Indirect Competitive Enzyme-linked Immunosorbent Assay Research Articles

Development of IgY-Based Indirect Competitive ELISA for the Detection of Fluoroquinolone Residues in Chicken and Pork Samples.

Fluoroquinolones (FQs) are among the antibiotics whose widespread use in farm-raised animals results in potentially harmful residues in the end products. Additionally, most Thai farmers use antibiotics. Amoxicillin and enrofloxacin were commonly used by pig farms, and hens were given enrofloxacin to prevent immunization side effects. Moreover, antibiotic overuse has harmed food safety in the long term, and the use of low-dose antibiotics causes bacterial resistance. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to make a fast, easy, sensitive, and cost-effective method for monitoring FQs residues. After immunizing hens with mixed multi-hapten ciprofloxacin-bovine serum albumin (CPFX-BSA) with norfloxacin-bovine serum albumin (NFX-BSA), the IgY antibody purified from egg yolk was used for the detection of FQs residues in chicken and pork samples. The efficiency of the IgY antibody showed excellent sensitivity, with 50% inhibitory concentration (IC50) of enrofloxacin at 0.05 µg/mL, far below the MRLs defined by the EU for muscle samples, which was not to exceed 100 µg/kg. The recovery range for chicken muscle samples spiked with ENFX concentrations of 1.00-0.01 µg/mL was 86.65-112.71%, similar to pork samples, which were 84.24-117.22.2%. This method has a lot of potential for analyzing fluoroquinolones in complex samples quickly, easily, and at a low cost on-site. The IgY-based ic ELISA was developed to detect ciprofloxacin (CPFX), norfloxacin (NFX), and enrofloxacin (ENFX) residues; it confirms that IgY could be a promising choice for the detection of antibiotic residues in food samples.

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10=6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n=10, sampled in 1994-1999, none MYXV-DNA positive, and n=28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p=.009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7vs 1.7% considering all the sample, p=.007), and lower during the winter than the autumn (p<.001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

A Novel Metabolite as a Hapten to Prepare Monoclonal Antibodies for Rapid Screening of Quinoxaline Drug Residues

Quinoxalines (Qx) are chemically synthesized antibacterial drugs with strong antibacterial and growth-promoting effects. Qx is heavily abused by farmers, resulting in large residues in animal-derived foods, which pose a serious threat to human health. Desoxyquinoxalines (DQx), which have the highest residue levels, have been identified as the major toxicant and have become a new generation of residue markers. In this study, we prepared monoclonal antibodies (mAb) based on a new generation metabolite (desoxymequindox, DMEQ) and establish an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the rapid determination of Qx residues in food. The mAb exhibited high sensitivity with half maximal inhibitory concentration (IC50) and a linear range of 2.84 µg/L and 0.8–12.8 µg/L, respectively. Additionally, the cross-reactivity (CR) of the mAb showed that it recognized multiple DQx to varying levels. The limits of detection (LOD), limits of quantification (LOQ), and recoveries for the ic-ELISA assay of pork, swine liver, swine kidney, chicken, and chicken liver were 0.48–0.58 µg/kg, 0.61–0.90 µg/kg, and 73.7–107.8%, respectively, and the coefficients of variation (CV) were less than 11%. The results of the ic-ELISA showed a good correlation with LC–MS/MS in animal-derived foods. This suggests that this analytical method can be used for the rapid screening of QX residues.

Significantly improved detection performances of immunoassay for ractopamine in urine based on highly urea-tolerant rabbit monoclonal antibody.

Highly sensitive and accurate screening of ractopamine (RAC) residue in animal urine is greatly needed to ensure food security. The detection performance of immunoassay for RAC was always seriously harmed by the antibody inactivation derived from urea. Here, we first discovered one rabbit monoclonal antibody (RmAb) to RAC with a high affinity of 0.007ngmL-1 and a surprising urea tolerance of 3M urea, which is beneficial for developing robustly developed immunoassay in urine without sample pretreatment. The limits of detection of developed indirect competitive enzyme-linked immunosorbent assay based on RmAb1 for RAC were 0.0042-0.014μgL-1 with the coefficient of variation below 11.7% in swine, sheep, and cow urine, significantly improved 10-100-fold in sensitivity. Moreover, the urea-tolerant mechanism of RmAb1 showed that more non-polar amino acids, more hydrogen bond donors on the surface, and preponderant Pi interaction of antibody-RAC all contributed to the stability of the RmAb1 in a high concentration of urea.

Development of enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for the rapid detection of gentamicin in chicken muscle and milk

• A specific monoclonal antibody against gentamycin was obtained. • Based on mAb, one-step ic-ELISA and GICA methods were developed. • Two immunoassays can be applied for the detection of gentamycin in real samples. A specific anti-gentamicin (GEN) monoclonal antibody (mAb) was used to develop an indirect competitive ELISA (ic-ELISA) and a colloidal gold immunochromatographic assay (GICA) for the determination of GEN residues in chicken muscle and milk. The result of ic-ELISA showed that the limits of detection (LOD) for GEN in chicken muscle and milk samples ranged from 0.54 µg kg −1 –0.57 µg kg −1 . As for the GICA method, the LOD of GEN in chicken muscle and milk were 10 µg kg −1 and 10 µg kg −1 , respectively. The GEN recovery of the two methods ranged from 83.8%–112.2%. In the actual samples detection, the ELISA and GICA methods had a good correlation with high-performance liquid chromatography (HPLC) results. These indicated that the methods established in this study could be a candidate tool for the rapid large-scale screening of GEN in animal-derived foods.

Development of a Reliable ic-ELISA with a Robust Antimatrix Interference Capability Based on QuEChERS Technology for the Rapid Detection of Zearalenone in Edible and Medical Coix Seeds and Subsequent Risk Assessments.

Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is an ideal immunoassay method for large-scale screenings to detect mycotoxin contaminants. However, the matrix effect of complicated samples has always been challenging when performing immunoassays, as it leads to false-positive or negative results. In this study, convenient QuEChERS technology combined with optimizing the dilution solvent was ingeniously used to eliminate interference from the sample matrix to greatly improve the detection accuracy, and reliable ic-ELISAs for the two official tolerance levels of 60 and 500 μg/kg were developed to screen zearalenone (ZEN) in edible and medical coix seeds without any further correction. Then, the 122 batches of coix seeds were determined, and the positive rate was up to 97.54%. The contaminated distribution was further analyzed, and risk assessment was subsequently performed for its edible and medical purposes. The findings indicated that consumption of coix seeds with higher ZEN contamination levels may cause adverse health effects for both medical and edible consumption in the adult population; even under the condition of average contamination level, ZEN from coix seeds was the more prominent contributor to the total risk compared to other sources when used as food; thus, effective prevention and control should be an essential topic in the future.

A Nanobody-Based Immunoassay for Detection of Ustilaginoidins in Rice Samples.

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins produced by the pathogen Villosiclava virens of rice false smut, which has recently become one of the most devastating diseases in rice-growing regions worldwide. In this research, the nanobody phage display library was established after an alpaca was immunized with the hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). Heterologous antigen selection and combing trypsin with competition alternant elution methods were performed for nanobody screening. Two nanobodies, namely, Nb-B15 and Nb–C21, were selected for the establishment of indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs). For Nb–B15 and Nb-C21, their IC50 values were 11.86 μg/mL and 11.22 μg/mL, and the detection ranges were at 3.41–19.98 μg/mL and 1.17–32.13 μg/mL, respectively. Two nanobodies had a broad spectrum to quantify the contents of total ustilaginoidins in rice samples according to cross-reactivity. The recognition mechanisms of Nb-B15 and Nb-C21 against ustilaginoidin A were elucidated by molecular modeling and docking. The key amino acid sites for the binding of Nb–B15 or Nb–C21 to ustilaginoidin A were mainly located in the FR1 and CDR1 regions. As Nb-B15 was superior to Nb–C21 in the aspects of protein expression, ELISA titer, and tolerance to organic solvents, it was selected for application in the detection of actual contaminated rice samples. The total ustilaginoidin contents of rice samples were analyzed by Nb–B15-based ic–ELISA and HPLC-DAD, between which the results were found to be consistent. The developed immunoassay based on the nanobody from the alpaca can be employed as a rapid and effective method for detection of total utilaginoidins in contaminated rice samples.

Development of an indirect competitive enzyme-linked immunosorbent assay for formononetin and its application in a cell-based assay using MC3T3-E1 cells

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53–1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.

Carboxyl retained hapten of quinclorac produces highly sensitive antibody and immunochromatographic assay

In order to obtain better anti-quinclorac antibody, we synthesized three novel quinclorac haptens de novo, including a hapten (H3) retains the carboxyl. As expected, H3 immunized mice showed the best immune responses and generated the anti-quinclorac monoclonal antibody (mAb 6D2F4) with the 50 % inhibition concentration (IC50) of 1.63 ng mL−1 in indirect competitive enzyme-linked immunosorbent assay, which is improved 30- to 245-fold than the reported anti-quinclorac antibodies, and no significant cross-reactivities with the analogues of quinclorac. Then mAb 6D2F4 was employed to develop the colloidal gold immunochromatographic assay (GICA) for the detection of quinclorac. The limit of detection (LOD) of naked eye observation is 8 ng mL−1, while the LODs of GICA in agro-products samples also can meet the requirements regulated by China. Besides, the detection results of blind samples by GICA show high consistency with those by high performance liquid chromatography analysis.

Analysis of residual monepantel sulfone in milk using icELISA based on a monoclonal antibody developed from an easy-synthesized and low-cost hapten

Monepantel (MNP) is a new class of aminoacetonitrile derivative antiparasitic drug and mainly metabolizes as monepantel sulfone in vivo. This study aimed at developing a novel monepantel sulfone (MNPS) detection method combining indirect competitive enzyme-linked immunosorbent assay (icELISA) and immunomagnetic separation. An easy-synthesized hapten was designed based on the partial structure of MNPS. A monoclonal antibody was obtained by immunizing mice, and applied to icELISA. The half inhibition concentration (IC50) was 1.36 ng/mL, the limit of detection (LOD) was 0.25 μg/L. The average recoveries of the icELISA ranged from 89.5% to 114.5% with the coefficient of variation lower than 13.2%. In summary, a rapid and reliable icELISA was developed for detecting MNPS in milk samples for the first time.

A Novel Lateral Flow Immunochromatographic Assay for Rapid and Simultaneous Detection of Aflatoxin B1 and Zearalenone in Food and Feed Samples Based on Highly Sensitive and Specific Monoclonal Antibodies.

Simultaneous aflatoxin (AFB1) and zearalenone (ZEN) contamination in agro-products have become widespread globally and have a toxic superposition effect. In the present study, we describe a highly sensitive and specific dual lateral flow immunochromatographic assay (dual test strip) for rapid and simultaneous detection of AFB1 and ZEN in food and feed samples based on respective monoclonal antibodies (mAbs). Two immunogens AFB1-BSA (an AFB1 and bovine serum albumin (BSA) conjugate) and ZEN-BSA (a ZEN and BSA conjugate) were synthesized in oximation active ester (OAE) and amino glutaraldehyde (AGA). The molecular binding ratio of AFB1:BSA was 8.64:1, and that of ZEN:BSA was 17.2:1, identified by high-resolution mass spectrometry (HRMS) and an ultraviolet spectrometer (UV). The hybridoma cell lines 2A11, 2F6, and 3G2 for AFB1 and 2B6, 4D9 for ZEN were filtered by an indirect non-competitive enzyme-linked immunosorbent assay (inELISA) and an indirect competitive enzyme-linked immunosorbent assay (icELISA), respectively. As AFB1 mAb 2A11 and ZEN mAb 2B6 had the lowest 50% inhibitive concentration (IC50) and cross-reactivity (CR), they were selected for subsequent experiments. By systematically optimizing the preparation condition of gold nanoparticles (AuNPs), AuNPs-labeled mAbs, and detection condition, the visual limit of detection (LOD) of the dual test strip was 1.0 μg/L for AFB1 and 5.0 μg/L for ZEN, whereas that of the test strip reader was 0.23 μg/L for AFB1 and 1.53 μg/L for ZEN. The high reproducibility and stability of the dual test were verified using mycotoxin-spiked samples. The dual test strips were highly specific and sensitive for AFB1 and ZEN, which were validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thus, the proposed AFB1 and ZEN dual test strip is suitable for rapid and simultaneous detection of AFB1 and ZEN contamination in food and feed samples.

Effect of hapten structures on development of novel antibody against capsaicin and dihydrocapsaicin

• Three novel capsaicinoids haptens were synthesized, and the antibody-eliciting properties of four hapten-protein conjugations were investigated. • Novel polyclonal antibodies against capsaicinoids were prepared, and the important role of molecular structure and electrostatic potential in producing specific antibodies was verified. • A competitive indirect ELISA (icELISA) was established for capsaicinoids detection in spicy foods. Capsaicinoids content as a basis for the evaluation of spicy taste has attracted much attention. Antibody, as an important component of immunoassay, plays an important role in the detection of capsaicinoids levels. The molecular structure of the hapten is the key factor affecting the quality of small molecule antibodies. In our study, six capsaicinoids haptens were selected with different junction arm of length and structure to analyzed immune effect by geometric structure and electrostatic potential (ESP). Among them, three haptens were capsaicinoids structural analogues, and others were designed and synthesized. The results showed that the retention of amide bond (–NH–CO–) and hydroxyl group in the benzene ring of capsaicinoids hapten as well as rigid spacer arm that linked proteins to hapten had a positive effect on the affinity of the antibody. It provided a theoretical basis for rapid screening of small molecular substance haptens. To test our hypothesis, four kinds of haptens were used to synthesize antigens and immunize animals. Comparing 28 antiserum/coating conjugates combinations tested by indirect competitive enzyme-linked immunosorbent assay (icELISA) method for capsaicinoids (i.e., capsaicin and dihydrocapsaicin), the homologous combination of Pa-B2/OVA-hapten B had been shown to be most suitable for the determination of capsaicin and dihydrocapsaicin, which was consistent with the ESP analysis. In short, ESP analysis can speed up haptens screening and reduce the workload while producing high-quality antibodies. Thus, an icELISA based on hapten B polyclonal antibody was developed with IC 50 value of 0.36 μg mL −1 and the limit of detections (LODs) of 20 µg kg −1 in spicy food samples. Moreover, these results were also validated by high performance liquid chromatography (HPLC), and showed good agreements.

Preparation and application of a specific single-chain variable fragment against artemether

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC50) value and the working range based on IC20 to IC80 were 4.33 ng mL−1 and 1.05–22.65 ng mL−1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.

Development of an immunochromatographic assay for the rapid screening of pendimethalin in potato and apple

Pendimethalin (PDM), a dinitroaniline group herbicide, is extensively used for controlling weeds in amounts of agricultural crops. In this work, Hapten-1 and Hapten-2 were synthesized for the preparation of an anti-PDM monoclonal antibody (mAb), which was used to establish an indirect competitive ELISA. Based on the anti-PDM mAb, we developed an immunochromatographic assay (ICA) for detection of PDM in potato and apple. Under optimal conditions, the ICA exhibited a cut-off value of 200 ng/g and the visible limit of detection was 10 ng/g for apple and potato samples in qualitative detection. Analysis of quantitative detection suggested that the limit of detection were 5.4 and 3.2 ng/g in apple and potato samples, respectively. Moreover, the results obtained from the developed ICA strip were shown to coincide with those of LC–MS/MS in the analysis of apple and potato samples. In addition, the ICA exhibited excellent performance with regard to sensitivity, speed, and portability in contrast to previous determination methods for PDM. Hence, the proposed ICA strip showed superior prospects in PDM residue determination.

Preparation of Monoclonal Antibody against Deoxynivalenol and Development of Immunoassays.

Fusarium toxins are the largest group of mycotoxins, which contain more than 140 known secondary metabolites of fungi. Deoxynivalenol (DON) is one of the most important compounds of this class due to its high toxicity and its potential to harm mankind and animals and a widespread contaminant of agricultural commodities, such as wheat, corn, barley, oats, bread, and biscuits. Herein, a hybridoma cell 8G2 secreting mAb against DON was produced by fusing the splenocytes with a tumor cell line Sp2/0. The obtained mAb had a high affinity (2.39 × 109 L/mol) to DON. An indirect competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) showed that the linear range for DON detection was 3.125–25 μg/mL, and the minimum inhibitory concentration (IC50) was 18.125 μg/mL with a limit of detection (LOD) of 7.875 μg/mL. A colloidal gold nanoparticle (AuNP) with 20 nm in diameter was synthesized for on-site detection of DON within 10 min with vLOD of 20 μg/mL. To improve the limit of detection, the gold nanoflower (AuNF) with a larger size (75 nm) was used to develop the AuNF-based strip with vLOD of 6.67 μg/mL. Compared to the vLOD of a convectional AuNP-based strip, the AuNF-based strip was three times lower. Herein, three immunoassay methods (ic-ELISA and AuNP/AuNF-based strips) were successfully developed, and these methods could be applied for the DON detection in agricultural products.

Bioimprinting as a Receptor for Detection of Kwakhurin

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

Detection of Acrylamide in Foodstuffs by Nanobody-Based Immunoassays.

Acrylamide is toxic aliphatic amide formed via the Maillard reaction between asparagine and reducing sugars during thermal processing of food. Herein, a specific nanobody termed Nb-7E against the acrylamide derivative xanthyl acrylamide (XAA) was isolated from an immunized phage display library and confirmed to be able to detect acrylamide. First, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for acrylamide with a limit of detection (LOD) of 0.089 μg/mL and working range from 0.23 to 5.6 μg/mL. Furthermore, an enhanced electrochemical immunoassay (ECIA) was developed based on the optimized reaction conditions. The LOD was as low as 0.033 μg/mL, threefold improved compared to that of ic-ELISA, and a wider linear detection range from 0.39 to 50.0 μg/mL was achieved. The average recoveries ranged from 88.29 to 111.76% in spiked baked biscuits and potato crisps. Finally, the analytical performance of the ECIA was validated by standard ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Rapid screening of imidacloprid residue in grains and medicinal herbs: A newly designed hapten and monoclonal antibody

Three different imidacloprid hapten structures were designed to conjugate with proteins (bovine serum albumin, BSA; ovalbumin, OVA; keyhole limpet hemocyanin, KLH) for screening the optimal immunogen and coating antigen. Among these, an unreported antigen (hapten 6-KLH) was selected as the optimal immunogen and coating antigen. In addition, an imidacloprid-specific and high titer monoclonal antibody (IMIB7C3) was obtained by using the above-selected immunogen. A sensitive ic-ELISA (indirect competitive enzyme-linked immunosorbent assay) with a half-maximal inhibitory concentration (IC50) of 1.3 ng mL−1 was established by using the IMIB7C3 antibody (only 1.2 ng per well) to detect the residues of imidacloprid in grains (wheat and maize) and different herbs (Notoginseng radix et rhizoma, Dioscoreae rhizoma, Lonicerae japonicae flos, Astragali radix, Jujubae fructus). The detection results of real samples by the developed immunoassay were confirmed by liquid chromatography-tandem mass spectrometry (LC–MS/MS), which proved the accuracy and reliability of the established ic-ELISA. These results indicate that the proposed ic-ELISA method is suitable for rapid and high-throughput detection of imidacloprid residues in agricultural products and medicinal herbs. Furthermore, a quantitative risk assessment was conducted for Lonicerae japonicae flos based on the detection results, which indicates an acceptable risk to human health after the intake of Lonicerae japonicae flos polluted by imidacloprid.

Detection and Occurrence of Microcystins and Nodularins in Lake Manatee and Lake Washington-Two Floridian Drinking Water Systems

Clean, fresh, and safe drinking water is essential to human health and well-being. Occasionally, biological contaminants taint surface water quality used for human consumption and recreation. Microcystins (MCs) and nodularins (NODs) are toxic monocyclic peptides produced by cyanobacteria in fresh and brackish water. These secondary metabolites can reach hazardous concentrations, impairing surface drinking water supplies and thereby increasing the risk of exposure to consumers. Inconsistent screening of MCs and NODs is not uncommon in drinking water systems and no provisional guidance value has been established in Florida to protect community and public health. We performed a case study of two Florida lakes supplying drinking water to adjacent communities for the purpose of monitoring MCs and NODs over the potential peak algae bloom season (June-August). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was employed to analyze concentrations of total MCs and NODs in environmental water samples from Lake Manatee and Lake Washington. Toxin concentrations were similar in each lake, averaging 0.19 μg/L. The highest toxin concentration (0.46 μg/L) was reported in Lake Manatee at Site 15, a location where other toxin concentrations demonstrated statistical significance with toxins detected at Sites 6 (p = 0.014) and 8 (p = 0.011). Inferential analyses from the Kruskal-Wallis H Test revealed a statistically significance difference in toxin concentrations by sampling month within the two Floridian drinking water systems (p &amp;lt; 0.001). Furthermore, phosphate and nitrite concentrations strongly correlated with total MCs and NODs in each lake (p &amp;lt; 0.01). Although results indicate a low probable health risk from cyanotoxins, more research is needed to understand the intrinsic nature of MCs and NODs by examining their prevalence, distribution, and dynamics in surface drinking water supplies serving nearby communities.

An Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Brigatinib and Gilteritinib Using a Specific Polyclonal Antibody.

Brigatinib and gilteritinib are oral tyrosine kinase inhibitors (TKIs). We aimed to develop a simple and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to quantify brigatinib and gilteritinib in various biological matrices. Antiserum against these TKIs was obtained from mice by using 3-methoxy-4-(-4-(4-methylpiperazin-1-yl) piperidin-1-yl) aniline as a hapten, which has a common substructure with these TKIs. The generated antibody was used to develop an indirect competitive ELISA for these TKIs in human serum. The lower limit of quantification of brigatinib and gilteritinib in human serum was 6.2 and 6.8 ng/mL, respectively. The developed ELISA was used to examine the pharmacokinetics of these TKIs after oral administration in mice and rats. This ELISA is expected to be a valuable tool in pharmacokinetic studies of these TKIs.