Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development.

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 µg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 µg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0 × 107  pfu mL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

Carba PBP: a novel penicillin-binding protein-based lateral flow assay for rapid phenotypic detection of carbapenemase-producing Enterobacterales.

Rapid phenotypic detection assays, including Carba NP and its variants, are widely applied for clinical diagnosis of carbapenemase-producing Enterobacterales (CPE). However, these tests are based on the acidification of the pH indicator during carbapenem hydrolysis, which limits test sensitivity and speed, especially for the detection of CPE producing low-activity carbapenem (e.g., OXA-48 variants). Herein, we developed a novel rapid and sensitive CPE detection method (Carba PBP) that could measure substrate (meropenem) consumption based on penicillin-binding protein (PBP). Meropenem-specific PBP was used to develop a competitive lateral flow assay (LFA) for meropenem identification. For the detection of carbapenemase activity, meropenem concentration was optimized using a checkerboard assay. The performance of Carba PBP was evaluated and compared with that of Carba NP using a panel of 94 clinical strains characterized by whole-genome sequencing and carbapenem susceptibility test. The limit of detection of PBP-based LFA for meropenem identification was 7 ng mL-1. Using 10 ng mL-1 meropenem as the substrate, Carba PBP and Carba NP could detect 10 ng mL-1 carbapenemase within 25 min and 1,280 ng mL-1 CPE in 2 h, respectively. The sensitivity and specificity were 100% (75/75) and 100% (19/19) for Carba PBP and 85.3% (64/75) and 100% (19/19) for Carba NP, respectively. When compared with Carba NP, Carba PBP showed superior performance in detecting all the tested CPE strains (including OXA-48-like variants) within 25 min and presented two orders of magnitude higher analytical sensitivity, demonstrating potential for clinical diagnosis of CPE. IMPORTANCE This study successfully achieved the goal of carbapenemase activity detection with both high sensitivity and convenience, offering a convenient lateral flow assay for clinical diagnosis of carbapenemase-producing Enterobacterales.

Antibody Production, Immunoassay Establishment, and Specificity Study for Flunixin and 5-Hydroxyflunixin.

Flunixin (FLU) is a nonsteroidal drug that is widely used in animals, causing severe drug residues in animal-derived foods and environment. The development of antibody-based rapid immunoassay methods is of great significance for the monitoring of FLU and its metabolite 5-hydroxyflunixin (5-FLU). We prepared monoclonal antibodies (mAbs) with different recognition spectra through FLU-keyhole limpet hemocyanin conjugates as immunogen coupled with antibody screening strategies. mAb5E6 and mAb6D7 recognized FLU with high affinity, and mAb2H5 and mAb4A4 recognized FLU and 5-FLU with broad specificity. Through evaluating the recognition of these mAbs against more than 11 structural analogues and employing computational chemistry, molecular docking, and molecular dynamics methodologies, we preliminarily determined the recognition epitope and recognition mechanism of these mAbs. Finally, an indirect competitive enzyme-linked immunosorbent assay for FLU based on mAb6D7 was developed, which exhibited limits of detection as low as 0.016-0.042 µg kg -1 (L-1) in milk and muscle samples.

A comprehensive review on the detection of Staphylococcus aureus enterotoxins in food samples.

Staphylococcal enterotoxins (SEs), the major virulence factors of Staphylococcus aureus, cause a wide range of food poisoning and seriously threaten human health by infiltrating the food supply chain at different phases of manufacture, processes, distribution, and market. The significant prevalence of Staphylococcus aureus calls for efficient, fast, and sensitive methods for the early detection of SEs. Here, we provide a comprehensive review of the hazards of SEs in contaminated food, the characteristic and worldwide regulations of SEs, and various detection methods for SEs with extensive comparison and discussion of benefits and drawbacks, mainly including biological detection, genetic detection, and mass spectrometry detection and biosensors. We highlight the biosensors for the screening purpose of SEs, which are classified according to different recognition elements such as antibodies, aptamers, molecularly imprinted polymers, T-cell receptors, and transducers such as optical, electrochemical, and piezoelectric biosensors. We analyzed challenges of biosensors for the monitoring of SEs and conclude the trends for the development of novel biosensors should pay attention to improve samples pretreatment efficiency, employ innovative nanomaterials, and develop portable instruments. This review provides new information and insightful commentary, important to the development and innovation of further detection methods for SEs in food samples.

A Proof-of-Concept Sandwich Enzyme-Linked Immunoassay Development for Small Molecules.

A sandwich immunoassay theoretically exhibits higher sensitivity and specificity compared to a competitive counterpart; however, it is extremely difficult to obtain a pair of antibodies that can bind to a small molecule simultaneously, which is always thought to be a single epitope. In the present study, abamectin (ABM) was selected to prove the effect of hapten design and antibody recognition properties on the development of a sandwich immunoassay for small molecules. First, the epitopes of ABM were roughly located, and epitope distances were determined. Then, two haptens were designed by introducing spacer arms at the C4″-OH and C5-OH of ABM, respectively, aiming to provide the longest epitope distances. A total of seven rabbit polyclonal antibodies (pAbs) and 21 mouse monoclonal antibodies (mAbs) with various recognition properties were obtained. Extensive combinatorial associations of antibody pairs for simultaneously binding to ABM were performed, and only two mAb-mAb pairs were observed to achieve a sandwich immunoassay for ABM with a total success rate of 0.27%. The best mAb pair for sandwich immunoassay was confirmed by surface plasmon resonance, used to develop a sandwich immunoassay, and then evaluated by cross-reactivities and molecular docking with structurally similar analogues and abamectin. Altogether, the study provided a theoretical foundation as well as practical experience and demonstrated the importance of careful hapten design and extensive antibody screening to successfully establish the sandwich immunoassay for small molecules.

Molecular Recognition Mechanism of an Anti-Amatoxins mAb and Its Application in Centrifugal Disk-Based Immunoassay.

Amatoxins are polypeptides that cause 90% of fatalities from accidental ingestion of poisonous mushrooms. Unfortunately, there are no specific antidotes against amatoxins poisoning, hence preparation of high-affinity antibodies, understanding the receptor (amatoxins) and ligand (antibody) mechanism, and establishing a straightforward screening approach are of great significance for confirming poison agents and clinical diagnosis. Here, anti-amatoxins monoclonal antibody (mAb) 9B2 was prepared and the recognition mechanism was investigated. The approach is useful for designing desirable immunogens, developing new antibodies with improved performance, and constructing effective immunoassays. Based on the mAb, we designed a centrifugal disk-like microfluidics chip and developed a fully automated immunoassay capable of detecting amatoxins poisoning in various samples including serum, urine, and mushrooms. The whole detection process could be automatically accomplished within 30 min, with a limit of detection of 0.08 to 0.12 µg/L for real samples, ∼30-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). Our platform not only provided a practical approach for performing poison agent confirmation and clinical diagnosis but also had important implications for improving the survival of patients with mushroom poisoning.

Precise isolation and structural origin of an ultra-specific nanobody against chemical compound.

Highly specific antibodies are the key reagents for developing immunoassays with a low false positive rate for environmental monitoring. Here, we provide evidence that nanobodies have the potential to achieve higher specificity than conventional antibodies and explain why from their structural features. Using sulfadimethoxine (SDM) as a model analyte, we constructed an immune phage display library and precisely isolated an ultra-specific nanobody (H1-17) by a crucial homologous antigen counter selection strategy. H1-17 showed no observable cross-reactivity (CR) with other structural analogs of 41 SDM tested, which has never been achieved by conventional antibodies. The structurally original specificity of H1-17 was illuminated and compared with that of one conventional antibody by homology modeling and site-directed mutagenesis validation. It was found that the noncanonical disulfide bond (C50-C104) of H1-17 helped CDR3 form a tailor-made binding pocket and divide it into two parts to accommodate the common structure of sulfonamides and the characteristic methoxyl group of SDM, respectively. Besides, the mutual-checking hydrogen bonds also played important roles in the specific recognition. Lastly, immunoassays with zero false positive rate were developed to screen SDM in water and milk samples, indicating that nanobodies could be reliable reagents for the accurate detection of chemical compounds.

A highly sensitive lateral flow immunoassay based on a group-specific monoclonal antibody and amorphous carbon nanoparticles for detection of sulfonamides in milk.

To meet high-throughput screening of the residues of sulfonamides (SAs) with high sensitivity toward sulfamethazine (SM2) in milk samples, a new highly sensitive lateral flow immunoassay (LFA) based on amorphous carbon nanoparticles (ACNs) was developed. First, a group-specific monoclonal antibody 10H7 (mAb 10H7) that could recognize 25 SAs with high sensitivity toward SM2 (IC50 value of 0.18 ng/mL) was prepared based on H1 as an immune hapten and H4 as a heterologous coating hapten. Then, mAb 10H7 was conjugated to ACNs as an immune probe for LFA development. Under the optimized conditions, the LFA could detect 25 SAs with the cut-off value toward SM2 of 2 ng/mL, which could meet the requirement for detection of SAs. In addition, the LFA developed was also used for screening SAs' residues in real milk samples, with results being consistent with HPLC-MS/MS. Thus, this LFA can be used as a high-throughput screening tool for detection of SAs.

Development of epitopephore-based rational hapten design strategy: A combination of theoretical evidence and experimental validation.

Antibody is the key biomolecule that governing the sensitivity and specificity of an immunoassay for chemical compound, also named hapten molecule. Obviously, predication of hapten effectiveness before chemical synthesis is beneficial to boost success, save cost and improve controllability. Here, we proposed and evaluated an epitopephore based rational hapten design (ERHD) to assist antibody production to chemical compound, combining theoretical evidence and then experimental validation by using dinitrocarbanilide (DNC) as a model analyte. Briefly, epitopephores of DNC were firstly generated by HipHop algorithm after features mapping. A homemade drug database also containing reported fragment haptens (HFR) and new designed full hapten (HFU) were constructed, and then was virtually screened by using generated epitopephore followed by structural analysis and visual inspection. The DNC haptens based on the selected hits were further identified by Density Functional Theory before total synthesis. To prove and clarify the usability of the ERHD, two retrieved HFU haptens, one non-retrieved HFU hapten and three non-retrieved HFR haptens were all selected to produce monoclonal antibodies (mAbs) for comparison purpose. A maximal 6000-fold increased affinity of mAb from retrieved HFU than HFR was observed, while, non-retrieved HFU failed to produce antibody to DNC. More importantly, mAbs from HFU haptens provided highly specificity to DNC, while, mAbs from HFR haptens could recognize 15 others analogues. We then constructed antibody structure and investigated molecular recognition of the mAbs to DNC, well supporting the rationality of the ERHD. Lastly, an icELISA was developed for DNC with an IC50 value as low as 0.19 ng mL-1 with high specificity, which has never achieved before.

How Exactly Do AIEgens Target Bacteria? Leveraging the Targeting Mechanism to Design Sensitive Fluorescent Immunosensors.

Aggregation-induced emission luminogens (AIEgens) are promising candidates for bacterial imaging and detection because they can "Light-Up" pathogenic bacteria without complicated labeling or washing steps. However, there have been few in-depth analyses of the intrinsic mechanism underlying their utility as fluorescence probes for targeting bacteria. Therefore, using large-scale molecular dynamics simulations, we investigated the mechanism of their bacterial "Light-Up" behavior with N,N-diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl) aniline functionalized with 1-bromoethane (TBP-1). We propose that the triphenylamine motif of TBP-1, rather than the positively charged pyridine group, first contacts the cell membrane. After TBP-1 completely inserts into the cell membrane, the hydrophobic triphenylamine motif localizes in the hydrophobic core of the cell membrane, restricting the molecular variation of TBP-1, which induces the fluorescent "turn-on" and bacterial "Light-Up." On this basis, we established a heterogeneous lateral flow immunoassay (LFIA) for the detection of foodborne pathogens. The LFIA system showed improved sensitivity with a limit of detection as low as 103 CFU mL-1 and strong specificity. Our protocol opened an effective shortcut to the design of more efficient AIEgens and novel AIEgens-based immunoassays.

The influence of hapten spacer arm length on antibody response and immunoassay development.

Antibodies against small molecules with high titer and high affinity are always pursued in the field of vaccines for drugs of abuse, antidotes to toxins and immunoassays in medical, environmental, and food safety. The exposure degree of the target molecule to the immune system is critical to induce a strongly specific antibody response, thus, the spacer arm length between the target molecule and carrier protein plays an important role. However, the influence of spacer arm length on antibody titer, affinity, and assay performance is not yet clear and highly demanded to be addressed. In the present study, we proposed a model study to answer the question by using two typical small molecules, melamine and p-nitroaniline, which were introduced by varied spacer arms with increasing alkane linear length from 2 to 12 carbon atoms brick by brick. The spacer arm lengths of the haptens were obtained by computational chemistry. The titer and affinity of mouse antisera were analyzed and compared, showing that all haptens with spacer arms of 6-8 carbon atoms, i.e. 6.3-8.8 Å in length, induced strong antibodies represented by the highest titer and affinity without exception, while the haptens with spacer arms of 2-4 carbon atoms and 10-12 carbon atoms, i.e. 1.5-3.9 Å and 11.3-13.9 Å in length, failed to induce high-quality antibody response. Moreover, the titer and sensitivity of the subsequently developed immunoassays were significantly affected by using coating haptens with different spacer arm lengths, and coating haptens with a spacer arm of 6.3-8.8 Å in length delivered the optimum detection performance. The antibody recognition mechanism study further confirmed that the hapten spacer arm length had a critical effect on the recognition properties of the induced antibody, which should be interactive with the spacer arm each other. This study showed that the hapten with appropriate spacer arm length is important to antibody response and immunoassay development, providing a valuable and general clue for the rational design of hapten.

Broad-specificity indirect competitive enzyme-linked immunosorbent assay for aristolochic acids: Computer-aided hapten design and molecular mechanism of antibody recognition.

Long-term dietary exposure of aristolochic acids (AAs)-contaminated food proved to be one of the main culprits of Endemic Nephropathy, renal failure; and urothelial cancer. The antibodies utilized in immunoassays for AAs suffer from low affinity and failure of recognition to the family of AAs. This study, we prepared a broad-specificity monoclonal antibody (mAb) 5H5 with highly and uniform affinity for AAs by help of computational chemistry fully exposing the AAs common structures of methoxy and hydroxyl groups. The mAb 5H5 exhibited half inhibitory concentrations of AAA, AAB, AAC, AAD were 0.03, 0.06, 0.05, 0.03 ng/mL. To explain the broad-specificity profile of mAb 5H5, molecular docking was performed. Results shown that multiple conformations of AAs can be flexibly oriented in the spacious cavity of single-chain variable fragment antibody (scFv) 5H5 and the specific hydron bonds were formed by ASN62 and GLY64 of scFV 5H5 to the nitro group of AAs which gave an explanation of the high cross-reactivity of mAb 5H5. The ELISA based on the broad-specificity mAb 5H5with detection limits of 0.04-0.11 µg/kg and 0.02-0.06 µg/kg for four AAs in flour and soil samples, respectively. The study provided a promising method for the family of AAs in environmental and food samples.

Dual-Wavelength Fluorescence Polarization Immunoassay for Simultaneous Detection of Sulfonamides and Antibacterial Synergists in Milk.

Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC50 values for 23 SAs below 100 µg L-1 and 5 ASGs below 50 µg L-1. The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.

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 0.007 ng mL-1 and a surprising urea tolerance of 3 M 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 0.0042-0.014 µg L-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.

A rare monoclonal antibody discovery based on indirect competitive screening of a single hapten-specific rabbit antibody secreting cell.

A rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, particularly for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody-secreting cells (ASCs) selection with 20.6 h and propose an efficient platform for RmAb production against small molecules within 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent poses a great threat to public health. We applied CSMN to select CAP-specific ASCs and produced one high-affinity RmAb, surprisingly showed extremely halophilic properties with an IC50 of 0.08 ng mL-1 in the saturated salt solution, which has not yet been seen for other antibodies. The molecular dynamic simulation showed that the negatively charged surface improved the stability of the RmAb structure with additional disulfide bonds compared with mouse antibodies. Moreover, the reduced solvent accessible surface area of the binding pocket increased the interactions of RmAb with CAP in a saturated salt solution. Furthermore, RmAb was used to develop an immunoassay for the detection of CAP in real biological samples with simple pretreatment, shorter assay time, and higher sensitivity. The results demonstrated that the practical and efficient CSMN is suitable for rare RmAb discovery against small molecules.

Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor.

Hybridoma technology is widely used for monoclonal antibody (mAb) discovery, whereas the generation and identification of single hybridomas by the limiting dilution method (LDM) are tedious, inefficient, and time- and cost-consuming, especially for hapten molecules. Here, we describe a single transgenic hybridoma selection method (STHSM) that employs a transgenic Sp2/0 with an artificial and stable on-cell-surface anchor. The anchor was designed by combining the truncated variant transmembrane domain of EGFR with a biotin acceptor peptide AVI-tag, which was stably integrated into the genome of Sp2/0 via a piggyBac transposon. To ensure the subsequent precise selection of the hybridoma, the number of on-cell-surface anchors of the transfected Sp2/0 for fusion with immunized splenocytes was further normalized by flow cytometry at the single cell level. Then the single antigen-specific transgenic hybridomas were precisely identified and automatically selected using a CellenONE platform based on the fluorescence assay of the on-cell-surface anchor with the corresponding secreted antigen-specific mAb. The STHSM produced 579 single chloramphenicol (CAP)-specific transgenic hybridomas with a positive rate of 62.7% in 10 plates within 2 h by one-step selection, while only 12 single CAP-specific hybridomas with a positive rate of 6.3% in 40 plates required at least 32 days using the LDM with multiple subcloning steps. The best affinity of mAbs from the STHSM was more than 2-fold higher than that of those from the LDM, and this was mainly due to the preaffinity selection based on the on-cell-surface anchors and more interactions between the mAb and CAP. Then the mAbs from the STHSM and LDM were used to develop an immunoassay for CAP in spiked and natural biological samples. The method displayed satisfactory sensitivity, accuracy, and precision, demonstrating that the STHSM we developed is a versatile, practical, and efficient method for mAb discovery.

Development of Fluorescence Polarization Immunoassay With scFv to Detect Fumonisin Bs in Maize and Simultaneous Study of Their Molecular Recognition Mechanism.

In this study, a fluorescence polarization immunoassay (FPIA) was developed based on the single-chain variable fragments (scFvs) for fumonisin Bs (FBs). The scFvs were prepared from FBs-specific monoclonal antibody secreting hybridomas (4F5 and 4B9). The established FPIA could determine the sum of fumonisin B1 (FB1) and fumonisin B2 (FB2) within a short time. The IC50 of FPIA for the detection of FB1 and FB2 were 29.36 ng/ml and 1,477.82 ng/ml with 4F5 scFv, and 125.16 ng/ml and 30.44 ng/ml with 4B9 scFv, so the 4B9 scFv was selected for detection of FB1 and FB2 in maize samples with a limit of detection of 441.54 µg/kg and 344.933 µg/kg. The recoveries ranged from 84.7 to 104.1% with a coefficient of variation less than 14.1% in spiked samples, and the result of the FPIA method was in good consistency with that of HPLC-MS/MS. To supply a better understanding of the immunoassay results, the interactions mechanism of scFvs-FBs was further revealed by the homology modelling, molecular docking, and molecular dynamic simulation. It was indicated that six complementarity-determining regions (CDRs) were involved in 4B9 scFv recognition, forming a narrow binding cavity, and FB1/FB2 could be inserted into this binding cavity stably through strong hydrogen bonds and other interactions. While in 4F5 scFv, only the FB1 stably inserted in the binding pocket formed by four CDRs through strong hydrogen bonds, and FB2 did not fit the binding cavity due to the lack of hydroxyl at C10, which is the key recognition site of 4F5 scFv. Also, the binding energy of FB2-4B9 scFv complex is higher than the FB2-4F5 scFv complex. This study established a FPIA method with scFv for the detection of FB1 and FB1 in maize, and systematically predicted recognition mechanism of FBs and scFvs, which provided a reference for the better understanding of the immunoassay mechanism.

'Three-To-One' multi-functional nanocomposite-based lateral flow immunoassay for label-free and dual-readout detection of pathogenic bacteria.

Sandwich lateral flow immunoassays (LFIAs) based on paired antibodies are the most frequently used platform for food-borne pathogen detection. Although label-free strategies are used in LFIAs to avoid the utilization of paired antibodies, challenges of probe design and detection reliability still remain. Here, we report a new label-free and dual-readout LFIA (LD-LFIA) mediated by a 'Three-To-One' multi-functional nanocomposite with a unique combination of magnetic-adhesion-color-nanozyme properties. The strengths of the new designed nanocomposite are: (i) the Fe3O4 magnetic core simplifies the separation processes; (ii) surface adherent polydopamine (PDA) films exhibit a strong adhesion to pathogenic bacteria and provide colorimetric detection signal; and (iii) the deposited platinum nanoparticles (Pt NPs) can function as nanozymes to generate an extra catalytic signal for constructing a dual-readout mode to improve the detection accuracy. The resulting Fe3O4@PDA@Pt nanocomposite-based LD-LFIA can detect highly pathogenic Escherichia coli O157:H7 with limits of detection of 102 and 10 CFU mL-1 for colorimetric and catalytic quantitative analyses, respectively. Systematic results also reveal that the proposed method exhibited high specificity and applicability for drinking water and chicken samples, serving as a promising tool for real bacterial sample testing. The multi-functional Fe3O4@PDA@Pt nanocomposite-based LD-LFIA can provide new ideas for designing new multi-functional probes for improving detection performance of conventional label-free LFIA and constructing more accurate and sensitive detection systems.

Comparison of two fluorescence quantitative immunochromatographic assays for the detection of amantadine in chicken muscle.

The two sensitive fluorescence quantitative immunochromatographic assays (FQICAs), background fluorescence quenching immunochromatographic assay (bFQICA) and time-resolved fluorescent immunochromatographic assay (TRFICA), play an important role increasingly in rapid detection technology for food safety. Amantadine (AMD), used extensively in virus infections in livestock and poultry, has been prohibited due to hazard concerns over public human health. Therefore, AMD was used as a model molecule in the FQICAs establishment and comparison based on the same bioreagents. The outstanding performance in technical parameters of the two FQICAs indicated that they could provide rapid, precise, reliable technical support for large-scale on-site screening for AMD detection. What's more, the systematic and comprehensive comparison of the two FQICAs would give useful suggestions for scientists and users in monitoring the harmful compounds.