Investigating the Role of KLF6 in the Growth of Bovine Preadipocytes: Using Transcriptomic Analyses to Understand Beef Quality.

Intramuscular fat is a crucial determinant of carcass quality traits like tenderness and taste, which in turn is influenced by the proliferation of intramuscular preadipocytes. This study aimed to investigate the Krüppel-like factor 6 (KLF6)-mediated proliferation of bovine preadipocytes and identify underlying molecular mechanisms. Down-regulation of KLF6 by siKLF6 resulted in a significant (p < 0.01) suppression of cell cycle-related genes including CDK1, MCM6, ZNF4, PCNA, CDK2, CCNB1, and CDK6. Conversely, the expression level of p27 was significantly (p < 0.01) increased. Moreover, EdU (5-ethynyl-20-deoxyuridine) staining revealed a significant decrease in EdU-labeled cells due to KLF6 down-regulation. Collectively, these findings indicate that KLF6 down-regulation inhibits adipocyte proliferation. Furthermore, RNA sequencing of preadipocytes transfected with siKLF6 and NC, followed by differential gene expression analysis, identified 100 up-regulated and 70 down-regulated genes. Additionally, the differentially expressed genes also significantly influenced various Gene Ontology (GO) terms related to cell cycle, nuclear chromosomes, and catalytic activity on DNA. Furthermore, the top 20 pathways enriched in these DEGs included cell cycle, DNA replication, cellular senescence, and homologous recombination. These GO terms and KEGG pathways play key roles in bovine preadipocyte proliferation. In conclusion, the results of this study suggest that KLF6 positively regulates the proliferation of bovine preadipocytes.

Isolation, structural, biological activity and application of Gleditsia species seeds galactomannans.

Gleditsia fruits have been known as a valuable traditional Chinese herb for tens of centuries. Previous studies showed that the galactomannans are considered as one of the major bioactive components in Gleditsia fruits seeds (GSGs). Here, we systematically review the major studies of GSGs in recent years to promote their better understanding. The extraction methods of GSGs mainly include hot water extraction, microwave-assisted extraction, ultrasonic extraction, acid extraction, and alkali extraction. The analysis revealed that GGSs exhibited in the form of semi-flexible coils, and its molecular weight ranged from 0.018 × 103 to 2.778 × 103 KDa. GSGs are composed of various monosaccharide constituents such as mannose, galactose, glucose, and arabinose. In terms of pharmacological effects, GSGs exhibit excellent activity in antioxidation, hypoglycemic, hypolipidemic, anti-inflammation. Moreover, GSGs have excellent bioavailability, biocompatibility, and biodegradability, which make them used in food additives, food packaging, pharmaceutical field, industry and agriculture. Of cause, the shortcomings of the current research and the potential development and future research are also highlighted. We believe our work provides comprehensive knowledge and underpinnings for further research and development of GSGs.

Advances of Predicting Allosteric Mechanisms Through Protein Contact in New Technologies and Their Application.

Allostery is an intriguing phenomenon wherein the binding activity of a biological macromolecule is modulated via non-canonical binding site, resulting in synchronized functional changes. The mechanics underlying allostery are relatively complex and this review is focused on common methodologies used to study allostery, such as X-ray crystallography, NMR spectroscopy, and HDXMS. Different methodological approaches are used to generate data in different scenarios. For example, X-ray crystallography provides high-resolution structural information, NMR spectroscopy offers dynamic insights into allosteric interactions in solution, and HDXMS provides information on protein dynamics. The residue transition state (RTS) approach has emerged as a critical tool in understanding the energetics and conformational changes associated with allosteric regulation. Allostery has significant implications in drug discovery, gene transcription, disease diagnosis, and enzyme catalysis. Enzymes' catalytic activity can be modulated by allosteric regulation, offering opportunities to develop novel therapeutic alternatives. Understanding allosteric mechanisms associated with infectious organisms like SARS-CoV and bacterial pathogens can aid in the development of new antiviral drugs and antibiotics. Allosteric mechanisms are crucial in the regulation of a variety of signal transduction and cell metabolism pathways, which in turn govern various cellular processes. Despite progress, challenges remain in identifying allosteric sites and characterizing their contribution to a variety of biological processes. Increased understanding of these mechanisms can help develop allosteric systems specifically designed to modulate key biological mechanisms, providing novel opportunities for the development of targeted therapeutics. Therefore, the current review aims to summarize common methodologies that are used to further our understanding of allosteric mechanisms. In conclusion, this review provides insights into the methodologies used for the study of allostery, its applications in in silico modeling, the mechanisms underlying antibody allostery, and the ongoing challenges and prospects in advancing our comprehension of this intriguing phenomenon.

Discrimination rancidity degree of infant formula rice flour based on Headspace Solid-Phase Microextraction combined with Gas Chromatography-Mass Spectrometry as an alternative to sensory evaluation.

To mitigating the serious threat of harmful volatile substances to the health of infants, an alternative method of odor evaluation were proposed based on Headspace solid-phase microextraction (HS-SPME) combined with Gas Chromatography-Mass Spectrometry (GC-MS) to discriminate the degree of rancidity of infant formula rice flour (IFRF). Inspectors can simply calculate the rancidity degree of infant formula rice flour according to the regression equation based on the concentration of rancidity markers. The results showed that the joint application of OPLS-DA, molecular sensory experiments, and unsaturated fatty acids (UFAs) degradation experiments could successfully recognize the rancidity markers without collinearity in multiple linear regression analysis. The rancidity markers curve fitting was helpful for the establishment of multivariate regression model of rancidity grading. The model had an accuracy of more than 92.90% by the verification of odor evaluation. The application of the model to investigate the market IFRF samples showed that about 3% of the samples collected in the experiment were unsuitable for infant feeding. Therefore, the established model was considered to be a robust and less workload method to replace the olfactory evaluation method for discriminating the rancidity degree of IFRF.

Preparation of bifunctional monomer molecularly imprinted polymer filled solid-phase extraction for sensitivity improvement of quantitative analysis of sulfonamide in milk.

To solve the problems of unstable adsorption performance and high organic solvent consumption of traditional molecular imprinting materials, we developed a water-resistant and highly adsorptive molecularly imprinted sulfamethazine polymer (MIP) through a novel synthetic strategy consisting of the application of mixed functional monomers combined with hydrophobic crosslinkers. The results showed that the imprinting factor of the prepared MIP was increased from 0.93 to 4.38, and that the relative selection coefficient k' was 5.59. With the application of the material to be developed as a solid-phase extraction (SPE) filler for sulfonamides (SAs) in milk combined with UPLC-MS/MS, the validated method showed a sensitive quantification limit (0.89 µg/kg), a steady recovery (95.55%-97.97%) and an excellent precision (0.08%-2.92% RSD). Moreover, after 5 times usage, the recovery rate of MIP-SPE was still above 90%. Therefore, the prepared materials showed high performance of molecular adsorption and were water-resistant, which was also considered an excellent filler of SPE for SAs extraction in food or other fields.

Integrating omics and network pharmacology reveals the anti-constipation role of chitosan with different molecular weights in constipated mice.

This study aimed to reveal the constipation-relieving role of chitosan (COS) with different molecular weights (1 kDa, 3 kDa and 244 kDa). Compared with COS3K (3 kDa) and COS240K (244 kDa), COS1K (1 kDa) more significantly accelerated gastrointestinal transit and defecation frequency. These differential effects were reflected in the regulation of specific gut microbiota (Desulfovibrio, Bacteroides, Parabacteroides and Anaerovorax) and short-chain fatty acids (propionic acid, butyric acid and valeric acid). RNA-sequencing found that the differential expressed genes (DEGs) caused by different molecular weights of COS were mainly enriched in intestinal immune-related pathways, especially cell adhesion molecules. Furthermore, network pharmacology revealed two candidate genes (Clu and Igf2), which can be regarded as the key molecules for the differential anti-constipation effects of COS with different molecular weights. These results were further verified by qPCR. In conclusion, our results provide a novel research strategy to help understand the differences in the anti-constipation effects of chitosan with different molecular weights.

Study on Anti-Constipation Effects of Hemerocallis citrina Baroni through a Novel Strategy of Network Pharmacology Screening.

Daylily (Hemerocallis citrina Baroni) is an edible plant widely distributed worldwide, especially in Asia. It has traditionally been considered a potential anti-constipation vegetable. This study aimed to investigate the anti-constipation effects of daylily from the perspective of gastro-intestinal transit, defecation parameters, short-chain organic acids, gut microbiome, transcriptomes and network pharmacology. The results show that dried daylily (DHC) intake accelerated the defecation frequency of mice, while it did not significantly alter the levels of short-chain organic acids in the cecum. The 16S rRNA sequencing showed that DHC elevated the abundance of Akkermansia, Bifidobacterium and Flavonifractor, while it reduced the level of pathogens (such as Helicobacter and Vibrio). Furthermore, a transcriptomics analysis revealed 736 differentially expressed genes (DEGs) after DHC treatment, which are mainly enriched in the olfactory transduction pathway. The integration of transcriptomes and network pharmacology revealed seven overlapping targets (Alb, Drd2, Igf2, Pon1, Tshr, Mc2r and Nalcn). A qPCR analysis further showed that DHC reduced the expression of Alb, Pon1 and Cnr1 in the colon of constipated mice. Our findings provide a novel insight into the anti-constipation effects of DHC.

PRDX1 stimulates non-small-cell lung carcinoma to proliferate via the Wnt/ß-Catenin signaling.

BACKGROUND: Previous studies have shown that PRDX1 is upregulated in some types of malignant tumors. The role of PRDX1 in non-small-cancer lung carcinoma (NSCLC) remains unclear. This study aims to identify the role of PRDX1 in influencing in-vitro biological functions of NSCLC and the molecular mechanism. METHODS: We collected 50 cases of fresh NSCLC and adjacent non-tumoral tissues for detecting differential expressions of PRDX1 by quantitative real-time polymerase chain reaction (qRT-PCR). Survival time of NSCLC patients, defined as the period from the operation to the latest follow-up or death due to recurrence or metastasis, was recorded for assessing the relationship between PRDX1 and prognosis in NSCLC. Using lentivirus transfection, PRDX1 level was downregulated in NSCLC cells. Subsequently, proliferative and apoptotic abilities, and expression levels of vital genes in the Wnt/ß-Catenin signaling were examined. Finally, the significance of activated Wnt/ß-Catenin signaling during PRDX1-regulated NSCLC proliferation was explored. RESULTS: Using GEPIA database and NSCLC tissues we collected, PRDX1 was detected to be upregulated in NSCLC samples than controls. PRDX1 level was related to tumor staging and prognosis in NSCLC. Knockdown of PRDX1 attenuated proliferative ability and stimulated apoptosis in NSCLC. Protein levels of Wnt5A was downregulated in H1299 and SPC-A1 cells with PRDX1 knockdown. Overexpression of ß-Catenin enhanced proliferative ability and inhibited apoptosis in NSCLC cells with PRDX1 knockdown. CONCLUSIONS: PRDX1 is upregulated in NSCLC samples, and linked to tumor staging and prognosis. It stimulates NSCLC to proliferate by activating the Wnt/ß-Catenin signaling.

Bayesian Fusion Model Enhanced Codfish Classification Using Near Infrared and Raman Spectrum.

In this study, a Bayesian-based decision fusion technique was developed for the first time to quickly and non-destructively identify codfish using near infrared (NIRS) and Raman spectroscopy (RS). NIRS and RS spectra from 320 codfish samples were collected, and separate partial least squares discriminant analysis (PLS-DA) models were developed to establish the relationship between the raw data and cod identity for each spectral technique. Three decision fusion methods: decision fusion, data layer or feature layer, were tested and compared. The decision fusion model based on the Bayesian algorithm (NIRS-RS-B) was developed on the optimal discrimination features of NIRS and RS data (NIRS-RS) extracted by the PLS-DA method whereas the other fusion models followed conventional, non-Bayesian approaches. The Bayesian model showed enhanced classification metrics (92% sensitivity, 98% specificity, 98% accuracy) that were significantly superior to those demonstrated by any of other two spectroscopic methods (NIRS, RS) and the two data fusion methods (data layer fused, NIRS-RS-D, or feature layer fused, NIRS-RS-F). This novel proposed approach can provide an alternative classification for codfish and potentially other food speciation cases.

Clinical diagnosis and etiology of patients with Chlamydia psittaci pneumonia based on metagenomic next-generation sequencing.

The incidence of severe Chlamydia psittaci (C. psittaci) pneumonia and coinfections is increasing. Early detection of this condition is needed to prevent negative outcomes, along with detailed descriptions of its associated clinical characteristics. Our study contributes by undertaking etiological analysis of patients with C. psittaci pneumonia based on metagenomic next-generation sequencing (mNGS). A retrospective analysis of 30 patients with C. psittaci pneumonia was undertaken and confirmed by mNGS or polymerase chain reaction (PCR). Clinical manifestations of the severe and non-severe C. psittaci pneumonia groups were compared for clinical reference. Etiological analyses were also performed to comprehensively understand pathogeny and coinfection with other respiratory pathogens in C. psittaci patients. The absolute value of lymphocytes (LYM) in the severe group was lower than in the non-severe group. At the same time, neutrophil-to-lymphocyte ratio (NLR), procalcitonin (PCT), alanine aminotransferase (ALT), D-II polymer, brain natriuretic peptide (BNP), myoglobin (MYO), and cardiac troponin I (cTnI) were significantly higher (P < 0.05) in the severe group. mNGS has a broader pathogen spectrum and can more sensitively detect C. psittaci and other low-abundance pathogens with a higher positive detection rate (100%, 13/13 vs. 46%, 6/13, P <0.05) than conventional culture methods. mNGS detected the following dominant species associated with C. psittaci in patients: bacteria (53.2%, 39% gram-positive, 61% gram-negative), fungi (12.9%), and viruses (33.9%). A total of 73.3% (11/15) of patients had suspected coinfections, with a coinfection rate of 91.7% (11/12) in the severe group. No coinfection or death occurred in the non-severe group. Prognosis in the severe group was poor, with a mortality rate of 27.3% (3/11) for patients with coinfection. Eight of 11 patients with coinfections (72.7%) recovered. In conclusion, the clinical symptoms of severe C. psittaci pneumonia manifested as abnormal inflammatory indicators, impaired liver function, myocardial injury, coagulation, and relatively low immune responses. The higher proportion of patients with coinfections in our study supports the use of mNGS for comprehensive early detection of respiratory infections in patients with C. psittaci pneumonia. Simultaneous early identification of coinfections would further improve the clinical treatment of these patients.

A high-resolution colorimetric immunoassay for tyramine detection based on enzyme-enabled growth of gold nanostar coupled with smartphone readout.

In this work, a specific monoclonal antibody against tyramine was produced based on a new hapten design. Then, we developed a high-resolution multicolor colorimetric immunoassay for tyramine based on this antibody by integrating enzyme-induced multicolor generation with smartphone-assistant signal readout. The multicolor generation is due to the shift of the local surface plasmon resonance band of gold nanostructure controlled by alkaline phosphatase-induced the growth of gold nanostars. Quantitative detection of tyramine was achieved via analyzing the red/blue channel values of assay solution's image taken by a smartphone with the support of a color recognizer application. The limit of detection of this immunoassay for tyramine detection in beef, pork and yoghurt was 19.7 mg/kg or L. The average recoveries were between 83 % and 103 %., and the results were validated by high performance liquid chromatography to be reliable. Overall, this developed immunoassay provides a promising platform for on-site detection of tyramine.

Untargeted metabolomics by liquid chromatography-mass spectrometry for food authentication: A review.

Food fraud is currently a growing global concern with far-reaching consequences. Food authenticity attributes, including biological identity, geographical origin, agricultural production, and processing technology, are susceptible to food fraud. Metabolic markers and their corresponding authentication methods are considered as a promising choice for food authentication. However, few metabolic markers were available to develop robust analytical methods for food authentication in routine control. Untargeted metabolomics by liquid chromatography-mass spectrometry (LC-MS) is increasingly used to discover metabolic markers. This review summarizes the general workflow, recent applications, advantages, advances, limitations, and future needs of untargeted metabolomics by LC-MS for identifying metabolic markers in food authentication. In conclusion, untargeted metabolomics by LC-MS shows great efficiency to discover the metabolic markers for the authenticity assessment of biological identity, geographical origin, agricultural production, processing technology, freshness, cause of animals' death, and so on, through three main steps, namely, data acquisition, biomarker discovery, and biomarker validation. The application prospects of the selected markers by untargeted metabolomics require to be valued, and the selected markers need to be eventually applicable at targeted analysis assessing the authenticity of unknown food samples.

Effective and repeatable chromatographic separation of 5 nucleotides in infant formula milk powder by ion-pair high-performance liquid chromatography-ultraviolet.

A robust method using HPLC-UV was developed to improve the accuracy and repeatability of a quantitative method to detect 5 nucleotides (cytidine-monophosphate, uridine monophosphate, adenosine monophosphate, guanine monophosphate, and inosine monophosphate) in infant formulas. The results showed that efficient separation could not be achieved without strict conditions. The proposed method displayed a strong linear relationship (R2 > 0.9999) of single nucleotide in infant formula milk powder in the range of 10 to 1,000 mg/kg, a steady recovery (80.0% ∼110.0%) with relative standard deviation from 0.5% to 3.5%, under strict conditions of hydrophilic C18 column with di-isopropyl at 62.5 ± 2.5°C (± standard deviation), 0.65 ± 0.1 mg/mL tetrabutylammonium bisulfate, and mobile phase of pH of 2.75 ± 0.02. By applying this method on a series of milk products in the Chinese market, we found a few of them exceeded the legal limits of nucleotides.

Prussian blue nanoparticles-enabled sensitive and accurate ratiometric fluorescence immunoassay for histamine.

Herein, a nanozyme-mediated ratiometric fluorescence immunoassay for histamine (HA) has been developed. Prussian blue nanoparticles (PBNPs) with outstanding peroxidase-like activity were labelled with goat anti-mouse IgG via a facile electrostatic adsorption to yield the nanozyme-antibody conjugate which acted as a bridge to link the ratiometric fluorescence readout with HA concentration. As substrate, o-phenylenediamine (OPD) was oxidized into 2,3-diaminophenazine (oxOPD) by H2O2 under the catalysis of PBNPs, producing a novel emission at 570 nm and quenching the fluorescence of carbon dots (CDs) at 450 nm simultaneously. Under optimal conditions, the ratio of fluorescence intensity at 570 nm and 450 nm (I570/I450) linearly correlated with HA concentration ranging from 1.6 ng/mL to 125 µg/mL, with a detection limit (LOD) of 1.2 ng/mL. In addition, analytical performances including specificity, accuracy and applicability were evaluated, which revealed that this ratiometric fluorescence immunoassay affords an effective platform for sensitive and accurate detection of HA.

Integration of Untargeted and Pseudotargeted Metabolomics for Authentication of Three Shrimp Species Using UHPLC-Q-Orbitrap.

In this work, an untargeted and pseudotargeted metabolomics combination approach was used for authentication of three shrimp species (Litopenaeus vanmamei, Penaeus japonicus, and Penaeus monodon). The monophasic extraction-based untargeted metabolomics approach enabled comprehensive-coverage and high-throughput analysis of shrimp tissue and revealed 26 potential markers. The pseudotargeted metabolomics approach confirmed 21 markers (including 9 key markers), which realized at least putative identification. The 21 confirmed markers, as well as 9 key markers, were used to develop PLS-DA models, correctly classifying 60/60 testing samples. Furthermore, DD-SIMCA and PLS-DA models were integrated based on the 9 key markers, with 59/60 and 20/20 samples of the species that were involved and uninvolved in model training correctly classified. The results demonstrated the potential of this untargeted and pseudotargeted metabolomics combination approach for shrimp species authentication.

Stabilization of maneb group by ethylenediamine and direct-determination by liquid chromatography tandem mass spectrometry.

A rapid, low-cost, and selective method for simultaneous and direct determination of maneb group residues (containing ethylenebis and propylenebis dithiocarbamates) in fruit by liquid chromatography coupled to tandem mass spectrometry was developed and validated in the current study. The results showed the maneb group could be melt and stabilized by 5 v% ethylenediamine for 60 days keeping in conventional refrigerators, in which a stable and ionizable pentadentate ligand complex was considered to be formed by the bidentate diamine and sulfhydryl followed by Density Functional Theory calculation. The validated method showed a sensitive quantification limits (0.03 mg/kg), a steady recovery (82.1%-91.0%) and an excellent precision (2.7%-4.3% RSD). This method is applied to analyze fruit samples and achieved satisfactory results. Therefore, this method can be proposed as a robust analytical method of maneb group in fruit, and can be adapted to detect other compounds with sulfhydryl group.

Detection of galactooligosaccharides with high lactose interference in infant formula using a simple single epimer chromatography.

Robust and more anti-interference enzymatic quantification of galactooligosaccharide (GOS) is important in consumer protection. However, many methods with harsh conditions could hardly separate GOS's hydrolysates galactose from its structurally similar isomer glucose, since each of them has double epimers, especially to determinate a trace of GOS from large amounts of lactose in the food matrix. The investigation was designed to solve the problem by using High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD), applying friendly mobile phase and column. Result showed the content of galactose was seldom affected even by a high content of glucose by integrating the peak area of an excellent resolution single epimer. Moreover, the method existed a good linearity and stability (recovery rate at 90.5-105.1%), which met the statutory limit requirement for the quantitative analysis of concentrated GOS in infant formula. It was also helpful for separating and quantifying other sugar or epimers.

Hypoglycemic effects and mechanism of different molecular weights of konjac glucomannans in type 2 diabetic rats.

Konjac glucomannan (KGM) is a hypoglycemic polysaccharide with a wide range of molecular weights. But study on hypoglycemic effects of KGMs relate to molecular weight is limited. In this study, KGMs with high and medium molecular weights, and the degraded KGMs were analyzed with physicochemical properties, hypoglycemic effects and mechanisms. Results showed that as the molecular weight KGMs decreased, the viscosity decreased, molecular flexibility increased, while chemical groups, crystal structures and main chains showed little change. KGMs with medium molecular weights (KGM-M1, KGM-M2) showed better effects on increasing body weight, decreasing levels of fasting blood glucose, insulin resistance, total cholesterol and low density lipoprotein cholesterol, and enhancing integrity of pancreas and colon, than KGMs with high or low molecular weights (KGM-H, KGM-L) in type 2 diabetic rats. Mechanism analysis suggested that KGM-M1 and KGM-M2 had higher antioxidant and anti-inflammatory activities on elevating superoxide dismutase, decreasing malondialdehyde and tumor necrosis factor-α levels. Moreover, KGM-M1 and KGM-M2 increased gut microbiota diversity, Bacteroidetes/Firmicutes ratio and Muribaculaceae, decreased Romboutsia and Klebsiella, and improved 6 diabetic related metabolites. Combined, KGM-M1 and KGM-M2 showed higher hypoglycemic effects, due to regulatory activities of antioxidant, anti-inflammatory, intestinal microbiota, and relieved metabolic disorders.

PVAm Nanofibers Fabricated by Rotary Jet Wet Spinning and Applied to Bisphenol A Recognition.

Poly(vinylamine) (PVAm) is an important polymer with the highest content of primary amine groups of any polymer. PVAm has a great potential in selective separation and smart materials. It is difficult to fabricate pure PVAm nanofibers by electrospinning and rotary jet spinning (RJS) without additional polymers. In this work, rotary jet wet spinning (RJWS) was applied to fabricate molecular imprinting nanofibers (MINFs) with polyelectrolyte for the first time. Initially, optimal parameters of spinning are investigated, including coagulation bath, solution viscosity, and rotation speed. The PVAm aqueous solution is sensitive to alcohol. To demonstrate RJWS application, PVAm-based MINFs for bisphenol A (one endocrine disruptor) recognition are prepared by adding dummy template, cross-linking, and template elution. The association constant (8.6 mg/L), equilibrium time (30 min), and binding sites utilization rate (80%) of MINFs are evaluated. Its adsorption amount and selectivity are little lower than those of MIPs prepared by bulk polymerization; however, its adsorption speed is faster than that of MIPs.

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine.

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.