Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 µg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 µg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine.

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

Characterization and application of quantum dot nanocrystal-monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay.

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot-MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen-antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC50) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10-100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1-9.2%.

Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine.

A lateral flow immunoassay (LFIA) device was developed and applied to testing urine samples for residues of the antimicrobial sulphamethazine (SMZ). This report describes the preparation of a rat monoclonal antibody to SMZ and its characterisation in an ELISA format. Apart from SMZ, the antibody showed high (> or =50%) cross-reactivity to N4-acetyl-sulphamethazine (55%), sulphamerazine (59%) and sulphisoxazole (50%) and lower cross-reactivity of 18% to sulphachlorpyridazine and sulphadiazine. The LFIA device consisted of a nitrocellulose membrane spotted with SMZ-ovalbumin and goat anti-mouse antibody as capture line and control line, respectively. Mouse anti-rat IgG F(ab')2 fragment specific antibody, adsorbed to colloidal carbon, was used as the detection ligand in the LFIA. The LFIA device had a cut-off value of 6.3 ng/ml in diluted (1/10) urine. Urine samples from SMZ-treated pigs, and bovine and porcine urine samples fortified with SMZ were used for a blind, four-laboratory evaluation of the performance of the LFIA device. Concentrations of SMZ in the test samples (n=29), as determined by LC-MS/MS, ranged from 0 (<3) to 1174 ng/ml. The evaluation of the LFIA device showed an overall sensitivity of 100%, a specificity of 71%, and positive and negative prediction values of 73% and 100%, respectively. The LFIA device has been fabricated as a test kit for determining SMZ residues in animals produced for slaughter.

Use of optical biosensor technology to study immunological cross-reactivity between different sulfonamide drugs.

Adverse reactions to medications account for a substantial number of hospitalizations and in some cases fatalities. The nature of the many drug-drug interactions caused by the inhibition of drug-metabolizing enzymes can now be predicted and examined with a greater deal of accuracy due to research developments in the understanding of the drug-metabolizing enzymes. However, the more troubling aspects of drug-drug interactions are the idiosyncratic reactions that are unpredictable and quite often life-threatening. These reactions are often caused by a prior sensitization of a person's immune system to a given drug or class of drugs. The following work offers a technique to examine in a medium-throughput system the cross-reactivity of drugs to antibodies in order to predict if structures share the same antigenic potential toward a sensitized individual. Two commercially important sulfonamide drugs, sulfamethazine and furosemide, were taken and their binding to their respective antibodies were tested in the presence of other structurally related sulfonamide drugs. The BIACORE 3000 biosensor was used for the study and the solution-phase equilibrium assay principle was employed. The data obtained help us determine which drugs can react, and to what extent, with sulfamethazine and furosemide, giving rise to possible allergic or hypersensitivity reactions. Though sulfamethazine and furosemide were used in this study; this principle and methodology can be applied to study any drug molecule-antibody pair.

Determination of sulfamethazine residues in milk by a surface plasmon resonance-based biosensor assay.

The use of antibiotics and chemotherapeutics in animal husbandry has led to the occurrence of veterinary drug residues in all types of food of animal origin. Due to the specification of toxicologically based maximum residue levels for a large number of substances, existing control strategies need even faster and more sensitive methods to meet new and more rigorous regulations. The applicability of an immunosensor device for biospecific interaction analysis was investigated and the development of an assay for analysis of sulfamethazine (SMZ) in milk is described. SMZ was covalently immobilized to a carboxymethyldextran-modified gold film. Spiked samples with known concentrations of SMZ were prepared in HBS buffer and skim and raw milk for construction of standard curves. Polyclonal antibodies against SMZ were added to the sample and the immobilized surface was used to determine the amount of free antibodies by surface plasmon resonance detection. After each measurement the surface was regenerated by NaOH and HCl. In milk, the mean relative standard deviation of the assay was approximately 2% and the limit of detection less than 1 ppb. By introduction of a secondary sheep anti-rabbit antibody, the use of specific antibody could be reduced. Milk samples from the individual cow, herd, and tanker levels were analyzed and the relative standard deviations within each sample category were 4.4, 2.4, and 2.2%, respectively. The effect of some potential interferences, e.g., high somatic cells, bacterial contamination, and preservatives, was investigated. The results were not influenced in such a way that the risk for so-called false-positive findings was obvious.

High-volume enzyme immunoassay test system for sulfamethazine in swine.

A high-volume enzyme immunoassay (EIA) system for slaughterhouse screening of sulfamethazine in swine plasma/serum has been developed. The system includes a robotic sample processor that performs most of the liquid handling requirements in the assay. The assay gives good correlation with the widely used thin layer chromatographic method for determination of sulfamethazine in serum and plasma (r = 0.826). Inter- and intra-assay coefficients of variation are less than 10%. Approximately 2,400 serum/plasma samples can be analyzed in a normal working day (8 h) with this system.

Determination of sulphadimidine in animal feedstuffs by an enzyme-linked immunoassay.

An enzyme immunoassay technique for the determination of sulphadimidine in animal feedstuffs has been developed. The antibody showed limited cross-reactivity with other drugs, including sulphonamides, used as feed additives. Using spiked samples recoveries of 80-88% were obtained. The limit of detection of the assay was 70 ng/g.