Evaluation and verification of the characteristic peptides for detection of Staphylococcus aureus in food by targeted LC-MS/MS.

The traditional methods for detection of Staphylococcus aureus in food have some shortcomings, such as fussy operation and dependence on separation of pure colony. A novel strategy for detection of pathogenic bacteria bases on targeted analysis of the characteristic peptides by LC-MS/MS. The key to the success of this strategy is to identify a combination of reliable characteristic peptides that afford high specificity. In this study, the candidate characteristic peptides of S. aureus were evaluated bioformatically and then verified experimentally. UniProt protein database were used to perform BLAST analysis on the candidate characteristic peptides, and then their specificity were compared via targeted analysis in S. aureus and other Staphylococcus strains by LC-MS/MS. Finally, suitable S. aureus characteristic peptide was determined, by using which 100 cfu/mL S. aureus in milk samples were successfully detected, meeting the detection limit requirement in actual application. The results demonstrated the feasibility of the detection strategy of S. aureus based on characteristic peptides. The exploratory work is expected to promote the methodology research of food-borne pathogen inspection and further improve the inspection efficiency and quality.

Rapid analysis of three ß-agonist residues in food of animal origin by automated on-line solid-phase extraction coupled to liquid chromatography and tandem mass spectrometry.

An automated online solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed and validated for the detection of clenbuterol, salbutamol, and ractopamine in food of animal origin. The samples from the food matrix were pretreated with an online solid-phase extraction cartridge by Oasis MCX for <5 min after acid hydrolysis for 30 min. The peak focusing mode was used to elute the target compounds directly onto a C18 column. Chromatographic separation was achieved under gradient conditions using a mobile phase composed of acetonitrile/0.1% formic acid in aqueous solution. Each analyte was detected in two multiple reaction monitoring transitions via an electrospray ionization source in a positive mode. The relative standard deviations ranged from 2.6 to 10.5%, and recovery was between 76.7 and 107.2% at all quality control levels. The limits of quantification of three ß-agonists were in the range of 0.024-0.29 µg/kg in pork, sausage, and milk powder, respectively. This newly developed method offers high sensitivity and minimum sample pretreatment for the high-throughput analysis of ß-agonist residues.

[Determination of 14 pesticide residues in crucian carp using on-line gel permeation chromatography-multi gas chromatography/mass spectrometry].

A new method was developed for the determination of pesticide residues in crucian carp using on-line gel permeation chromatography-multi gas chromatography/mass spectrometry (GPC-MDGC/MS). The pesticide residues were extracted with cyclohexane-ethyl acetate (1:1, v/v) for twice. After being frozen, the extract was filtered by 0.22 microm member, and then cleaned up and analyzed by online GPC-MDGC/MS. The pesticides were selectively transferred from the 1st column to the 2nd column by means of heart cutting. As a result, selective transfers of some pesticides from the first to the second dimension were at times essential to avoid overlapping. The selected separation conditions from the study with standards were applied to crucian carp spiked with some pesticide standards. The performance of this method was examined by recovery, linearity and precision. Inter-standard quantitative method was used for calculation. Good linear relationship was achieved in the range of 0.01-0.9 mg/L for the 14 pesticides with correlation coefficients above 0.99. The recoveries of the method ranged from 83.0% to 112.9% with the relative standard deviations (RSDs) among 3.2%-12.0% at 3 spiked levels of 0.01, 0.05 and 0.1 mg/kg. This is a simple, rapid method for the analysis of multiple pesticide residues in crucian carp with high accuracy and sensitivity. The results prove that the employment of a multidimensional analysis technique permits to avoid interferences of the analytes with matrix components.

[Determination of 14 mycotoxins in Chinese herbs by liquid chromatography-tandem mass spectrometry with immunoaffinity purification].

A method was developed for the determination of 14 mycotoxins, aflatoxins, T-2, HT-2, fumonisins, ochratoxin A, zearalenone, etc. in Chinese herbs by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). The sample was extracted with phosphate buffer solution (PBS) and methanol in turn, and then purified by a high selective multi-functional immunoaffinity column. The column was washed by PBS (containing 0.1% Twain) and water, and then eluted by methanol. The eluate was dried under nitrogen, dissolved in methanol-10 mmol/L NH4Ac (40 : 60, v/v) solution. The mycotoxins were separated on a Waters Xterra C18 MS column (100 mm x 2.1 mm, 3.5 microm) and detected by MS/MS. The limits of quantification (LOQs) of the 14 mycotoxins were from 1.0 to 5.0 microg/kg. The average recoveries of the 14 mycotoxins spiked in Chinese herbs (Ginseng, Campanulaceae, Radix and Ophiopogonis) ranged from 71.9% to 99.7% at the three spiked levels of 1.0, 5.0, 10.0 microg/kg, and the relative standard deviations (RSDs, n = 6) were between 4.8% and 15.8%. The method is rapid, sensitive and accurate, and suitable for the determination of the 14 mycotoxins in Chinese medicines. The quantification limits of aflatoxins can meet the domestic and foreign requirements.

Production and characterization of monoclonal antibody against recombinant human erythropoietin.

OBJECTIVE: To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. METHODS: rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot. RESULTS: The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. CONCLUSIONS: The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column.

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.

Screening of carbohydrate-specific phage antibodies against recombinant human erythropoietin (rhuEPO) using a phage display antibody library: preliminary study.

This paper is a preliminary report on development of a screening method for carbohydrate-specific phage antibodies against recombinant human erythropoietin (rHuEPO), using a phage display antibody library. rHuEPO was oxidized with sodium periodate or treated with 1,4-dithiothreitol and guanidine hydrochloride for detecting the specificity of obtained phage antibodies. Of 100 phage clones, three initially showed higher carbohydrate-related specificity. One of them (No. 62) bound specifically to the carbohydrate chains of rHuEPO, while the other two (Nos. 63 and 83) might recognize the steric conformation related to both the carbohydrate and the polypeptide chain of rHuEPO. These phage antibodies may serve as useful capture ligands for future development of efficient analytical methods for rHuEPO.

Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library.

The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B2 was expressed in soluble form in Escherichia coli DH5alpha F' and purified by His-bond nickel affinity chromatography with a yield of about 1-2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0 x 10(8) L mol(-1) based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.

Production and characterization of anti-recombinant human erythropoietin (rhEPO) monoclonal antibody.

Anti-rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti-rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme-linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS-PAGE, and Western-blotting. Experimental results showed that the subclass and the light chain of anti-rhEPO McAb was IgG1 and kappa light chain. The molecular weight of anti-rhEPO McAb was 166,000 Daltons. The affinity constant (K(aff)) of anti-rhEPO McAb with coated antigen was 5.0 x 10(5)L/mol.

Pharmacokinetics and tissue distribution of iv injection of polyphase liposome-encapsulated cisplatin (KM-1) in rats.

AIM: The pharmacokinetics and biodistribution of cisplatin encapsulated in polyphase liposome (KM-1) were compared with those of free drug in rats. METHODS: The platinum levels in serum and normal organs, after a single dose of iv injection of free or encapsulated cisplatin to rats, were determined by induced coupled plasma atomic emission spectrometry. RESULTS: Serum platinum concentration-time curve after a single iv dose of KM-1 4.5 mg/kg in rats was fitted with an open three-compartment model. The pharmacokinetic parameters were as follows: Vc=0.10 L/kg, T1/2pai=0.3 h, T1/2alpha=3.5 h, T1/2beta=2.7 h, AUC=265 mg.h.L(-1), and CL(s) =0.02 g.L.h(-1). KM-1 was cleared from the circulation much more slowly than free cisplatin. Liver and spleen had the highest concentration of platinum after KM-1 treatment. CONCLUSION: KM-1 remained in the bloodstream longer than its free drug, and was taken mainly by the reticuloendothelial system.