Preparation Of Anti-furosemide Monoclonal Antibodies And Immunochromatographic Strip Based On Aggregation-induced Luminescence Nanobeads

Slimming dietary supplements has been widely used in the obese or fat control people because of its functions of lowering fat and losing weight.To rapid losing weight,several synthetic drugs have been illegally added to slimming dietary supplements,and these chemical drugs pose a serious health risk to consumers,such as electrolyte disorders,cardiovascular and cerebrovascular diseases,liver and kidney function damage,and even leading to death.Furosemide（LASIX）is an effective diuretic drug because it can accelerate or inhibit cell metabolism by changing the activity of related enzymes or the permeability of cell membrane to accelerate the discharge of water in the body,thereby resulting in weight loss.Illegal addition of LASIX in slimming dietary supplements could cause a series of side effects,including vomiting,dizziness,kidney function damage and hypokalemia.Up to now,various instrument-dependent methods have been established for LASIX detection.However,these methods commonly require complex sample pretreatment,are also time consuming,high cost input and unsuitable for on-site test in the field area.In recent years,the lateral flow immunoassay（LFIA）method has been widely used for the rapid detection of bacteria,viruses,proteins and small molecules due to its simple operation,and low cost.Thus,the preparation of anti-LASIX monoclonal antibodies with high affinity and sensitivity in LFIA is of great significance for the realization of rapid field screening detection for the prohibited use of LASIX.Aggregation-induced emission luminogens（AIEgens）have the properties of aggregation-induced emission.Aggregation-induced emission fluorescent microspheres（AIEFMs）are prepared by loading aggregation-induced emission dyes,can achieve a highly luminescent signal intensity by loading more AIEgens because it has effectively overcome the aggregation-caused quenching（ACQ）effect of conventional organic fluorescent dyes.In addition,the AIEFMs also have some unique advantages,such as light bleaching resistance,high luminous intensity,good stability and large Stokes shift,and thereby have attracted extensive attention from researchers in the fields of bioimaging,biosensing and food safety monitoring.In this study,the activated ester method（EDC/NHS）has been used to synthesize the LASIX and keyhole hemocyanin（KLH）conjugates.After several rounds of immunization of Balb/c mice with LASIX-KLH,the serum titer of mouse was determined by using an enzyme linked immunosorbent assay（ELISA）method,and two anti-LASIX monoclonal antibodies（mAbs）were obtained by cell fusion.Subsequently,AIEFMs and mAbs conjugates（AIEFM@mAbs）were prepared by coupling the anti-LASIX mAbs on the surface of AIEFMs,and established for the rapid,and sensitive determination of LASIX,and the applicability and feasibility of AIEFMs-LFIA in the detection of LASIX in the samples of slimming dietary supplements were also demonstrated.Firstly,LASIX-KLH was synthesized by directly coupling the carboxyl group of LASIX with amino groups of KLH,and two anti-LASIX mAbs,named 3H8 and 6D1,were successfully obtained by immune,serum assay,cell fusion,hybridoma cell screening and cell subcloning.The titers of 3H8-mAbs and 6D1-mAbs were determined by an ELISA method,and their isotypes were analyzed using the mouse immunoglobulin isotyping ELISA kit.The results showed that the both of mAbs belonged to the IgG₁,and their titers were 1:243000（3H8）and 1:81000（6D1）.The affinities of 3H8-mAbs and 6D1-mAbs were 3.48×10⁹L/mol and 3.63×10⁸L/mol,respectively.Under the optimal conditions,the detection performance of 3H8-mAbs were assessed by the ELISA method,and the results showed that the 50%competitive inhibitory concentration(IC₅₀),quantitative detection of LASIX range and detection of limit（LOD）of 3H8-mAbs were 2.71 ng/m L,0.39～18.82 ng/m L and 0.204 ng/m L,respectively.In addition,the 3H8-mAbs showed a negligible cross-reaction（<0.5%）to the LASIX analogues,including Torasemide（TOR）,Chlorothiazide（THI）,Benzthiazide（BEN）,Hydrochlorothiazide（HYD）and Chlorthalidone（TRA）.Secondly,an ultra-bright red AIEFMs was introduced as signal amplified probe to construct a novel fluorescent LFIA for the highly sensitive detection of LASIX in slimming dietary supplements.Under the optimal conditions,AIEFMs-LFIA exhibited a good dynamic linearity with the LASIX concentrations ranging from 0.08ng/m L to 1.22 ng/m L.The IC₅₀and LOD values were 0.31 ng/m L and 0.049 ng/m L,respectively.The average recoveries of intra-and inter-assays for the LASIX spiked slimming tea samples were in a range of 88.71%to 111.78%with the coefficient of variation in a range of 3.74%to 13.08%,indicating that the AIEFMs-LFIA had good precision and accuracy for the LASIX quantitative determination in real slimming tea samples.In addition,the developed AIEFMs-LFIA also showed a good specificity with a negligible cross-reaction with the LASIX analogues.Finally,the reliability of the proposed method was evaluated by analyzing eighteen of LASIX artificially contaminated slimming tea and slimming capsule samples,and the results of AIEFMs-LFIA also showed a good agreement with those of ELISA kit.In conclusion,the ultra-bright red AIEFMs can be used as a good signal amplified probe to enhance the sensitivity of LFIA,and the developed AIEFMs-LFIA also showed a good detection performance for the LASIX rapid and sensitive detection in the slimming dietary supplements.