Indirect Competitive Enzyme-linked Immunosorbent Assay Research Articles

Re-assessment of monoclonal antibodies against diclofenac for their application in the analysis of environmental waters.

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is an important environmental contaminant occurring in surface waters all over the world, because, after excretion, it is not adequately removed from wastewater in sewage treatment plants. To be able to monitor this pollutant, highly efficient analytical methods are needed, including immunoassays. In a medical research project, monoclonal antibodies against diclofenac and its metabolites had been produced. Based on this monoclonal anti-DCF antibody, a new indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed and applied for environmental samples. The introduction of a spacer between diclofenac and the carrier protein in the coating conjugate led to higher sensitivity. With a test midpoint of 3 μg L-1 and a measurement range of 1-30 μg L-1, the system is not sensitive enough for direct analysis of surface water. However, this assay is quite robust against matrix influences and can be used for wastewater. Without adjustment of the calibration, organic solvents up to 5%, natural organic matter (NOM) up to 10 mg L-1, humic acids up to 2.5 mg L-1, and salt concentrations up to 6 g L-1 NaCl and 75 mg L-1 CaCl2 are tolerated. The antibody is also stable in a pH range from 3 to 12. Cross-reactivity (CR) of 1% or less was determined for the metabolites 4'-hydroxydiclofenac (4'-OH-DCF), 5-hydroxydiclofenac (5-OH-DCF), DCF lactam, and other NSAIDs. Relevant cross-reactivity occurred only with an amide derivative of DCF, 6-aminohexanoic acid (DCF-Ahx), aceclofenac (ACF) and DCF methyl ester (DCF-Me) with 150%, 61% and 44%, respectively. These substances, however, have not been found in samples. Only DCF-acyl glucuronide with a cross-reactivity of 57% is of some relevance. For the first time, photodegradation products were tested for cross-reactivity. With the ELISA based on this antibody, water samples were analysed. In sewage treatment plant effluents, concentrations in the range of 1.9-5.2 μg L-1 were determined directly, with recoveries compared to HPLC-MS/MS averaging 136%. Concentrations in lakes ranged from 3 to 4.4 ng L-1 and were, after pre-concentration, determined with an average recovery of 100%.

De novo synthesis of a novel hapten and development of a monoclonal antibody–based immunoassay for the detection of dichlorvos and trichlorfon

The absence of high-affinity antibodies has hindered the development of satisfactory immunoassays for dichlorvos (DDVP) and trichlorfon (TCP), two highly toxic organophosphorus pesticides. Herein, the de novo synthesis of a novel anti-DDVP hapten was introduced. Subsequently, a specific anti-DDVP monoclonal antibody (Mab) was produced with satisfying affinity to DDVP (IC50: 12.4 ng mL−1). This Mab was highly specific to DDVP, and TCP could readily convert into DDVP under mild alkaline conditions. Leveraging this insight, an indirect competitive ELISA was successfully developed for simultaneous detection of DDVP and TCP. The limit of detection in rice, cabbage and apple for DDVP /TCP was found to be 12.1/14.6 μg kg−1, 7.3/8.8 μg kg−1 and 6.9/8.3 μg kg−1, respectively. This study not only provides an effective strategy for producing a high-quality anti-DDVP Mab but also affords a reliable and cost-effective tool suitable for high-throughput detection of DDVP and TCP in food samples.

Hapten synthesis, monoclonal antibody production and immunoassay development for analyzing spiropidion residues

Spiropidion developed by Syngenta shows high insecticidal and acaricidal activity against a wide range of sucking pests. In this study, according to the structure of spiropidion, two haptens were synthesized by introducing carboxyl groups from the ester group. After cell fusion, a monoclonal antibody (mAb 8B5) of spiropidion was obtained. The IC50 of the established heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was 7.36 ng/mL, and its working range was 1.75–34.92 ng/mL. The average recoveries were 76.05–124.78% in the Yangtze River and citrus samples. Moreover, the ic-ELISA results of 15 citrus samples agreed well with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Overall, the established ic-ELISA could be applied for the spiropidion residue monitor in food and agricultural samples.

Hapten design, monoclonal antibody preparation and immunoassay development for direct detection of the toxic metabolite of nitrovin without derivatization from fish

Nitrovin, classified as a nitrofuran drug, has been banned in numerous countries due to its carcinogenic effect on human health. However, existing analytical techniques for its detection are laborious, requiring an overnight derivatization process lasting 16 h. In this study, novel haptens were designed to prepare antibodies against aminogunidine (AGD), the metabolite of nitrovin, and then developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect AGD from fish samples without derivatization for the first time. Computer-assisted molecular modeling predicted the efficiency of novel haptens by conformational studies and electronic analysis. The results were consistent with the predicted results in producing high-affinity monoclonal antibody (mAb) against AGD. The half-inhibition concentration (IC50) value of icELISA was 0.80 ng mL−1 for AGD, with a limit of detection of 0.27 μg kg−1 and recoveries of 89.8%–97.2%. The results showed that this new hapten design and synthesis provided a great prospect to obtain high-affinity mAb for AGD. This developed icELISA substantially avoided the conventional and lengthy derivatization process and was an effective method for the direct detection of AGD in fish tissue without derivatization.

Fluorescence immunosensor based on a specific monoclonal antibody for highly sensitive and rapid detection of gizzerosine in feed

Gizzerosine is a biogenic amine produced in fish meal drying process and posted higher mortality due to gizzard erosion in poultry than histamine. However, it is difficult to obtain gizzerosine and achieve sensitive practical detection due to its simple structure. Herein, a monoclonal antibody (mAb) specific to gizzerosine was generated based on the new structural design and a fluorescence immunosensor for sensitive and on-site detection of gizzerosine in feed was first established. Molecular modeling of the three-dimensional (3D) structure and surface electrostatic potential of gizzerosine indicated that the carbonyl group of gizzerosine hapten might affect the important sites of antigen-antibody interactions. The proposed structure was used to obtain the sensitive and specific mAb with IC50 of 3.88 ng/mL in indirect competitive ELISA which was approximately 100-fold lower than that of direct competitive ELISA. Considering the practical application scenarios, a fluorescence immunosensor based on microporous dry method integrated with independent quality control line was established to improve detection stability. Under the optimum conditions, the proposed immunosensor showed a good linear relationship from 1.10 to 19.78 ng/mL and provided a low detection limit of 50 ng/g which was approximately 80-fold lower than the maximum recommended amount (0.4 mg/kg) of gizzerosine in feed. The recoveries of 6 kinds of feed ranged from 83.1% to 114.3%, which was in good consistence with that of UHPLC–MS/MS. Overall, this work provides a fast, cost-effective and reliable on-site tool for rapid screening of gizzerosine residues in feed samples.

Optimisation of fermentation for developing a novel low‐allergenic fermented milk beverage with reduced antigenicity

In pursuit of expanding dietary options for those with allergies, we aimed to develop a low‐allergenic fermented milk beverage. We adopted a combination fermentation strategy to enhance the allergy mitigation efficacy of 4 strains of lactic acid bacteria. Our fermentation conditions were optimised with an equal ratio (1:1:1:1) of strains, a total 5% inoculation rate and fermentation at 40°C for 6.5 h. Under conditions, the antigen reduction rates for α‐lactalbumin (α‐LA), β‐lactoglobulin (β‐LG), α‐casein and β‐casein were impressive, ranging from 43.54 to 70.51%. A 28‐day storage assessment showed that the reduction rates of the 4 antigens ranged from 38.98% to 78.06%. Most importantly, it was found through indirect competitive ELISA that the binding ability of immunoglobulin E to α‐LA and β‐LG, which we are concerned about, was significantly reduced compared to the unfermented sample. Notable, the beverage maintained favorable physicochemical properties throughout the storage period.

Development of time-resolved fluorescence immunochromatography and ic-ELISA based on monoclonal antibodies specifically recognizing fumonisin B1

Fumonisins (FBs) are mycotoxins primarily synthesized by Fusarium moniliformis. Among these, FB1 exhibits not only high toxicity towards humans and animals but also carcinogenic properties. The global prevalence of FB1 contamination in cereals and related products, particularly maize, is alarmingly significant. Consequently, the accurate determination of FB1 levels in cereals holds immense importance. In this study, highly sensitive monoclonal antibodies specifically targeting FB1 were prepared and utilized for the establishment of a time-resolved fluorescence immunochromatographic assay (TRFIC) to detect FB1. The parameters of antibody labeling with time-resolved fluorescent microspheres were optimized. The detection time was significantly reduced to 6 min. The limits of detection (LOD) for corn, rice, and feed were determined as 0.496–0.844 μg/kg, and the quantification (LOQ) was 0.788–1.322 μg/kg. In addition, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was successfully developed. Under optimized conditions, the half inhibitory concentration (IC50) value for FB1 was determined as 2.137 μg/L. A strong correlation between the results obtained from these two methods and HPLC-MS/MS analysis was observed in the same samples tested. In conclusion, both immunological methods developed in this work are highly suitable for rapid FB1 detection in real field samples.

Development of a Monoclonal Antibody-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Gallic Acid.

Gallic acid (GA) is closely related to the quality of herbal medicines and other agricultural products. In order to facilitate the rapid detection of GA, we developed a monoclonal antibody-based ic-ELISA method. Antigens with and without connecting arms were prepared. It was found that the introduction of connecting arms (linear carbon chain) was beneficial for immune response. By utilizing hybridoma technology, a specific mAb (anti-GA-M702) was screened and identified, which exhibited a 1:40,500 antibody titer and IgG2b antibody subtype. The ic-ELISA assay was established based on anti-GA-M702. The optimal working concentrations of the encapsulated antigen and antibody were 0.5 µg/mL and 0.67 µg/mL, respectively. The ic-ELISA method showed a linear detection range of 297.17-2426.61 ng/mL for GA with a sensitivity of 849.18 ng/mL. It displayed a good applicability for the determination of GA in Galla chinensis. In conclusion, the ic-ELISA method provides an efficient approach to the rapid detection of GA in products.

Nanozyme-assisted molecularly imprinted polymer-based indirect competitive ELISA for the detection of marine biotoxin

Saxitoxin (STX), which is produced by certain dinoflagellate species, is a type of paralytic shellfish poisoning toxin that poses a serious threat to human health and the environment. Therefore, developing a technology for the convenient and cost-effective detection of STX is imperative. In this study, we developed an affinity peptide-imprinted polymer-based indirect competitive ELISA (ic-ELISA) without using enzyme–toxin conjugates. AuNP/Co3O4@Mg/Al cLDH was synthesized by calcining AuNP/ZIF-67@Mg/Al LDH, which was obtained by combining AuNPs, ZIF-67, and flower-like Mg/Al LDH. This synthesized nanozyme exhibited high catalytic activity (Km = 0.24 mM for TMB and 132.5 mM for H2O2). The affinity peptide-imprinted polymer (MIP) was imprinted with an STX-specific template peptide (STX MIP) on a multi-well microplate and then reacted with an STX-specific signal peptide (STX SP). The interaction between the STX SP and MIP was detected using a streptavidin-coated nanozyme (SA-AuNP/Co3O4@Mg/Al cLDH). The developed MIP-based ic-ELISA exhibited excellent selectivity and sensitivity, with a limit of detection of 3.17 ng/mL (equivalent: 0.317 μg/g). Furthermore, the system was validated using a commercial ELISA kit and mussel tissue samples, and it demonstrated a high STX recovery with a low coefficient of variation. These results imply that the developed ic-ELISA can be used to detect STX in real samples.

An Enzyme Immunoassay Developed for the Determination of Methylene Blue in Aquatic Products Based on a Novel Hapten

Methylene blue (MB) is a chemical dye which is used as an alternative drug for malachite green. In this study, based on computational chemistry, a novel hapten (TM) of MB was designed by comparing the conformational and electronic properties of MB. TM was synthesized and further conjugated with bovine serum albumin (BSA) and Ovalbumin (OVA). Then, the polyclonal antibodies were obtained by immunization with the immunogen of TM-BSA. Under optimal conditions, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the determination of MB, with an IC50 value of 41.5 μg L−1 and displaying a cross reaction of 78.2~88.9% with three different MB metabolins. The limit of detection (LOD) and the limit of quantitation (LOQ) for MB were validated to be 4.8 µg/kg and 6.0 µg/kg, respectively. Spiking experiments showed recoveries between 82.3% and 84.3%, with a relative standard deviation (RSD) of ≤6%. The results showed the generated polyclonal antibodies from the new hapten TM were reliable and could be used for detecting MB in fishery productions.

An automated fluorescent immunoassay for on-site screening of AFM1 in raw milk at the ppt level

In the dairy industry, the quality of raw milk as it is collected from dairy farmers must be strictly ensured. Therefore, when on-site screening of typical carcinogens in raw milk, this technology must simultaneously be convenient to operate and highly sensitive. Here, an automated and fluorescent immunoassay system for screening trace amounts of aflatoxin M1 (AFM1) in raw milk was developed. The whole immunoassay procedure can be processed in a true “sample-to-results” paradigm, avoiding the tedious manual operation of the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method. In addition, we designed an integrated fluorescent spectrometer that can quantitatively measure fluorescent signals with high sensitivity. The automated and fluorescent immunoassay system can screen AFM1 in raw milk samples with an ultra low limit of detection (4.7 pg/mL in raw milk). The half maximal inhibitory concentration (IC50) was 8.3 pg/mL, which is approximately 4-fold lower than that of traditional ic-ELISA. When the system was applied to screen actual raw milk samples, the recovery rates ranged from 79.4% to 104.6%, with a coefficient of variation ranging from 8.9% to 15.2%. Overall, we believe that the automated and fluorescent immunoassay system can provide a one-stop solution that is user-friendly and highly sensitive for screening trace AFM1 contaminants in raw milk.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Simultaneous identification of three emergent stimulant laxative adulterants in slimming foods using only one antibody.

Stimulant laxatives were recently found to be abused in slimming foods, resulting in harmful effects on consumers. To ensure the safety of relative products, sensitive yet multiplex immunoassays are crucial in rapid screening of stimulant laxatives. However, there are few immunoassays for these substances, and even less for broad-specific recognition. Thus, in this work, four theoretically promising haptens of emerging stimulant laxative bisacodyl were rationally designed using molecular modeling and synthesized to immune animals, whose feasibility was confirmed by the obtained broad-specific antibody. Based on this unique antibody, a highly sensitive multiplex competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with low limits of detection for bisacodyl, sodium picosulfate, and BHPM (0.23, 13.68, and 0.11 ng/mL). In spiked sample recovery test and real sample detection, this ciELISA exhibited acceptable consistency with the validation method, demonstrating high accuracy and applicability of our method. This reliable multiplex ciELISA proceeds the rapid screening of stimulant laxatives in slimming foods.

On-site rapid detection of perfluorooctanoic acid by visual immunochromatographic strip biosensor in domestic water and real human samples

The International Agency for Research on Cancer (IARC) classifies PFOA as a Class 1 carcinogen. Here, a new naked-eye PFOA immunochromographic strip was developed to recognize PFOA in domestic water and real human samples within 10 min based on a novel custom designed anti-PFOA monoclonal antibody (mAb) 2A3, which was firstly an immune rapid detection method for PFOA has been proposed. Using computer simulation techniques such as quantum computing to assist in designing the structural formula of PFOA semi antigen, which hapten was firstly proposed. The half maximal inhibitory concentration of PFOA monoclonal antibody (mAb) 2A3 was 2.4 μg/mL. Using mAb 2A3, we developed an immunochromatographic strip (ICS) for detecting PFOA in real samples. The developed method generated results in 10 min, with visual detection limits of 20, 20, and 200 μg/mL and limit of detection of 50, 200, and 500 μg/mL for water, blood and urine samples, respectively. The established ICS and indirect competitive enzyme-linked immunosorbent assay were used to analyze the actual samples, and the results were confirmed by LC-MS/MS. Our study findings showed that the ICS and ic-ELISA can quickly detect PFOA in actual samples.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen–antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25–23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%–118.8% and 77.5%–113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

The hapten rigidity improves antibody performances in immunoassay for rifamycins: Immunovalidation and molecular mechanism

The immunogenicity of haptens determines the performance of the resultant antibody for small molecules. Rigidity is one of the basic physicochemical properties of haptens. However, few studies have investigated the effect of hapten rigidity on the strength of an immune response and overall antibody performance. Herein, we introduce three molecular descriptors that quantify hapten rigidity. By using of these descriptors, four rifamycin haptens with varied rigidity were designed. The structural and physicochemical feasibility of the designed haptens was then assessed by computational chemistry. Immunization demonstrated that the strength of induced immune responses, i.e., the titer and affinity of antiserum, was significantly increased with increased rigidity of haptens. Furthermore, molecular dynamic simulations demonstrated conformation constraint of rigid haptens contributed to the initial binding and activation of naïve B cells. Finally, a highly sensitive indirect competitive enzyme-linked immunosorbent assay was developed for detection of rifaximin, with an IC50 of 1.1 μg/L in buffer and a limit of detection of 0.2–11.3 μg/L in raw milk, river water, and soil samples. This work provides new insights into the effect of hapten rigidity on immunogenicity and offers new hapten design strategies for antibody discovery and vaccine development of small molecules.

Development of a colloidal gold immunochromatographic strip for rapid detection of ribavirin in pig urine

ABSTRACT A specific and sensitive monoclonal antibody (mAb) for ribavirin (RBV) was generated by immunizing BALB/c mice with RBV – bovine serum albumin. Using the mAb, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic (CGI) test strip were developed for detecting RBV in pig urine. Under optimum assay conditions, IC50 was 0.08 ng/mL in 0.05 M phosphate-buffered saline and the recovery rate in real-world samples ranged between 77.3% and 99%. The cut-off limit of the CGI test strip for RBV was 5 ng/mL in pig urine. The cross-reactivity rate of the test strip to the tested antiviral drugs including acyclovir and moroxydine hydrochloride and the metabolites 1,2,4-triazole-3-carboxamide and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylic acid was below 0.1%. The results indicate that ic-ELISA and the CGI test strip have high sensitivity and specificity and that these methods are suitable for the rapid detection of RBV in pig urine.

Development of gold nanoparticle-based lateral flow immunoassay for the determination of tebufenozide in agro-products

Tebufenozide is a non-steroidal insect growth regulator, which has been widely used in agriculture to control pests. Despite being recognized as an eco-friendly pesticide, its residues still can pose potential health hazards to humans. In this study, two haptens of tebufenozide were synthesized to prepare the monoclonal antibodies (mAbs). Out of the eight mAbs, mAb 5C5B6 exhibited the highest sensitivity (half maximal inhibitory concentration = 0.15 ng/mL) in the heterologous indirect competitive enzyme-linked immunosorbent assay, and was utilized to develop gold nanoparticle lateral flow immunoassay (LFIA). The cut-off value and visual limit of detection of the LFIA were 10 and 1.25 ng/mL. The detection results of both the spiked and blind samples demonstrated that the proposed LFIA exhibits high accuracy in detecting tebufenozide in agricultural products.

Quantitative Determination of Aflatoxin B1 in Maize and Feed by ELISA and Time-Resolved Fluorescent Immunoassay Based on Monoclonal Antibodies.

In this study, a highly sensitive monoclonal antibody (mAb) was developed for the detection of aflatoxin B1 (AFB1) in maize and feed. Additionally, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluorescence immunoassay assay (TRFICA) were established. Firstly, the hapten AFB1-CMO was synthesized and conjugated with carrier proteins to prepare the immunogen for mouse immunization. Subsequently, mAb was generated using the classical hybridoma technique. The lowest half-maximal inhibitory concentration (IC50) of ic-ELISA was 38.6 ng/kg with a linear range of 6.25-100 ng/kg. The limits of detections (LODs) were 6.58 ng/kg and 5.54 ng/kg in maize and feed, respectively, with the recoveries ranging from 72% to 94%. The TRFICA was developed with a significantly reduced detection time of only 21 min, from sample processing to reading. Additionally, the limits of detection (LODs) for maize and feed were determined to be 62.7 ng/kg and 121 ng/kg, respectively. The linear ranges were 100-4000 ng/kg, with the recoveries ranging from 90% to 98%. In conclusion, the development of AFB1 mAb and the establishment of ic-ELISA for high-throughput sample detection, as well as TRFICA for rapid detection presented robust tools for versatile AFB1 detection in different scenarios.