Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

Colloidal Gold Immunochromatographic Assay for Rapid On-Site Detection of Tetracycline in Seawater

Recently increasing concerns from the scientists and public have been paid for seawater pollution due to tetracycline(TC)overuse in maricultural area.However,there are few methods or instruments that can be used for specific and rapid detection of this antibiotic in seawater.In this study,the colloidal gold immunochromatographic assay(CG-ICA)was used to achieve this goal.A commercialized monoclonal antibody against TC(anti-TC mAb)was selected because of its higher sensitivity(half-maximal inhibitory concentration of 2.38μgL^(-1)).The prepared CG particles(average diameter of 20 nm)were used to label anti-TC mAb at pH 8.0.The conjugate pad was formed by spraying the CG-labeled anti-TC mAb on a glass fibre membrane followed by proper dryness.The test pad was made by immobilizing artificial antigen and anti-mouse mAb in the test line and the control line,respectively,in a nitrocellulose membrane.The test strip,assembled with sample pad,conjugate pad,test pad and absorbent pad,could be used to detect TC during seawater sample flowing through these components in turn.The results could be observed by the naked eye in 10min.The visible limit of detection(vLOD)was 20μgL^(-1) for TC in seawater.The CG-ICA test results were in good agreement with those of liquid chromatography-tandem mass spectrometry(LC-MS/MS).The assay also showed that,oxytetracycline(OTC)and chlortetracycline(CTC),as the structural analogues of TC,did not interfere with TC determination.Furthermore,the TC concentration given by test strip could not be affected by the fluctuation of temperature(10℃–30℃),pH(7–9)and salinity(0–40)of seawater.Therefore,CG-ICA is a suitable tool for rapid,on-site,and semi-quantitative detection of TC in seawater.

Effects of 105 traditional Chinese medicines on the detection ofβ-agonists in medicine extracts and swine urine based on colloidal gold immunochromatographic assay

Colloidal gold immunochromatographic assay(CGIA)is commonly used for the on-site detection ofβ-agonists that are sometimes used illegally as feed additives in swine diets.However,few studies have evaluated the causes of false-positive results that sometimes occur when applying CGIA in agricultural settings.In this study,we investigated if this false-positive phenomenon is related to the addition of certain traditional Chinese medicines(TCMs)to swine feed.We established and verified an extraction method for TCMs,and then applied CGIA to detectβ-agonists in the extracts of 105 TCMs and in the urine of swine dosed with TCMs,respectively.Liquid chromatography-tandem mass spectrometry was used to validate the results of the urine samples tested positive forβ-agonists using CGIA.The results were also verified using TCMs and colloidal gold test strips produced by different manufacturers.The extracts of Citri Reticulatae Pericarpium Viride,Citri Reticulatae Pericarpium,Magnoliae Officinalis Cortex,Chaenomelis Fructus,and Rhodiolae Crenulatae Radix Et Rhizoma were tested positive forβ-agonists.Meanwhile,the addition of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium to swine feed resulted in false-positive results forβ-agonists in swine urine.The results provide a new way to explain false-positive CGIA results and provide valuable information for livestock feeding programs.

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.

Development of a colloidal gold-based immunochromatographic strip for rapid detection of Rice stripe virus

Rice stripe virus(RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies(MAbs) 16E6 and 11 C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper(SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20 480(w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560(individual SPBH/μL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.

An immunochromatographic assay for rapid and simultaneous detection of levonorgestrel and methylprednisolone in water samples

Synthetic contraceptive levonorgestrel （LNG） and glucocorticoid methylprednisolone （MP） residues are eventually discarded to environmental water system and function as environmental hormones, displaying potential risk to humans and ecosystems, thus there is an urgent need for fast, sensitive and simultaneous detection of these compounds in water samples. In this study, a competitive immunochromatographic assay （ICA） using colloidal gold-labeled polyclonal antibodies as probes for rapid and simultaneous detection of LNG and MP in water samples was developed. The visual detection limits of LNG and MP in water samples were 10 ng/mL. The detection process could be completed within 10 min. There was no cross-reactivity of the ICA with other seven compounds. The strips could be stored at 4 ℃ for 10 weeks without significant loss of activity. The assay is a suitable tool for rapid and semi- quantitative detection of LNG and MP in water samples on site.