Preparation of monoclonal antibodies recognizing pharmacologically active metabolites of metamizole based on rational hapten design and their application in the detection of animal-derived food.

Metamizole (MET) is an antipyretic and analgesic drug, the illegal use of which can result in residues of MET metabolites in edible tissues of animals. In this study, a computational chemistry-assisted hapten screening strategy was used to screen for the optimal immunogenic hapten-A and the optimal coating antigen hapten-G-OVA. A monoclonal antibody capable of recognizing two pharmacologically active metabolites of MET, 4-methylamidinoantipyrine (MAA) and 4-aminoantipyrine (AA), was prepared from the hapten-A. The antibody showed excellent specificity for MAA and AA and almost no cross-reactivity with the pharmacologically inactive metabolites 4-formamidinoantipyrine (FAA) and 4-acetamidinoantipyrine (AAA). An ic-ELISA was developed for the simultaneous detection of MAA and AA in animal-derived food, the limits of detection for MAA ranged from 0.93 to 1.18 µg/kg, while those for AA ranged from 1.74 to 4.61 µg/kg. The recovery rate fell within the range of 82 %-110 %, with a coefficient of variation less than 16.39 %.

Advancements in the preparation technology of small molecule artificial antigens and their specific antibodies: a comprehensive review.

Small molecules find extensive application in medicine, food safety, and environmental studies, particularly in biomedicine. Immunoassay technology, leveraging the specific recognition between antigens and antibodies, offers a superior alternative to traditional physical and chemical analysis methods. This approach allows for the rapid and accurate detection of small molecular compounds, owing to its high sensitivity, specificity, and swift analytical capabilities. However, small molecular compounds often struggle to effectively stimulate an immune response due to their low molecular weight, weak antigenicity, and limited antigenic epitopes. To overcome this, coupling small molecule compounds with macromolecular carriers to form complete antigens is typically required to induce specific antibodies in animals. Consequently, the preparation of small-molecule artificial antigens and the production of efficient specific antibodies are crucial for achieving precise immunoassays. This paper reviews recent advancements in small molecule antibody preparation technology, emphasizing the design and synthesis of haptens, the coupling of haptens with carriers, the purification and identification of artificial antigens, and the preparation of specific antibodies. Additionally, it evaluates the current technological shortcomings and limitations while projecting future trends in artificial antigen synthesis and antibody preparation technology.

Methods and applications of noncompetitive hapten immunoassays.

Hapten immunoassays have found extensive application across various domains such as disease diagnostics, environmental monitoring, as well as the evaluation of food and pharmaceutical safety. These techniques traditionally rely on competitive assay formats and often face challenges with sensitivity and specificity. This review focuses on the emergent noncompetitive immunoassay technologies that promise to transcend these limitations through innovative approaches. Noncompetitive immunoassays, leveraging novel elements such as anti-idiotype antibodies, anti-immunocomplex (IC) antibodies, and the strategic use of nanomaterial-enhanced signal detection, are setting new benchmarks for analytical performance. These advancements not only enhance the detection capabilities but also significantly improve specificity inherent in traditional methods. Moreover, the integration of novel materials and binding reagents in these assays offers substantial improvements in assay dynamics, providing faster, more accurate, and reliable results. This review consolidates the latest methodologies and their applications, underlining the transformative impact of noncompetitive technologies in the sensitive detection of haptens across various fields.

A rapid spectrophotometric test for assessing skin sensitization potential of chemicals by using N-acetyl-L-cysteine methyl ester in chemico.

During the key event 1 of skin sensitization defined as covalent binding or haptenization of sensitizer to either thiol or amino group of skin proteins, a sensitizer not only covalently binds with skin proteins but also interacts with nucleophilic small molecules such as glutathione (GSH). Although GSH would not be directly associated with skin sensitization, this interaction may be applied for developing an alternative test method simulating key event 1, haptenization. Thus, the aim of the present study was to examine whether N-acetyl-L-cysteine methyl ester (NACME), a thiol-containing compound, was selected as an electron donor to determine whether NACME reacted with sensitizers. Following a reaction of NACME with a sensitizer in a 96-well plate, the remaining NACME was measured spectrophotometrically using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions with two different vehicles, such as acetonitrile (ACN) and dimethyl sulfoxide (DMSO), 64 test chemicals were tested to determine the predictive capacity of current NACME test method. The results obtained showed, the predictive capacity of 94.6% sensitivity, 88.9% specificity, and 92.2% accuracy utilizing DMSO as a vehicle with a cutoff NACME depletion of 5.85%. The three parameters were also over 85% in case of ACN. These values were comparable to or better than other OECD-approved test methods. Data demonstrated that a simple thiol-containing compound NACME might constitute as a reliable candidate for identifying reactive skin sensitizers, and that this method be considered as practical method as a screening tool for assessing a chemical's tendency to initiate skin sensitization.

Rapid Screening of Emergent Febuxostat Adulteration in Functional Foods Based on a Simulation-Inspired High-Quality Antibody.

Due to the potential health risks of adulterated febuxostat in uric-acid-lowering foods, it is urgent to develop rapid detection methods. However, there are no fast analytical techniques for febuxostat yet. Herein, an efficient hapten simulation strategy was proposed to successfully produce a highly sensitive and selective monoclonal antibody toward febuxostat. Based on such a robust recognition element, easy colorimetric and ultrasensitive fluorescent lateral flow immunochromatographic immunoassays were first established, which can detect febuxostat as low as 60 µg/kg by the naked eye or 1.01 µg/kg by a commercial test strip reader with acceptable stability. Furthermore, in the recovery test and blind sample analysis, consistent results between our methods and the authorized liquid chromatography-tandem mass spectrometry method suggested the high accuracy and practicality of this work. The present work not only proposes a rational hapten design idea but also provides favorable tools for the rapid screening of febuxostat in functional foods.

Opioid-Based Haptens: Development of Immunotherapy.

Over the past decades, extensive preclinical research has been conducted to develop vaccinations to protect against substance use disorder caused by opioids, nicotine, cocaine, and designer drugs. Morphine or fentanyl derivatives are small molecules, and these compounds are not immunogenic, but when conjugated as haptens to a carrier protein will elicit the production of antibodies capable of reacting specifically with the unconjugated hapten or its parent compound. The position of the attachment in opioid haptens to the carrier protein will influence the specificity of the antiserum produced in immunized animals with the hapten-carrier conjugate. Immunoassays for the determination of opioid drugs are based on the ability of drugs to inhibit the reaction between drug-specific antibodies and the corresponding drug-carrier conjugate or the corresponding labelled hapten. Pharmacological studies of the hapten-carrier conjugates resulted in the development of vaccines for treating opioid use disorders (OUDs). Immunotherapy for opioid addiction includes the induction of anti-drug vaccines which are composed of a hapten, a carrier protein, and adjuvants. In this review we survey the design of opioid haptens, the development of the opioid radioimmunoassay, and the results of immunotherapy for OUDs.

Shrimp hemocyanin elicits a potent humoral response in mammals and is favorable to hapten conjugation.

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development.

A Glycolipid-Peptide-Hapten Tricomponent Conjugate Vaccine Generates Durable Antihapten Antibody Responses in Mice.

Eliciting an antihapten antibody response to vaccination typically requires the use of constructs where multiple copies of the hapten are covalently attached to a larger carrier molecule. The carrier is required to elicit T cell help via presentation of peptide epitopes on major histocompatibility complex (MHC) class II molecules; as such, attachment to full-sized proteins, alone or in a complex, is generally used to account for the significant MHC diversity in humans. While such carrier-based vaccines have proven extremely successful, particularly in protecting against bacterial diseases, they can be challenging to manufacture, and repeated use can be compromised by pre-existing immunity against the carrier. One approach to reducing these complications is to recruit help from type I natural killer T (NKT) cells, which exhibit limited diversity in their antigen receptors and respond to glycolipid antigens presented by the highly conserved presenting molecule CD1d. Synthetic vaccines for universal use can, therefore, be prepared by conjugating haptens to an NKT cell agonist such as α-galactosylceramide (αGalCer, KRN7000). An additional advantage is that the quality of NKT cell help is sufficient to overcome the need for an extra immune adjuvant. However, while initial studies with αGalCer-hapten conjugate vaccines report strong and rapid antihapten antibody responses, they can fail to generate lasting memory. Here, we show that antibody responses to the hapten 4-hydoxy-3-nitrophenyl acetyl (NP) can be improved through additional attachment of a fusion peptide containing a promiscuous helper T cell epitope (Pan DR epitope, PADRE) that binds diverse MHC class II molecules. Such αGalCer-hapten-peptide tricomponent vaccines generate strong and sustained anti-NP antibody titers with increased hapten affinity compared to vaccines without the helper epitope. The tricomponent vaccine platform is therefore suitable for further exploration in the pursuit of efficacious antihapten immunotherapies.

Protein Haptenation and Its Role in Allergy.

Humans are exposed to numerous electrophilic chemicals either as medicines, in the workplace, in nature, or through use of many common cosmetic and household products. Covalent modification of human proteins by such chemicals, or protein haptenation, is a common occurrence in cells and may result in generation of antigenic species, leading to development of hypersensitivity reactions. Ranging in severity of symptoms from local cutaneous reactions and rhinitis to potentially life-threatening anaphylaxis and severe hypersensitivity reactions such as Stephen-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), all these reactions have the same Molecular Initiating Event (MIE), i.e. haptenation. However, not all individuals who are exposed to electrophilic chemicals develop symptoms of hypersensitivity. In the present review, we examine common chemistry behind the haptenation reactions leading to formation of neoantigens. We explore simple reactions involving single molecule additions to a nucleophilic side chain of proteins and complex reactions involving multiple electrophilic centers on a single molecule or involving more than one electrophilic molecule as well as the generation of reactive molecules from the interaction with cellular detoxification mechanisms. Besides generation of antigenic species and enabling activation of the immune system, we explore additional events which result directly from the presence of electrophilic chemicals in cells, including activation of key defense mechanisms and immediate consequences of those reactions, and explore their potential effects. We discuss the factors that work in concert with haptenation leading to the development of hypersensitivity reactions and those that may act to prevent it from developing. We also review the potential harnessing of the specificity of haptenation in the design of potent covalent therapeutic inhibitors.

Quantitative determination of prothioconazole in wheat grain, soybean, and pond water based on a polyclonal antibody.

Prothioconazole and its metabolite are considered a potential threat to human health and environmental safety. Thus, the development of a sensitive and rapid detection method for prothioconazole is crucial to ensure the safety of agricultural products. In this study, a new hapten of prothioconazole was designed and synthesized, and a selective polyclonal antibody with high affinity against prothioconazole was produced, which was obtained from immunized New Zealand white rabbits. Based on the polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) were developed for detecting prothioconazole pesticides. Under optimized experimental conditions, the limit of quantification (LOQ) values for ic-CLEIA and ic-ELISA were 1.8 and 10.7 ng mL-1, respectively. The results demonstrated that the sensitivity (LOQ) achieved by ic-CLEIA was more than five times higher compared to that obtained with ic-ELISA. In addition, the recoveries obtained by adding standard prothioconazole to wheat grain, soybean, and pond water samples were in the range of 81.9 to 104.7% for ic-ELISA and 89.0 to 118.0% for ic-CLEIA.

In-Depth Structural Simplification of Celangulin V: Design, Synthesis, and Biological Activity.

Celangulin V is a novel botanical insecticide with significant bioactivity and a unique molecular target, but its complex polyol ester structure hinders its broader application in agriculture. To discover new analogues of celangulin V with a simpler structure and enhanced biological activities, we initiated a research project aimed at simplifying its structure and assessing insecticidal efficacy. In this study, a series of novel 1-tetralone derivatives were designed via a structure-based rational design approach and synthesized by a facile method. The biological activities of the target compounds were determined against Mythimna separata (M. separata), Plutella xylostella, and Rhopalosiphum padi. The results revealed that most of the synthesized compounds exhibited superior activities compared to celangulin V. Remarkably, the insecticidal activity of compound 6.16 demonstrated 102-fold greater stomach toxicity than celangulin V against M. separata. In addition, certain compounds showed significant contact toxicity against M. separata, a finding not reported previously in the structural optimization studies of celangulin V. Molecular docking analysis illustrated that the binding pocket of compound 6.16 with the H subunit of V-ATPase was the same as celangulin V. This study presents novel insights into the structural optimization of botanical pesticides.

General hapten skeleton motivated duplex-immunoassay for emergent bisoxatin adulterants in slimming foods.

Adulterating hazardous bisoxatin (BSO) and bisoxatin acetate (BSOA) in slimming foods poses a threat to public health. A rapid synchronous detection method is urgently needed. Herein, the precise design of four novel haptens based on the general skeleton of BSO and BSOA was driven by computer-chemical visualization strategy, which was used to raise monoclonal antibody (mAb) toward both target compounds. The generated mAb 1F1 recognized BSO and BSOA with maximal half-inhibitory concentration (IC50) of 0.26 and 16.85 ng/mL, respectively. The molecular mechanism governing the duplex-recognition of mAb was elucidated by homology modeling and molecular docking. Finally, an immunochromatography (ICA) was developed for identifying BSO and BSOA, demonstrating a detection capability for screening (CCß) estimated to be 10-500 ng/g in candy tablets, jellies, and oral liquids. This study provides a robust approach for determining adulteration in food and offers insights into hapten design to improve antibody recognition spectrum.

Preparation of high-affinity and high-specificity monoclonal antibody and development of ultra-sensitive immunoassay for the detection of 1-aminohydantoin, the metabolite of nitrofurantoin.

1-Aminohydantoin (AHD), the residual marker of nitrofurantoin, is usually detected after derivatisation using the derivatisation reagent 2-nitrobenzaldehyde. Avoiding the antibody recognition of the derivatisation reagent is essential for the accurate detection of AHD residues. In this paper, a novel hapten called hapten D was designed, and then, a monoclonal antibody that did not recognise 2-nitrobenzaldehyde was prepared based on this novel hapten. An ultra-sensitive indirect competitive enzyme linked-immunosorbent assay (icELISA) was established under optimal conditions. The 50% inhibition concentration and limit of detection of AHD were 0.056 and 0.0060 ng/mL, respectively, which improved the sensitivity by 9-37-fold compared with the previously reported icELISA methods. The average recovery rates were 88.1%-97.3%, and the coefficient of variation was <8.6%. The accuracy and reliability of the icELISA were verified using liquid chromatography-tandem mass spectrometry. These results demonstrated that the developed icELISA is a useful and reliable tool.

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.

Establishment of enzyme-linked immunosorbent assay for aristolochic acid.

In recent years, the nephrotoxicity and carcinogenicity of aristolochic acid have attracted worldwide attention, and the traditional Chinese medicine containing this ingredient has been banned in many places, affecting the TCM industry. To meet this challenge, researchers have developed various detection methods, such as high-performance liquid chromatography, gas chromatography-mass spectrometry and thin-layer chromatography. A rapid detection method must therefore be developed to ensure safety. A polyclonal antibody capable of recognizing aristolochic acid was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the amount of aristolochic acid in the sample to be measured. Methods Using 1-(4-chlorophenyl) cyclobutylamine as a hapten, immunogens and coating antigens were obtained by coupling with bovine serum albumin (BSA) and chicken ovalbumin (OVA) using the active ester method. UV scanning confirmed the successful coupling of the conjugate, and New Zealand white rabbits were immunized. The obtained antibody serum was screened for the best antibody by ic-ELISA detection. Use the chessboard method to determine three optimal combinations of original coating concentration and antibody dilution ratio, establish a standard curve for each combination to obtain the best combination, and establish a rapid detection method. Finally, the standard aristolochic acid A was added to the purchased apple vinegar and canned coffee for recycling experiments to verify the detection method.By changing the antigen antibody concentration, the antibody showed the highest sensitivity to aristolochic acid standard at the original coating, 1000-fold dilution, IC50 of 24.88 ng/mL, limit of detection IC10 of 3.19 ng/mL, and detection range IC20-IC80 of 6.81-90.91 ng/mL. The recovery experiments under this conditions yielded a recovery rate of 92%-105%, within reasonable limits, indicating the success of the ELISA rapid detection method. Conclusion The enzyme-linked immunoassay method established in this paper can quickly detect the content of aristolochic acid in the sample to be tested, and the antibody prepared by this method has good broad-spectrum and can detect other aristolochic acid, such as aristolochic acid A, aristolochic acid B, aristolochic acid C, and aristolochic acid D.

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.

Computerized analysis of haptens for the ultrasensitive and specific detection of Pyriftalid.

Pyriftalid (Pyr) is one of the most commonly used herbicides and due to its widespread and improper use, it has led to serious pollution of groundwater, soil and other ecosystems, threatening human health. A rapid method to detect Pyr was urgently needed. A high specific monoclonal antibody (mAb) against Pyr with IC50 values of 4.7 ng/mL was obtained by mAb screening technique and method with enhanced matrix effect. The study firstly proposed colloidal gold immunochromatographic test strips (CGIA) for Pyr, which enables rapid qualitative and quantitative determination of a large number of samples anytime and anywhere, so as to effectively monitor Pyr in environment and grain samples. Based on the properties of the desired Pyr antibody, the hapten Pyr-hapten-4 with high structural similarity to Pyr molecule, similar electrostatic potential distribution, and the ability to expose Pyr functional groups was screened out from five different Pyr haptens, which was consistent with mouse antiserum test. The CGIA quickly analyze the Pyr content in positive samples such as water samples, soil samples, paddy samples, brown rice samples within 10 min, the LOD for Pyr by CGIA as low as 1.84 ng/g, the v LOD value as low as 6 ng/g, and the extinction value as low as 25 ng/g. The content of positive samples detected by CGIA was consistent with the quantitative results of LC-MS/MS, the relative accuracy was within the range of 97-103 %. The recovery rate range for Pyr by CGIA was 92.0-99.7 %, and the coefficient of variation was between 1.30-8.56 %. It indicated Pyr-targeted CGIA test strip was an efficient and fast detection method to detect real environment and food samples.

Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Evaluation of a hapten conjugate vaccine against the "zombie drug" xylazine.

Xylazine has emerged as a primary adulterant in fentanyl, exacerbating the complexity of the opioid crisis. Yet, there is no approved drug that can reverse xylazine's pathophysiology. As a prelude to monoclonal antibodies being assessed as a viable therapeutic, a vaccine inquiry was conducted evaluating the immune response in reversing xylazine induced behavior effects.

Central Chirality and Axial Chirality Recognition of the Enantioselective Antibodies to Herbicide Metolachlor.

Enantioselective antibodies have emerged as efficient tools in the field of chiral chemical detection and separation. However, it is complicated to obtain a highly stereoselective antibody due to the unclear recognition mechanism. In this study, the hapten of metolachlor was synthesized and enantio-separated. The absolute configuration of the four haptens obtained was identified by the computed and experimental electronic circular dichroism comparison. Five polyclonal antibodies against the Rac-metolachlor and its enantiomers were generated by immunization. The cross-activity of all the 5 antibodies with 44 structural analogues, including metolachlor enantiomers, was tested. It demonstrated that antibodies have higher specificity to recognize central chirality than axial chirality. Especially, αRR-MET-Ab exhibited excellent specificity and stereoselectivity. Accordingly, 3D-QSAR models were constructed and revealed that paired stereoisomers exhibited opposite interactions with the antibodies. It is the first time that the antibodies against four stereoisomers were prepared and analyzed, which will be conducive to the rational design of the stereoselective antibodies.