An ultrasensitive immunochromatographic assay for non-pretreatment monitoring of chloramphenicol in raw milk

A non-pretreatment monitoring method based on immunochromatographic test (ICT) strips was developed to test for chloramphenicol (CAP) in raw milk. This assay was designed to measure competitive binding affinities for CAP molecules in raw milk and CAP antigen immobilized on strips with colloidal gold nanoparticles (GNPs, average diameter of 30 nm) labeled with anti-CAP monoclonal antibody (CAP mAb). Several working conditions, including the CAP solvent, different types and concentrations of CAP mAb, coating antigen, and GNP, were optimized for performance. After optimization, the detection process was carried out in 8 min with a visual limit of detection (LOD) of 0.3 ng/mL for qualitative detection and a LOD of 0.064 ng/mL for semi-quantitative detection. No cross-reactivity was detected toward CAP analogs. Based on these results, this simple and ultrasensitive assay could be thoroughly applied in the on-site testing of CAP in raw milk.     

Competitive ELISA of Chloramphenicol: Influence of Immunoreagent Structure and Application of the Method for the Inspection of Food of Animal Origin

An indirect competitive ELISA for the detection of chloramphenicol (CAP) in food of animal origin (milk, meat, eggs) is described. Influence of immunoreagent structure and composition on the assay sensitivity and specificity was investigated. Two CAP derivatives were used for conjugation with proteins: CAP succinate and a diazo derivative of CAP. Molar incorporation of CAP into the coating conjugates was also varied. To eliminate matrix effect on the assay results, a special casein-containing buffer was used for milk samples, whereas for meat and egg samples a 50-fold dilution of the buffer extracts was needed. The method developed permits CAP concentrations to be determined in the range 0.08-100 μg 1^-1. The detection limit is 0.08 μg kg^-1. Recovery in different food samples averages between 70 and 130%. The method can be applied for inspection of food of animal origin for CAP residues.     

Design and Use of a General Capturing Surface in Optical Biosensor Analysis of Sulphamethazine in Milk

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 microg kg -1 and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cow's milk. The advantages of the new assay format include analyte-independent immobilisation and regeneration. Furthermore, the assay enables measurements with covalent interactions between analyte and detecting molecule. The main disadvantage is the requirement of a conjugate between analyte and the hapten H1. Moreover, it is likely that the antibody surface will have a shorter life span than a surface with the antimicrobial immobilised.     

ELISA of streptomycin in buffer and milk: Effect of reagents' structure and analysis format on assay performance

The influence of immunoreagents' structure on assay performance was investigated and a range of ELISAs for streptomycin in direct and indirect format was developed. Streptomycin was conjugated with proteins (bovine serum albumin (BSA) for immunization and ovalbumin (OVA) for immobilization on a plate) by two different methods. Streptomycin was derivatized with carboxymethoxylamine (CMO) and then coupled to a protein or the protein was activated with adipic acid dihydrazide (ADH) and then coupled with streptomycin. A conjugate with horse-radish peroxidase was synthesized using streptomycin-ADH derivative. With the indirect ELISA the most sensitive assay for polyclonal antisera against streptomycin-oxime-BSA in combination with homologous and heterologous conjugates (limit of detection 2.5 ng ml^-1) was developed, whereas for a combination 'antisera against streptomycin-ADH-BSA/heterologous conjugate' higher background level of a calibration curve was observed. Besides the level was very high (about 60%) for homologous conjugate. In a direct ELISA similar sensitivity was achieved only for antisera against streptomycin-oxime-BSA (limit of detection 3.0 ng ml^-1). Chemiluminescent detection allowed to increase the assay sensitivity by several times (limit of detection 0.5 ng ml^-1) but led to the worse reproducibility (CV 16%). A sensitive and simple direct ELISA for analysis of streptomycin in milk products without preliminary sample preparation was developed (limit of detection 3.2 ng ml^-1). In the indirect ELISA an influence of fat content of a milk product on assay performance was observed.     

Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens

BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied. OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects. METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques. RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon. CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.     

Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk

Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from na?ve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.     

A Sensitive Streptavidin‐Biotin Enzyme‐linked Immunosorbent Assay for Rapid Screening of Residues of Chloramphenicol in Milk

A sensitive streptavidin‐biotin enzyme‐linked immunosorbent assay (ELISA) for the detection of chloramphenicol (CAP) in milk is described. This test is based on a procedure developed earlier for the direct detection of CAP in crude aqueous meat extracts. Milk samples were defatted by centrifugation, filtered and directly submitted to the ELISA procedure. The results were compared with the values obtained by analysis of a part of the sample from which CAP was removed by an immobilized monoclonal antibody preparation. In spiked samples the presence of CAP at concentrations of 1·0 μg/kg and higher can be easily demonstrated. The possibility of the interpretation of the ELISA results with a statistical method, i.e. the so‐called standard deviation ratio (SDR) procedure, was also investigated.     

An ultrasensitive chemiluminescent ELISA for determination of chloramphenicol in milk,milk powder,honey, eggs and chicken muscle

A competitive, direct, chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for chloramphenicol (CAP) residues in milk, milk powder, honey, eggs and chicken muscle has been developed. The method gave a detection limit of 0.7 ng L^?1 and a linear range of 2.1–92.4 ng L^?1, with the IC₅₀ of 13.6 ng L^?1 under optimal conditions, dramatically better than any previously reported ELISA method for CAP detection. Spiked at levels of 5–60 ng L^?1 in different food samples, recoveries were in the range of 72.1–116.0%, with coefficient of variations of 4.2–20.2%. In a study of incurred residues, the chicken muscle samples diluted 5-, 10- and 20-fold, results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector and traditional ELISA. The developed CL-ELISA method is, therefore, suitable for rapid screening trace CAP residues in food samples.     

Substitution of antibody with molecularly imprinted 96-well plate in chemiluminescence enzyme immunoassay for the determination of chloramphenicol residues

A direct competitive chemiluminescence enzyme immunoassay was developed by using the molecularly imprinted 96-well plate as an artificial antibody to detect chloramphenicol (CAP). The artificial antibody was synthesised on the well surface of MaxiSorp polystyrene 96-well plate and CAP was conjugated with the horseradish peroxidase. The results showed that the imprinted plate exhibited antibody-like binding ability. The plate showed fast adsorption rate, 66% adsorption was finished within 20 min. The cross-reactivity for CAP, florfenicol and thiamphenicol were 100%, 1.25% and 2.08%, respectively. And the imprinted plate could be reused for many times without loss of sensitivity. The IC₅₀ and the detection limit values under optimum conditions were 30±2 µg·L^?1 and 0.9±0.01 µg·L^?1, respectively. The plate was used to detect CAP in sea cucumber, which showed excellent recoveries ranging from 89% to 98.7%. And the result correlated well with that obtained by the CAP enzyme-linked immunosorbent assay (ELISA) kit.     

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg^-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.     

Development of a quantitative fluorescence-based lateral flow immunoassay for determination of chloramphenicol,thiamphenicol and florfenicol in milk

A competitive lateral flow immunoassay (LFA) using fluorescent microspheres (FMs) as label was developed for the quantitative detection of chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) in milk. The limit of detection (LOD) for CAP, TAP and FF in milk was 0.08, 0.8 and 1.9?μg?L^?1, respectively. The recovery of intra and inter-assay ranged from 87.7% to 107.0% and 82.5% to 112.3%, with corresponding coefficient of variations less than 11.3% and 14.5%, respectively. The sensitivity of the FMs lateral flow assay (FMs-LFA) was comparable to those of the colloidal gold lateral flow assay. A quantitative comparison of the FMs-LFA and LC–MS/MS analysis of milk samples indicated good agreement between the two methods. The FMs-LFA can be used as a reliable, rapid and cost-effective method for food safety analysis.     

A novel method to chemically immobilize antibody on nylon and its application to the rapid and differential detection of two Vibrio parahaemolyticus toxins in a modified enzyme-linked immunosorbent assay.

A new method of chemically immobilizing antibody on nylon was developed. The method consists of serial treatments with HCl, polyethylene imine, and maleic anhydride methylvinyl ether copolymer, which resulted in the stable immobilization of sufficient amounts of antibodies on nylon. This principle was used to differentially detect two immunologically related but nonidentical hemolysins (thermostable direct hemolysin [TDH] and TDH-related hemolysin [TRH]) of Vibrio parahaemolyticus in a modified enzyme-linked immunosorbent assay with antibodies immobilized on nylon slips (NSIT). The results (dark purple color on nylon slips) were easily evaluated by the naked eye. The results with NSIT were compatible with those obtained by using DNA probes or a conventional bacterial culture test, not only with cultured specimens but also with clinical specimens (diarrheal stool samples). Furthermore, the NSIT differentially detected TDH and TRH in a single test. The antibody immobilization method developed here is applicable to various immunological detection methods and may improve their sensitivity and specificity.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Peripheral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody/antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.     

Preparation of an anti-dexamethasone monoclonal antibody and its use in development of a colloidal gold immunoassay

A lateral flow colloidal gold (CG) immunoassay strip has been developed for detection of dexamethasone (DEX) residues in milk samples. For this purpose, an anti-DEX monoclonal antibody (McAb), based on a DEX succinic anhydride derivative hapten, was prepared and characterized. The McAb showed a high specificity to DEX, the half inhibitory concentration of the antibody was 0.095?ng/mL, its limit of detection (LOD) was 0.017?ng/mL, and its linear range of detection was 0.034–0.265?ng/mL. The developed CG immunoassay had a visual cut-off value of 0.3?ng/mL in phosphate buffered saline (PBS) and 0.5?ng/mL in milk samples. Each test requires 10 min. Analysis of DEX in milk indicated that the results of strip assay had a strong agreement with indirect competitive enzyme-linked immunosorbent assay. Therefore, the CG immunoassay is a sensitive screening method for semi-quantitative and qualitative detection of DEX residues in milk samples.     

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibodies to porcine parvovirus (PPV). Antisera to PPV were raised in pigs, for which PPV grown on PK15 cells was used for primary intranasal inoculations, and PPV cultured on autologous kidney cells for booster immunisations. A competition ELISA was developed, based on the principle of a double antibody sandwich assay, using immunoglobulin fractions prepared from these sera. The ELISA was compared with a haemagglutination inhibition (HI) test. The tests were equally sensitive for detecting antibodies early after infection and for detecting a significant increase in antibody titre between paired sera. A high correlation was found between antibody titres of field sera measured by the two tests (r = 0.91). We conclude that ELISA is preferable to the HI test, because it is labour-saving and can be standardised better and automated.     

A novel separation-free assay technique for serum antibodies using antibody bridging assay principle and two-photon excitation fluorometry

A new technique for separation-free detection of antigen-specific antibodies is presented. The new technique employs antibody bridging assay principle and the recently developed ArcDia TPX fluorescence detection technology. According to the assay scheme, antibody molecules from the sample bind with one arm to an antigen on polymer microspheres and with the other arm to a fluorescently labeled secondary antigen reagent. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique, an assay for anti-adenovirus antibodies was constructed. The function of the assay method was tested both with monoclonal anti-adenovirus antibody preparation (standard analyte), and with positive serum samples. Standard class-specific ELISA was used as a reference method. The new assay method provides comparable sensitivity and precision, and wider dynamic range for IgG antibodies than the ELISA method. The standard curve showed linear response (R(2)=0.999) with a dynamic range of three orders of magnitude, detection limit (mean+3S.D.) of 8 pM, and intra-assay signal precision of 5%. Applicability of the new method for clinical serodiagnostics is discussed.     

Low prevalence of Chlamydia pneumoniae in adults with community-acquired pneumonia

The incidence of community-acquired pneumonia (CAP) due to Chlamydia pneumoniae was determined in a prospective study of 546 adult patients with CAP included in the German CAP Competence Network (CAPNETZ) project. Three different PCR protocols for detection of C. pneumoniae in respiratory specimens were compared by a multicenter, inter-laboratory comparison involving three laboratories. A case was defined as a patient with a respiratory sample positive by PCR in at least two laboratories. CAP was caused by C. pneumoniae in 5/546 cases (0.9%). Antibody testing by microimmunofluorescence was done in 376 of 546 patients. All patients were negative for IgM antibodies. In the five PCR-positive patients, neither specific IgG nor IgA antibodies were found. Patients with CAP caused by C. pneumoniae had a lower median age (36 years) than the general study population (62 years). C. pneumoniae is currently a rare cause of CAP in adult patients in Germany. Analysis of a single serum sample is not useful for diagnosis of acute C. pneumoniae infection in CAP.     

Two-site enzyme immunometric assays for determination of native and denatured β-lactoglobulin

Two enzyme immunometric assays suitable for measuring native and denatured β-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cow's milk and cow's milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cow's milk in goat's milk.     

Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk

Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug for animals, which has antipyretic, analgesic, and anti-inflammatory effects. The drug, which was originally used to relieve inflammation in horses, musculoskeletal disorders, and pain, has been approved for use in endotoxemia, infectious diseases in swine, etc. A sensitive anti-FM monoclonal antibody 2H4 was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay for the detection of FM in milk. The complete antigen and coating antigen were conjugated with bovine serum albumin and ovalbumin, respectively. The monoclonal antibody 2H4, with a half inhibition concentration of 0.29?ng/mL, had a limit of detection of 0.432?ng/mL and a linear range of detection of 0.08664–0.97226?ng/mL. A sensitive and convenient immunochromatographic strip assay was developed with an FM cutoff value of 0.29?ng/mL. The developed methods were suitable for the detection of FM in milk.