Optical Fiber-Mediated Immunosensor with a Tunable Detection Range for Multiplexed Analysis of Veterinary Drug Residues.

We describe herein a newly developed chemiluminescent optical fiber immunosensor (OFIS) with a tunable detection range for multiplexed analysis of veterinary drug residues with vastly different concentrations in milk samples. The optical fiber probe is used as a carrier of biorecognition element as well as a transducer, enabling a low-cost compact design, which makes this system suitable for cost-effective on-site detection of the target analytes. Importantly, the synergy between modulation of the length of the optical fiber sensing region and the number of fibers allows performing multiplexed immunoassays in an easily controllable manner over a tunable detection range from pg/mL to µg/mL analyte concentrations. By combining the optical fiber sensor with a nanocomplex signal amplification system, a highly sensitive chemiluminescent OFIS system is demonstrated for the multiplexed assaying of veterinary drug residues in milk samples with linear ranges of 10-(2 × 104) pg/mL for chloramphenicol, 0.5-500 ng/mL for sulfadiazine, and 0.1-300 µg/mL for neomycin. This controllable strategy, based on modulation of the fiber probe, provides a versatile platform for multiplexed quantitative detection of both low-abundance and high-abundance targets, which shows great potential for on-site testing in food safety.

Nanoparticles-Enabled Surface-Enhanced Imaging Ellipsometry for Amplified Biosensing.

The main issues of imaging ellipsometry-based biosensing for small molecules are the low sensitivity and narrow detection range due to the low molecular weight of small molecules that results in a negligible signal. To meet this challenge, we theoretically investigated the deciding factors of the ellipsometry signal and further applied the theory to guide the design of ellipsometry-based biosensor using metal nanoparticles that have a high dielectric constant. Significant signal amplification effects can be achieved by using nanoparticle labels including magnetic nanoparticles and gold nanoparticles. Guided by the theory, we have developed a sensitive surface-enhanced imaging ellipsometry (SEIE)-biosensor for detecting chloramphenicol in real milk sample with high sensitivity (with a limit of detection of 6 pg/mL) and broaden detection range. This nanoparticles-enabled SEIE not only greatly improves the sensitivity of conventional imaging ellipsometry-based biosensors but also retains the advantages of conventional methods in terms of automated and convenient operation, providing an effective strategy for detection of trace small molecules in complex samples that holds great promise in scientific research, clinical diagnosis, and food safety.

A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples.

A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1 µg L(-1). The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement.

Development of a homogeneous immunoassay based on the AlphaLISA method for the detection of chloramphenicol in milk, honey and eggs.

BACKGROUND: A homogenous light-induced chemiluminescence immunoassay was developed using AlphaLISA technology for the detection of chloramphenicol (CAP). This technology is based on two different kinds of bead, namely light-sensitive donor beads and beads containing chemiluminescers, also called acceptor beads. A competitive CAP AlphaLISA method was established using artificial antigen-coated acceptor beads, polyclonal antibodies, biotinylated goat anti-rabbit IgG and streptavidin-coated donor beads. RESULTS: The sensitivity of detection was 0.0086 ng mL⁻¹ and the working range was from 0.0096 to 25 ng mL⁻¹. The intra- and inter-assay coefficients of variation were both below 10%. The average recovery rates at spiked levels of 0.05-10 ng mL⁻¹ were 103.2, 108.4 and 91.6% for milk, honey and eggs respectively. The data obtained from the samples showed good correlation with ELISA results. CONCLUSION: The CAP AlphaLISA method is highly sensitive, specific and rapid and is suitable for screening large quantities of samples.

Label-free immunoassay for chloramphenicol based on hollow gold nanospheres/chitosan composite.

A novel label-free electrochemical immunosensor for rapid determination of chloramphenicol (CAP) was fabricated by entrapping monoclonal antibody to chloramphenicol (anti-CAP) in hollow gold nanospheres (HGNs)/chitosan composite modified on a glassy carbon electrode. The hollow gold nanospheres (HGNs) were prepared by using Co nanoparticles as sacrificial templates and characterized by transmission electron microscopy (TEM). The changes of the electrode behavior after each fabrication step were investigated by electrochemical impedance spectroscopy (EIS) technique. Under optimal conditions, the proposed immunosensor has a sensitive response to CAP in a linear range of 0.1-1000 ng mL(-1) with the detection limit of 0.06 ng mL(-1). Accurate detection of CAP in real meat samples was demonstrated by comparison with conventional HPLC method. The proposed method was proven to be a feasible quantitative method for CAP analysis with the properties of simple preparation, stability, high sensitivity and selectivity.

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode.

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10⁻¹6 M, and a very low determination limit of 1.0 × 10⁻¹6 M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10⁻¹5 M, and SATUM electrode, 4.7 × 10⁻¹4 M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.

Competitive immunoassay by capillary electrophoresis with laser-induced fluorescence for the trace detection of chloramphenicol in animal-derived foods.

A competitive immunoassay using CE with an LIF detector was developed for the detection of chloramphenicol (CAP). The method was based on the competitive reactions between fluorescently labeled CAP hapten and free CAP, with a limited amount of anti-CAP antibody. The poly(N-isopropylacrylamide) (pNIPA) hydrogel was added in the separation buffer as a dynamic modifier to reduce adsorption and enhance reproducibility. The linear range and LOD for CAP were 0.008-5 microg/L and 0.0016 microg/L, respectively. An ELISA using the same immuno-reagents was also developed for the analysis of CAP, with an LOD of 0.03 microg/L. The sensitivity of this CE immunoassay (CEIA)-LIF was almost 20 times greater than that of the ELISA. Using CEIA-LIF, equilibrium was reached in 15 min and the analytical results were obtained within 5 min by CE separation. Sample preparation for CEIA-LIF was not time-consuming and the matrix effect was easy to remove. An LOD of 0.1 microg/kg CAP in food matrices was easily achieved. This method is thus proposed as a fast and sensitive means of detecting trace amounts of CAP residues in animal-derived foods.

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies.

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC50 values ranged from 0.3 ng mL(-1) to 5.5 ng mL(-1) by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL(-1) to 1.7 ng mL(-1) by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserum G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using D1). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (G1) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1).

A free energy calculation study of the effect of H-->F substitution on binding affinity in ligand-antibody interactions.

Changes in binding affinity to catalytic antibody 6D9 of chloramphenicol phosphonate derivatives (CPDs) containing H or F were investigated by performing free energy calculations based on molecular dynamics simulations. We calculated the binding free energy, enthalpy, and entropy changes (DeltaDeltaG, DeltaDeltaH, and -TDeltaDeltaS) attributable to H-->F substitution by comparing results for CPDs containing a trifluoroacetylamino group (CPD-F) or an acetylamino group (CPD-H). The calculated DeltaDeltaG, DeltaDeltaH, and -TDeltaDeltaS values were -2.9, -6.3, and 3.5 kcal mol(-1) and close to experimental values observed for a series of similar ligands, chloramphenicol phosphonates with F and H (-1.4, -3.5, and 2.1 kcal mol(-1)). Therefore, CPD-F binds more strongly to 6D9 than does CPD-H. To clarify the origin of the large difference in DeltaDeltaG, we apportioned the calculated values of DeltaDeltaG and DeltaG for the associated and dissociated states into contributions from various atomic interactions. We found that the H-->F substitution increased the binding affinity mainly by decreasing the hydration free energy and not by increasing favorable interactions with the antibody. The decreased hydration free energy of the ligand was mainly due to unfavorable coulombic interactions between the trifluoroacetylamino group and solvent waters, which increased the free energy of the dissociated state (by about 3.7 kcal mol(-1)). Also, the trifluoroacetylamino group slightly increased the free energy level of the associated state (about 0.8 kcal mol(-1)) because favorable van der Waals interactions compensated for unfavorable coulombic interactions with antibody atoms. In addition, the enthalpy and entropy changes, DeltaDeltaH and -TDeltaDeltaS (computationally -6.3 and 3.5 kcal mol(-1)), originated mainly from a decrease in hydration free energy in the dissociated state. The CPD-F and CPD-H ligands had substantially different structures in the dissociated and complexed states.

Directed evolution governed by controlling the molecular recognition between an abzyme and its haptenic transition-state analog.

The catalytic antibody, 6D9, was subjected to directed evolution in the phage-display system using two structurally related transition-state analogs (TSAs) for panning. One analog, TSA 3, was originally used for immunization, and the other, TSA 4, a derivative of TSA 3, was designed to optimize the differential affinity for the transition state relative to the ground state so as to provide variants with improved reaction rates. We previously reported that by panning with TSA 4, we could obtain variants with highly improved catalytic rate enhancement (k(cat)/k(uncat)), and Tyr (L27e) seemed to play a key role in stabilizing the transition-state structure [Nat. Biotechnol. 19 (2001) 563]. Here, we examined in detail a large number of the variants selected by these haptens, in order to elucidate the mechanism of the directed evolution driven by them. ELISA with 3- and 4-bovine serum albumin (BSA) showed that variants selected by these TSAs exhibited distinct binding patterns. All the variants whose rate enhancement was greater than five-fold of that of 6D9 had Tyr (L27e) and were obtained from the library panned with TSA 4, but not from the library panned with TSA 3. Kinetic studies showed that TSA 4 could efficiently select variants with increased differential binding affinity for the transition state relative to the ground state, and these variants exhibited improved rate enhancements. This study verified the difference of in vitro evolution driven by the two structurally related TSAs and stresses the importance of designing an appropriate hapten for panning.

[Immunogenic properties of the colloidal gold]. / Immunogennye svoistva kolloidnogo zolota.

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or Freund's adjuvant). Application of colloidal gold increased nonspecific immune responses as well: lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The obtained antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens as well as complete antigens was shown to induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. In addition, antigen quantities for animal immunization with colloidal gold was by one order of magnitude lower as compared to the complete Freund's adjuvant immunization. This fact can point to direct adjuvant activity of colloidal gold.

Development of a direct-binding chloramphenicol sensor based on thiol or sulfide mediated self-assembled antibody monolayers.

A batch-type antibody-immobilized quartz crystal microbalance (QCM) system for detecting chloramphenicol (CAP) was developed. To bind an anti-CAP antibody onto the gold electrode surface of piezoelectric crystals, self-assembled monolayers (SAMs) of different thiols or sulfides were formed by a chemisorption procedure. Then, the anti-CAP antibody was covalently linked to the pre-formed monolayers by an activation procedure using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide. The antibody-immobilized QCM chip thus prepared was installed in a well holder and was measured for sensor response. Compared with the bare QCM chip and the QCM chip only coated with 3-mercaptopropionic acid (MPA), the antibody-immobilized sensor showed greatly enhanced frequency shifts by 10-50-fold after CAP injection. In this case, CAP detection which was indicated by steady-state resonant frequency shift was accomplished within 10 min. When CAP solution was injected into the reaction cell in 50mM concentration, the frequency shifts obtained were, respectively, 530 and 505 Hz in case of thiosalicylic acid and MPA immobilization. Repeated use of the sensor chips up to eight times was possible after 1 min regeneration with 0.1M NaOH. This system demonstrated a potential application of thiol or sulfide mediated SAMs as the pre-coatings of a real-time detection on CAP in solution.

Molecular features determining lymphocyte reactivity in allergic contact dermatitis to chloramphenicol and azidamphenicol.

BACKGROUND: We report on two cases of allergic contact dermatitis to chloramphenicol and azidamphenicol respectively, with in vivo and in vitro lymphocyte reactivity to both compounds. The molecular features determining lymphocyte reactivity were explored because chloramphenicol, azidamphenicol, and thiamphenicol exhibit almost identical chemical structures. METHODS: With chloramphenicol, azidamphenicol, and the chemically related thiamphenicol, we performed patch tests and lymphocyte transformation tests with both patients. Furthermore, the interleukin-5 and interferon-gamma concentrations in the cultures of peripheral blood mononuclear cells of one patient were determined. RESULTS: Patch tests showed delayed hypersensitivity reactions to chloramphenicol and azidamphenicol, but not to thiamphenicol. These results were confirmed by lymphocyte transformation tests with peripheral blood mononuclear cells of the patients, showing a proliferative T-cell response to azidamphenicol and chloramphenicol. Moreover, lymphocytes from one patient secreted large amounts of interleukin-5, but not of interferon-gamma upon coculture with azidamphenicol. CONCLUSIONS: Since lymphocyte reactivity was observed to chloramphenicol and azidamphenicol, but not to thiamphenicol, the epitope(s) recognized by the allergen-reactive T cells may be formed by the nitro-group of the benzene ring shared by chloramphenicol and azidamphenicol.

Lymphocyte activation in allergic reactions elicited by small-molecular-weight compounds.

The involvement of cytochrome-P450-dependent metabolism of small-molecular-weight compounds as a prerequisite for an optimal lymphocyte response to those compounds is discussed on the basis of studies with compound such as p-phenylenediamine, sulfamethoxazole, chloramphenicol and fragrances. Subsequently, data on cytochrome P450 activity and CYP gene expression in monocytes and MAC-6 cells for cytochromes P450 2E1 and 3A4 are presented.

Antimicrobial resistance of enteropathogenic Escherichia coli strains from a nosocomial outbreak in Kenya.

The majority of the 78 enteropathogenic (EPEC) and the 151 non-EPEC Escherichia coli strains isolated from preterm neonates during an outbreak of gastroenteritis in a hospital in Nairobi, Kenya, were resistant to trimethoprim-sulfamethoxaxole, chloramphenicol, oxytetracycline and ampicillin, but only a few strains were resistant to cefazolin, cefamandole, cefotaxime, amikacin and nalidixic acid. Fourteen different antimicrobial resistance patterns were observed in the 229 strains of E. coli analysed. Eighty-two percent of the EPEC strains belonged to two resistance pattern compared with 79% of non-EPEC strains which exhibited three resistance patterns. There was no consistent relationship between plasmid profile group and antimicrobial resistance pattern, although one resistance pattern was more frequently observed in EAF-positive strains belonging to the dominant plasmid profile group. Nine percent of the EPEC strains were resistant to gentamicin compared to 37% in the non-EPEC group. No correlation was observed between administration of gentamicin and percentage of resistant strains isolated. None of the nine neonates receiving gentamicin died during the outbreak. Gentamicin resistance was observed in E. coli strains from six out of these nine neonates. Five out of fourteen neonates who received other antimicrobials, or no antibiotic treatment at all, died.

Penicillin-resistant pneumococci in a Merseyside hospital.

Strains of Streptococcus pneumoniae (N = 915) from clinical specimens were examined for penicillin resistance over a 2-year period. The prevalence of resistance [minimum inhibitory concentration (MIC) greater than 0.1 mg l-1] increased from 1.4 to 2.5% per year during this time. In addition, 83% of penicillin-resistant pneumococci (PRP) showed resistance to chloramphenicol. Most PRP were isolated from uninfected children colonized with the organism, but two out of the three adult cases were clinically infected, one by cross-infection between in-patients. In only two cases was there an association with foreign travel. Three children showed prolonged carriage providing a potential reservoir of infection for other members of the community. The percentage of strains showing high level resistance (MIC greater than 1 mg l-1) increased from 0.7% to 1.9% of all isolates during the 2-year study period. This high prevalence of high level resistance has not been reported previously in the UK and if the trend continues, it will have serious implications for the management of invasive pneumococcal infection, particularly meningitis.

Pharmacokinetic assessment of immunosuppressive activity of antibiotics in human plasma by a modification of the mixed lymphocyte reaction.

Antibiotics may impair the development and expression of specific or nonspecific immune responses. Prophylactic administration of antibacterial antibiotics is widely used in ICUs. We studied the immunosuppressive activities of cefotaxime, chloramphenicol, gentamicin, metronidazole, and rifamycin as a function of time after the administration of these drugs to ICU patients, finding that the last 4 drugs had an immunosuppressive activity detectable up to 8 h by a mixed lymphocyte reaction. When these antimicrobial agents were added to normal pooled plasma in concentrations similar to those obtained in vivo, a similar degree of inhibition was observed.