pH = 8.5. The reaction
mixture was stirred overnight at room temperature. The
mixture was then dialyzed progressively beginning with a
Optical densities of developed assays were read with a 96-well
Microplate Reader, Bio-Rad model 3550 Microplate reader
(
Bio-Rad Laboratories, Hercules, CA). The Bio-Rad Reader
solution of DMF (50%, v/v) in H
was decreased in three increments until only RO H
2
O. The concentration of DMF
driver software version 1.0 (for Power Macintosh 6100/60) was
2
O was
used at the settings: 8 × 12 format, automix (3 min). Reverse
used, and the dialysis was conducted at 4 °C using Spectra/
Por 4 dialysis membrane tubing, MWCO 12 000-14 000.
BSA Conjugate. The plate coating antigen was produced by
dissolving Sulfo-NHS (15.4 mg, 0.071 mmol) in dry DMF (1
mL), followed by addition of a solution of PNA-C (22.6 mg,
0.072 mmol) in dry DMF (0.25 mL). To this solution was added
DCC (14.1 mg, 0.068 mmol) and dry DMF for a final volume
of 2.5 mL DMF. After the sample was stirred for 2 h at room
temperature, the reaction mixture was added to a solution of
2
osmosis water, pyrogen free (RO H O), was produced on site
by a reverse osmosis system obtained from Millipore Corp.
(Bedford, MA) and used for all cELISA experiments.
Syn th esis of p-Nitr osu ccin a n ilic Acid [CAS Registr y
No. 5502-63-6]. p-Nitrosuccinanilic acid was synthesized as
described by Beier and Stanker (39). Briefly, p-nitroaniline (0.1
g, 0.724 mmol) and succinic anhydride (0.29 g, 2.898 mmol)
were dissolved in EtOAc (4 mL). Addition of 2 drops of 2,6-
lutidine started the reaction, and it was held at a temperature
of 70 °C overnight (19 h). The reaction mixture was cooled to
2
BSA (43.1 mg) in H O (6 mL, pH 8.5). During the addition,
NaOH (0.1 N) was added to keep the pH = 8.5. The reaction
mixture was stirred overnight at room temperature. The
mixture was then dialyzed against a solution of DMF (20%,
v/v) in H O and three times against RO H O at 4 °C using
2 2
Spectra/Por 4 dialysis membrane tubing, MWCO 12 000-
14 000.
4
°C, and the solvent was decanted from the solid products.
The products were rinsed with cold EtOAc (1 mL), and a crude
product was obtained by removing some impurities by thin-
layer chromatography (TLC) using Kieselgel 60 F254 HPTLC
plates using solvent system CHCl
3
/EtOAc/MeOH/formic acid,
8
6:10:4:1%. Following TLC, the material that remained at the
cELISA Solu tion s. Detergent wash buffer was made by
origin was eluted with DMF. After removal of DMF with a
stream of argon, the product was made basic using NaOH (4
N). The final product was collected from HPLC using a 15 cm
adding Tween 20 (0.05%, v/v) to RO H
consisted of Na CO (0.015 M), NaHCO
(0.002 M) in RO H O, pH 9.6. Phosphate-buffered saline (PBS-
9) contained Na HPO (0.01 M) and NaCl (0.15 M) in RO H O,
pH 9. Blocking buffer consisted of NFDM (3%, w/v) in PBS-9.
Assay buffer consisted of adding part A (495 mL) and part B
(5 mL). Part A contained per 1 L of water: 11.4 g of TRIZMA
hydrochloride, 3.32 g of TRIZMA base, and 8.7 g of NaCl in
2
O. Carbonate buffer
2
3
3
(0.035 M), and MgCl
2
2
×
4.6 mm, 5 µm, Supelcosil LC-ABZ column with a solvent
system of MeOH (30%, v/v) at a flow rate of 1 mL/min at 254
nm. Sodium p-nitrosuccinanilate (PNA-S) (C10 NaO ) was
2
4
2
H
9
N
2
5
obtained in a yield of 59%. High-resolution mass measurement
was obtained by fast atom bombardment mass spectrometry
on a double-focusing VG 70-250 EHF spectrometer of the
2
RO H O adjusted to pH (6.8, 7.0, 7.2, 7.4, 7.6, 7.75) using HCl
double sodium ion and was observed at m/z 283.0326 (calcu-
or NaOH. Part B contained per 95 mL of water: 1 g of NFDM
and 0.5 mL Tween-20.
+
lated for C10
H
9
N
2
Na
2
O
5
, 283.0307).
Syn t h esis of p -Nit r o-cis-1,2-cycloh exa n ed ica r b oxa -
n ilic Acid [CAS Registr y No. 17716-19-7]. The desired
product was made by dissolving p-nitroaniline (0.1 g, 0.724
mmol) and cis-1,2-cyclohexanedicarboxylic anhydride (CHDA,
Assa y P la te Coa tin gs. Nunc-Immuno plates with a Max-
iSorp surface were washed with a solution of Tween-20 (0.05%,
v/v) in RO H O followed by a RO H O rinse to clean the plates
2 2
prior to coating. Assay plates were coated overnight at 4 °C
with a solution containing nicarbazin (141 ng/100 µL/well) (an
equivalent of 100 ng 4,4′-dinitrocarbanilide per 100 µL) in
MeOH (50%, v/v) containing Tween-20 (0.02%, v/v) in part A
of assay buffer; plate coating 1 (PL-1). The nicarbazin solution
was made for coating plates by initially dissolving nicarbazin
(2.4 mg) in MeOH (100 mL) with 0.1 mL Tween-20 (Std-1).
Five hundred milliliter quantities of the coating solution were
made by adding Std-1 (29.4 mL), MeOH (220.6 mL), and assay
buffer part A to a 500 mL volumetric flask.
0
2
.446 g, 2.896 mmol) in EtOAc (4 mL). Addition of 2 drops of
,6-lutidine started the reaction, and it was held at a temper-
ature of 70 °C overnight (19 h). The reaction vessel was cooled
to 4 °C, and the solvent was decanted from the solid products.
The products were rinsed with cold EtOAc (1 mL). A crude
product was obtained by removing some impurities by TLC
on Kieselgel 60 F254 HPTLC plates using the solvent system
3
CHCl /EtOAc/MeOH/formic acid, 86:10:4:1%. Following TLC,
the material that remained at the origin was eluted with DMF.
After removal of the DMF with a stream of argon, the product
was made basic using NaOH (4 N). The final product was
separated and collected from HPLC using a 15 cm × 4.6 mm,
Assay plates also were coated with the PNA-C-BSA conju-
gate. Washed and rinsed plates were coated overnight at 4 °C
with a solution containing 100 ng/100 µL/well PNA-C-BSA in
pH 9 carbonate buffer. After the plates were coated overnight,
5
µm, Supelcosil LC-ABZ column with a solvent system of
MeOH (30%, v/v) at a flow rate of 1 mL/min at 254 nm. Sodium
p-nitro-cis-1,2-cyclohexanedicarboxanilate (PNA-C) (C14
they were washed with RO H
2
O containing Tween-20 (0.05%,
H
15
N
2
-
v/v) followed by a RO H O rinse. Assay plates were incubated
2

Monoclonal Antibodies to Nicarbazin
J. Agric. Food Chem., Vol. 49, No. 10, 2001 4545
with blocking buffer (15 g of nonfat dry milk in 500 mL PBS-
incubation at 37 °C, the plates were washed with Tween-20
9
) (300 µL/well), for 1 h at room temperature, and then washed
(0.05%, v/v) and rinsed with RO H
µL/well) was added to each well and incubated at room
temperature for 30 min. A stop solution of 2 N H SO (50 µL)
was added to each well, and optical density measurements (450
nm) were taken on a Bio-Rad model 3550 microplate reader.
The mean of 100 background OD measurements was 0.09 (
0.008.
2
O. K-Blue substrate (100
and rinsed with RO H O.
2
Cell Gr ow th Med ia . Iscoves Modified Dulbecco’s Medium
2
4
was used containing the following additives: L-glutamine (100
mL/10 L media) and NaHCO (3.7 g/L). The media solution
3
was sterile filtered into 500 mL bottles and incubated over-
night at 37 °C to ensure sterility. Media was stored at 4 °C.
Complete media was used for growing the SP2/0 myeloma cells
and for all other cell maintenance applications and consisted
of the addition of fetal bovine serum (FBS, 25 mL/500 mL)
and a penicillin/streptomycin (Pen/Strep) solution (5 mL/500
mL) resulting in a concentration of 100 U/mL and 100 µg/mL,
respectively. HAT (2×) was used only for the newly fused cells
to select against unfused cells. Two vials were reconstituted
to 10 mL each and added to complete media (480 mL). The
concentration of added chemicals in the HAT (2×) media was
Im m u n iza tion of Mice. The immunogen was prepared for
injection by addition of PNA-S-KLH and sterile isotonic saline
to the contents of one vial of Ribi-Corixa adjuvant system for
a final volume of 2 mL. The mixture was emulsified to produce
a solution of PNA-S-KLH conjugate in Ribi-Corixa adjuvant
(75 µg/0.15 mL). BALB/c mice were immunized i.p. with 0.15
mL of the mixture at a minimum of three times at two-week
intervals. One week after the third injection, blood was
collected from the tail vein to determine which mouse had the
highest anti-hapten titer and best competition for nicarbazin.
The selected mouse received a booster injection i.p. of 75 µg/
0.15 mL of only the PNA-S-KLH conjugate (no Ribi-Corixa
adjuvant) in sterile isotonic saline 3-4 days prior to the day
of fusion.
Mon oclon a l An tibod y (Ma b) P r od u ction . Fusion of
SP2/ 0 Myeloma Cells and Spleen Cells. Some of the fusion
and cloning conditions were described by Stanker et al. (42),
with the following modifications: splenocytes were fused with
SP2/0 myeloma cells cultured in 96-well Costar plates contain-
ing a macrophage feeder cell layer. Macrophages were obtained
from ICR mice treated with pristane. SP2/0 myeloma cells
were passed through media containing 8-azaguanine and then
grown for 4 to 5 days immediately before the fusion. On the
day of fusion, the mouse (that received the booster injection)
was sacrificed by cervical dislocation, and the spleen was
removed aseptically. The spleen was disrupted through a
tissue sieve into serum free Iscoves Modified Dulbecco’s media,
and the resulting cell suspension was strained through a
sterile nylon cloth sieve w/100 µm pore size into a sterile
beaker. SP2/0 myeloma cells were fused to splenocytes by using
poly(ethylene glycol) (43).
-
4
-7
2
1
× 10 M hypoxanthine, 8 × 10 M aminopterin, and 3.2 ×
-5
0
M thymidine. The cell suspension in HAT (2×) media was
pipetted (100 µL/well) into 30 macrophage coated 96-well
Costar 96w plates. Final concentration of HAT on the fusion-
coated plates was 1×. HT media supplement, Hybri-Max,
following reconstitution to 10 mL and addition to complete
media (490 mL) resulted in a concentration of hypoxanthine
and thymidine of 1 × 10 M and 1.6 × 10 M, respectively.
Solven t System s Requ ir ed for Solu bility of Nica r ba -
zin . Because of the low solubility of nicarbazin or 4,4′-
-
4
-5
2
dinitrocarbanilide in various solvents including H O, a study
of various concentrations of solvents in assay buffer was
undertaken that would lead to the appropriate solubility of
nicarbazin, while still allowing the cELISA to work.
Solvent System Used in Titration of Mouse Sera or Media.
For titration of mouse sera or media, a solution containing
DMF (3%, v/v) and ACN (10%, v/v) in assay buffer was used.
This solution also was used in the cELISA control wells.
Nicarbazin Standard and Other Chemicals Used for Com-
petitions. Standards of nicarbazin and all other chemicals used
for competition in the cELISA were prepared with a solution
containing DMF (12%, v/v), ACN (40%, v/v), and Tween-20
(
0.1%, v/v) in part A of assay buffer. These standards were
Screening of the Cell Fusion. Cells usually are ready for their
initial screening in 10 to 14 days. In the initial screen, the
supernatant from each well of 30 fusion-coated plates was
evaluated for antibody binding to the coating conjugate. Cells
from positive wells (approximately 144) were transferred to
24-well Costar plates to allow expansion of cells and production
of enough antibody to further test for anti-hapten titer and
complete competition studies with the target chemical (nicar-
bazin). After a grow-out period of 7-10 days, the supernatants
from the 24-well Costar plates were checked for antibody titer
levels, followed by competition studies. Those wells that exhibit
competition with the target chemical were then further
submitted to hybridoma cloning.
Hybridoma Cloning. Following initial hybridoma screening,
HT media was used for cell maintenance until the final picks
were made. Screening for antibody producing hybridomas
made use of the cELISA described above. Hybridoma cells from
wells showing antibody competition to nicarbazin were ex-
panded and subcloned three times by limiting dilution. The
wells picked for expansion were viewed under a microscope
to confirm the presence of a single cell source in the well,
ensuring their monoclonal origin. After the final picks were
made, those selected cell lines were weaned off HT media and
on to complete media.
then pipetted (100 µL) into column 2 of the 96-well plates
containing 100 µL of assay buffer.
Solvent System Used during Competitions with cELISAs. A
solution containing DMF (6%, v/v) and ACN (20%, v/v) in assay
buffer was used for serial dilutions of the standards on 96-
well plates. For competitions, the mouse sera or media or their
dilutions were layered on top of the above diluted standards
resulting in solutions with a final solvent concentration of 3%
DMF (v/v) and 10% ACN (v/v) in assay buffer.
Com p etitive ELISA. A cELISA was developed to evaluate
antibody specificity and to quantify nicarbazin in solution.
Assay plate preparation and coating follow those steps de-
scribed above under Plate Coating Antigens. Early work
during this study utilized nicarbazin (PL-1) as plate coating
material, and later work utilized PNA-C-BSA (PL-2) as plate
coating material. The competitor (nicarbazin/100 µL) solution
in DMF (12%, v/v), ACN (40%, v/v), and Tween-20 (0.1%, v/v)
was added to assay buffer (100 µL) in column 2 of the 96-well
assay plate. Final solvent concentration in column 2 resulted
in DMF (6%, v/v) and ACN (20%, v/v). This solution was
diluted 1:2 across the plate in DMF (6%, v/v) and ACN (20%,
v/v) in assay buffer (100 µL) to form a concentration gradient
of the competitor. Antibody diluted in assay buffer (100 µL)
was added to all wells except column 1 (solvent control) making
a final concentration of DMF (3%, v/v) and ACN (10%, v/v).
Wells in column 1 contain the solvent control, DMF (3%, v/v),
and ACN (10%, v/v) in assay buffer. The amount of antibody
used was that amount that produced a minimum optical
density reading of 0.45 absorbance units after background
subtraction. The sample antibody mixture was incubated for
Mab Production. Rather than producing antibodies by
ascites tumor production as described in Stanker et al. (42),
the Mabs were produced in vitro production. In vitro produc-
tion of Mabs in high concentration was accomplished in a
reusable modular minifermenter for high-density culture of
hybridoma cells, the miniPERM bioreactor (44, 45). In those
cases where the antibody has an isotype of IgG, a protein G
column can be used for Mab purification.
1
h at 37 °C, and the plates were washed with Tween-20
(
0.05%, v/v) in RO H
2
O and rinsed with RO H O. Then, goat
2
Titr a tion Stu d ies w ith Va r iou s Assa y Bu ffer p H Va l-
u es. Studies were carried out using both PL-1 and PL-2 plate
coatings. Assay buffer part A was formulated at various pH
values (6.8, 7.0, 7.2, 7.4, 7.6, 7.75) as was described in the
anti-mouse IgG (whole molecule) horseradish peroxidase
conjugate diluted 1:500 in assay buffer, plus NFDM (2%, w/v),
was added (100 µL) to each well. Following a second 1-h

4546 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Beier et al.
section titled “cELISA Solutions”. Titrations at various pH
values were carried out using a solvent mixture similar to that
described in the section titled “Solvent System used in Titra-
tion of Mouse Sera or Media”. Competitions at various pH
values were carried out using a solvent mixture similar to that
described in the section titled “Solvent System used during
Competitions with cELISAs,” and the cELISA was accom-
plished as is described in the section titled “Competitive
ELISA.”
Ch a r a cter iza tion of An tibod ies. Isotype and Affinity.
Antibody isotypes were determined using reagents supplied
in the commercially available kit SBA Clonotyping System/
AP. Relative affinity of the four Mabs for nicarbazin was
measured by determining the 35% inhibition of control values
compete. Therefore, another hapten was synthesized
(PNA-C, Figure 2). Greirson et al. (47) used different
bridging moieties during conjugation to overcome un-
wanted cross-reactivity associated with antibody bind-
ing with 13-hydroxylupanine. They used a succinate-
KLH derivative of 13-hydroxylupanine as the immunogen
and the cis-1,2-cyclohexanedicarboxylate derivative with
BSA for the plate coating antigen. Likewise, we used
cis-1,2-cyclohexanedicarboxylic anhydride to react with
PNA at the same elevated temperature conditions used
with succinic anhydride and produced PNA-C (Figure
2
). PNA-C linked to BSA gave a suitable plate coating
antigen (Figure 3) that did not suffer from unwanted
cross-reactivity.
(IC35, center of curve). IC35 was used for Nic 6, Nic 8, and Nic
9
Mabs, and IC30 was used for the Nic 7 Mabs at an antibody
dilution producing an absorbance minus control of 0.45 in the
absence of competitor.
Antibody Specificity. Nicarbazin [4,4′-dinitrocarbanilide (DNC)
P r od u ction of Mon oclon a l An tibod ies to Nica r -
ba zin . Ten days following the fusion, growing hybrido-
mas were observed in many of the 2880 wells of the
seeded 96-well plates. At this time, all wells were tested
for anti-nicarbazin activity using nicarbazin (PL-1)
bound plates in an ELISA described above. Hybridoma
cultures from 120 wells with the highest anti-nicarbazin
activity were selected for further evaluation. The hy-
bridomas in these wells were expanded in 24-well plates.
The supernatants were titrated on PL-1 coated plates
and were evaluated for competition against nicarbazin.
Seven picks were made that showed good to poor
competition and were grown up after a limiting dilution.
Those wells that showed binding to PL-1 were ex-
panded, and titration and competition were completed
arriving at picks from three of the initial wells. Selection
of clones from these cultures by limiting dilution led to
four stable hybridoma cell lines. These monoclonal
cultures and their corresponding Mabs were named Nic
6, Nic 7, Nic 8, and Nic 9.
+
2-hydroxy-4,6-dimethylpyrimidine (HDP)], DNC, HDP, PNA-
S, and PNA-C were used in cross-reactivity studies. Other
chemicals used were arrived at in two different ways. A
number of chemicals are structurally similar to PNA-S: 2-
nitroaniline, 3-nitroaniline, 4-nitroaniline, 2-nitrobenzyl alco-
hol, 3-nitrobenzyl alcohol, 4-nitrobenzyl alcohol, 4-nitrophen-
ethyl alcohol, 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol.
A number of compounds with structural similarities to 4,4′-
dinitrocarbanilide were obtained through a structural frag-
ment-based computer search of available organic compounds
from Aldrich Chemical Co., Inc. (Milwaukee, WI): 1-benzyl-
3
-(4-nitrophenyl)urea, 1,3-bis(4-nitrophenyl)urea (DNC), 1-(3-
chlorophenyl)-3-(2-methoxy-5-nitrophenyl)urea, 1-(3-chlorophe-
nyl)-3-(4-methoxy-3-nitrophenyl)urea, 1-(4-chlorophenyl)-3-(4-
nitrophenyl)urea, 1-(2-fluorophenyl)-3-(2-methoxy-4-nitrophen-
yl)urea and 1-(3-methoxyphenyl)-3-(3-nitrophenyl)urea. Each
of the compounds studied were dissolved in a solution contain-
ing DMF (12%, v/v), ACN (40%, v/v), and Tween-20 (0.1%, v/v)
in part A of assay buffer, and examined as potential competi-
tors using the cELISA described above. Structural information
regarding these compounds is presented in Table 1.
Solu bility of Nica r ba zin . Nicarbazin is known for
its very difficult solubility, both in water and in some
organic solvents. It is well understood that some solvent
in the ELISA can be tolerated. A general example was
the work by Li et al. (48). In that study, their Mabs
worked well in solutions of acetone or DMSO (2%, v/v)
and up to 5% methanol (v/v). Also, the work by Wa-
tanabe et al. (49) showed the effect of MeOH concentra-
tions on the reactivity and sensitivity of a Mab with
various concentrations of imazalil. That work showed
a depression of the observed absorbance with 10% or
higher MeOH concentrations. They used 10% MeOH (v/
v) in their ELISA analysis, which decreased the absor-
bance by about 20%. That solvent tolerance was suffi-
cient for analysis of their samples. These amounts of
solvents in their ELISA seem to be what is most
commonly observed, perhaps with 10% MeOH (v/v)
being on the high end. In our case, these quantities of
solvent in the ELISA were not enough for solubility of
nicarbazin.
RESULTS AND DISCUSSION
Syn th esis of Mim ic Ha p ten s a n d Con ju ga tes.
Antibodies useful for the detection of nicarbazin have
not been previously produced. In an initial approach to
making antibodies against nicarbazin, we attempted to
make the hydrazone of DNC (Figure 1) using 4-hydrazi-
nobenzoic acid. This approach was discussed in Beier
and Stanker (35). Antibodies obtained from that hapten
never competed with “free” DNC. Molecular modeling
was used to study this hapten and others to help
understand the dynamics of the problem (35). Molecular
modeling gave the electrostatic potential energy isos-
urfaces of DNC and other possible haptens. The hapten
that had surface charges nearly identical to those of
DNC was the mimic succinate derivative of p-nitro-
aniline (referred to as PNA-S, Figure 2). Although this
mimic is smaller than DNC, it is identical to each half
of DNC bisected at the carbonyl group of the urea. It is
well-known that antibodies can be produced to mol-
ecules of this size (46).
PNA-S was synthesized as described by Beier and
Stanker (39). Temperature was very critical during the
reaction. It was found that heating the reaction mixture
to 70 °C overnight resulted in a good product yield.
PNA-S was linked to KLH using an NHS-enhanced,
carbodiimide-mediated coupling reaction (Figure 3), the
conjugate was used as the immunogen and injected into
BALB/c mice. However, using the conjugate of PNA-S
linked to BSA by the same method resulted in nonspe-
cific binding and “free” nicarbazin or DNC would not
It was determined that a concentration of 3% DMF
(v/v) and 10% ACN (v/v) were required to provide
adequate solubility to nicarbazin to allow standard
curve applications. Nicarbazin did not have high enough
solubility in MeOH buffer solutions to use MeOH in the
assay. Standards were made in DMF (12%, v/v), ACN
(40%, v/v), and Tween-20 (0.1%, v/v) in part A of assay
buffer. These standards were added to equal volumes
of assay buffer on the microtiter plates resulting in a
solution of 6% DMF (v/v) and 20% ACN (v/v). Concen-
tration gradient dilutions were made on the microtiter

Monoclonal Antibodies to Nicarbazin
J. Agric. Food Chem., Vol. 49, No. 10, 2001 4547
Ta ble 1. cELISA In h ibition Stu d ies w ith Nitr op h en yls a n d Oth er 4,4′-Din itr oca r ba n ilid e Rela ted Com p ou n d s

4548 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Beier et al.
Ta ble 1. (Con tin u ed )
a
Maximum concentration of compound used in cross-reactivity studies. b Percentage of cross-reactivity was calculated according to
c
d
the formula: (IC35 of nicarbazin)/(IC35 of other compound) × 100. The IC30 was used for Nic 7, which was the center of the curve. The
e
Nic 9 curve was depressed by solvent interactions and values were calculated at the IC20, Nic 9-nicarbazin IC20 ) 0.66 nmol/mL. No
f
cross-reactivity was observed at the concentrations of chemical used. Competition was observed at the limit of detection (LOD) of Nic 6
at IC15 ) 0.33 nmol/mL nicarbazin. Determined from average of 3 runs. h “C” Competition was observed at the LOD of Nic 8 at IC15 )
g
j
0
.34 nmol/mL nicarbazin. i Competition was observed at the LOD of Nic 7 at IC10 ) 0.4 nmol/mL nicarbazin. Competition was observed
k
at the LOD of Nic 9 at IC15 ) 0.5 nmol/mL nicarbazin. The Nic 7 curve was depressed by solvent interactions, and values were calculated
at the IC20, Nic 7-nicarbazin IC20 ) 0.69 nmol/mL.
plates in 6% DMF (v/v) and 20% ACN (v/v). Equal
volumes of antibody were then added to the wells
resulting in a final concentration of 3% DMF (v/v) and
of solvent did depress the cELISA results, as observed
in attempted trials with less solvent (data not shown).
In general, inhibition curves ranged from 0 to 70%
inhibition. Therefore, the center of the curves (IC35) were
1
0% ACN (v/v) used in the cELISA. This concentration

Monoclonal Antibodies to Nicarbazin
J. Agric. Food Chem., Vol. 49, No. 10, 2001 4549
F igu r e 3. Synthetic pathway for producing the immunogen
(PNA-S-KLH) or the plate coating antigen (PNA-C-BSA).
F igu r e 5. Standard curves for nicarbazin with Mabs Nic 6
and Nic 7 were obtained using assay buffer fortified with DMF
(
3%, v/v) and ACN (10%, v/v). Each point represents the mean
standard deviation from 13 and 12 determinations, respec-
(
tively, over an eight-week period. Each data point was
calculated from the absorbance at 450 nm using the equation
(1 - (B/B°)) × 100.
We utilized the heterologous assay by using nicarbazin
or PNA-C-BSA as a coating antigen and PNA-S-KLH
as the immunogen to help overcome unwanted cross-
reactivity. Nicarbazin inhibition curves in 3% DMF (v/
v) and 10% ACN (v/v) for the Nic 6 and Nic 7 Mabs are
shown in Figure 5. Figure 5 shows the inhibition of
antibody binding by nicarbazin in the cELISA with
PNA-C-BSA coated plates. The IC35 for Nic 6 is 78.5 ng/
well (0.92 nmol/mL), and the IC30 for Nic 7 is 89.7 ng/
well (1.1 nmol/mL) (see Table 1).
Ch a r a cter iza tion of Ma bs. Isotype and Affinity.
Immunoglobulin isotype of the four Mabs Nic 6, Nic 7,
Nic 8, and Nic 9 were found to be IgMκ. The concentra-
tions of nicarbazin resulting in 35% inhibition of control
activity (i.e., wells with no competitor present) were
F igu r e 4. Evaluation of the effect of assay buffer pH on the
binding of the Mabs Nic 6-9: (A) PNA-C-BSA is the plate
coating antigen, and (B) nicarbazin is the plate coating
antigen.
used for analysis points, and the limit of detection (LOD)
usually was found at IC15.
Titr a tion Stu d ies w ith Va r iou s Bu ffer p H Va l-
0
.92, 1.2, and 1.4 nmol/mL for Nic 6, Nic 8, and Nic 9,
u es. Titration studies were carried out with both PL-1
respectively. The concentration of nicarbazin resulting
in 30% inhibition of control for the Nic 7 antibody was
(
4
nicarbazin, Figure 4B) and PL-2 (PNA-C-BSA, Figure
A) coatings. The assay buffer was formulated at pH
values of 6.8, 7.0, 7.2, 7.4, 7.6, and 7.75 with 3% DMF
v/v) and 10% ACN (v/v). The y-axis is the dilution of
1
.1 nmol/mL. The Nic 6 and Nic 7 dosage-inhibition
curves are shown in Figure 5.
(
Specificity. Cross-reactivity studies with all four Mabs
yielded similar results in most cases. Table 1 shows all
candidate cross-reacting compounds examined. All cross-
reactivity studies were carried out using PNA-C-BSA
coated plates. The results with nicarbazin was selected
to be used as 100% cross-reactivity value since it is
nicarbazin that the Mabs will be detecting in feed
samples. Percentage of cross-reactivity was calculated
according to the formula: (IC35 of nicarbazin)/(IC35 of
other compound) × 100. The IC30 was used for Nic 7
experiments. For those cases where the solvent played
a larger role in depressing the inhibition curves, the
cross-reactivity was calculated at the LOD for both
nicarbazin and the other compound. Only two com-
pounds out of the 21 tested showed no cross-reactivity
with nicarbazin; the compound HPD, that is used to
form the complex with DNC in nicarbazin, and 1-(2-
fluorophenyl)-3-(2-methoxy-4-nitrophenyl)urea. The Mab,
antibody required to produce an OD450 reading of 0.45
after subtraction of the solvent background. The four
Mabs (Nic 6, Nic 7, Nic 8, and Nic 9) binding on PL-1
plates oscillated across the pH range with peaks at
approximately pH 7.0, 7.4, and 7.75. Whereas, use of
PL-2 plates resulted in the highest antibody binding at
approximately pH 7.3-7.5.
The use of nicarbazin coated directly on the assay
plates without the use of a protein conjugate is novel.
As seen in Figure 4B, the nicarbazin coating worked
well at pH 7.0, 7.4, and 7.75. However, the PNA-C-BSA
conjugate coating produced results that were more
consistent from plate to plate than that seen on the
nicarbazin-coated plates.
cELISA Develop m en t. Heterologous assays are
well-known to help improve immunoassay sensitivity
(50, 51) and overcome unwanted cross-reactivity (47).

4550 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Beier et al.
Nic 6, showed different cross-reactivity patterns than
the other Mabs in many cases. Nic 6 showed cross-
reactivity with 2-nitroaniline (37%), 4-nitrophenol (69%),
boxanilate; PNA-S, p-nitrosuccinanilate; RO H2O, re-
verse osmosis water, pyrogen free; NaOH, sodium
hydroxide; Sulfo-NHS, sulfo-N-hydroxy succinimide;
TLC, thin-layer chromatography;
4
3
-nitrophenethyl alcohol (2%), and 1-(4-chlorophenyl)-
-(4-nitrophenyl)urea (47%), and the other Mabs showed
no cross-reactivity to these four candidates. Also, the
Nic 7 and Nic 8 Mabs showed cross-reactivity to 4-ni-
troaniline, 4-nitrobenzyl alcohol, 2-nitrobenzyl alcohol,
ACKNOWLEDGMENT
We thank Dr. Abilio C. Tardin of Mogiana Alimento
S.A., Rlia Das Magn o´ lias, Das Bandeiras, for generously
supplying the DNC, HDP and nicarbazin standards.
3
-nitrobenzyl alcohol, and 1-(3-chlorophenyl)-3-(4-meth-
oxy-3-nitrophenyl)urea, while the Nic 6 Mabs showed
no cross-reactivity to these candidates. Other candidates
such as DNC, PNA-S, PNA-C, 3-nitroaniline, 2-nitro-
phenol, 3-nitrophenol, and 1-(3-chlorophenyl)-3-(2-meth-
oxy-5-nitrophenyl)urea were bound for the most part by
all Mabs. Nic 7 was the only Mab that showed better
cross-reactivity to some candidates, 4-nitroaniline (121%),
LITERATURE CITED
(1) Long, P. L., Ed. The Biology of the Coccidia, University
Park Press: Baltimore, MD, 1982.
(2) McDougald, L. R.; Reid, W. M. Coccidiosis. In Diseases
of Poultry, 10th ed.; Calnek, B. W., Ed.; Iowa State
University Press: Ames, IA, 1997; pp 865-883.
1
1
-(3-chlorophenyl)-3-(2-methoxy-5-nitrophenyl)urea (172%),
-(3-chlorophenyl)-3-(4-methoxy-3-nitrophenyl)urea (107%),
(
(
(
3) Cuckler, A. C.; Malanga, C. M.; Basso, A. J .; O’Neill, R.
C. Antiparasitic activity of substituted carbanilide com-
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and 1-(3-methoxyphenyl)-3-(3-nitrophenyl)urea (144%),
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4) Cuckler, A. C.; Malanga, C. M.; Ott, W. H. The anti-
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CONCLUSIONS
1
09.
Mabs were developed to the highly water insoluble
coccidiostat nicarbazin. Four Mabs were developed, Nic
5) McDougald, L. R. Control of coccidiosis: Chemotherapy.
In Coccidiosis of Man and Domestic Animals; Long, P.
L., Ed.; CRC Press: Boca Raton, FL, 1990; pp 307-320.
6
, Nic 7, Nic 8, and Nic 9, and had an isotype of IgMκ.
These Mabs detected the active component in nicarba-
zin, DNC, and had no cross-reactivity to the nonactive
component, HDP. PNA-C-BSA or nicarbazin used as
plate coating antigens overcame unwanted cross-
reactivity. Because of the poor solubility of nicarbazin,
rather harsh solvent restrictions were placed on the
cELISA. A cELISA solvent system of 3% DMF (v/v) and
(6) Cannavan, A.; Ball, G.; Kennedy, D. G. Determination
of nicarbazin in feeds using liquid chromatography-
electrospray mass spectrometry. Analyst 1999, 124,
1
431-1434.
(
7) Razmi, G. R.; Kalideri, G. A. Prevalence of subclinical
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47-253.
1
0% ACN (v/v) was used during these studies. These
(
8) Campbell, W. C.; Cuckler, A. C. Inhibition of egg
production of Schistosoma mansoni in mice treated with
nicarbazin. J . Parasitol. 1967, 53, 977-980.
added solvents did depress the inhibition curves for
nicarbazin. It was demonstrated that a pH range from
7
.3 to 7.5 showed highest antibody binding using PNA-
(9) Ball, S. J .; Pittilo, R. M.; Yvor e´ , P.; Mancassola, R.; Long,
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C-BSA coated microtiter plates. The two Mabs with the
best detection for nicarbazin was Nic 6 and Nic 7. The
IC35 for Nic 6 was 78.5 ng/well (0.92 nmol/mL) and the
IC30 for Nic 7 was 89.7 ng/well (1.1 nmol/mL). These
Mabs are good candidates for the use in a cELISA for
the determination of nicarbazin in animal feeds. Nic 6
and Nic 7 have been deposited with ATCC, resulting in
ATCC designations of PTA-2647 and PTA-2648, respec-
tively.
(
2
27-233.
(
11) McDougald, L. R.; Seibert, B. P. Residual activity of
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ABBREVIATIONS USED
6
9, 19-29.
ACN, acetonitrile; ATCC, American Type Culture
Collection; BSA, bovine serum albumin; BSA-hapten,
BSA conjugated to hapten; CHCl3, chloroform; CHDA,
cis-1,2-cyclohexanedicarboxylic anhydride; DCC, N,N-
dicyclohexylcarbodiimide; DMF, dimethylformamide;
DNC, 4,4′-dinitrocarbanilide; cELISA, competitive en-
zyme-linked immunosorbent assay; EtOAc, ethyl ac-
etate; FSIS, Food Safety and Inspection Service; HAT,
hypoxanthine, aminopterin, and thymidine; HDP, 2-hy-
droxy-4,6-dimethylpyrimidine; HPLC, high performance
liquid chromatography; HT, hypoxanthine and thymi-
dine; H2SO4, sulfuric acid; i.p., intraperitoneal; KLH,
keyhole limpet hemocyanin; KLH-hapten, KLH conju-
gate to hapten; Mab, monoclonal antibody; MeOH,
methanol; MS, mass spectrometry; MWCO, molecular
weight cutoff; NFDM, nonfat dry milk; NHS, N-hydroxy-
succinimide; PBS-9, phosphate buffered saline pH 9; PL-
(
13) Daugschies, A.; G a¨ sslein, U.; Rommel, M. Comparative
efficacy of anticoccidials under the conditions of com-
mercial broiler production and in battery trials. Vet.
Parasitol. 1998, 76, 163-171.
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M.; Leanza, W. J .; Nichols, J . R.; Ostlind, D. A.; Rodino,
T. M. Nicarbazin complex yields dinitrocarbanilide as
ultrafine crystals with improved anticoccidial activity.
Science 1983, 222, 630-632.
(
(15) Porter, C. C.; Gilfillan, J . L. The absorption and excre-
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(
16) Anon. Nicarbazin. In Code of Federal Regulations; 21
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(
1
, plate coating 1 (nicarbazin); PL-2, plate coating 2
PNA-C-BSA); PNA-C, p-nitro-cis-1,2-cyclohexanedicar-
(

Monoclonal Antibodies to Nicarbazin
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(
18) Michielli, R. F.; Downing, G. V., J r. Differential pulse
polarographic determination of nicarbazin in chicken
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(
19) Wood, J . S., J r.; Downing, G. V. Modified pulse polaro-
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Received for review February 16, 2001. Revised manuscript
received J uly 12, 2001. Accepted J uly 12, 2001. Mention of a
trade name, proprietary product, or specific equipment does
not constitute a guarantee or warranty by the U.S. Depart-
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J F010208J