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The generation of antibody-drug conjugates with optimal functionality depends on many parameters. These include binder epitope, antibody format, linker composition, conjugation site(s), drug-to-antibody ratio, and conjugation method. The production of matrices that cover all possible parameters is a major challenge in identifying optimal antibody-drug conjugates. To address this bottleneck, we adapted our Format Chain Exchange technology (FORCE), originally established for bispecific antibodies, toward the generation of binder-format-payload matrices (pair-FORCE). Antibody derivatives with exchange-enabled Fc-heterodimers are combined with payload-conjugated Fc donors, and subsequent chain-exchange transfers payloads to antibody derivatives in different formats. The resulting binder-format-conjugate matrices can be generated with cytotoxic payloads, dyes, haptens, and large molecules, resulting in versatile tools for ADC screening campaigns. We show the relevance of pair-FORCE for identifying optimal HER2-targeting antibody-drug conjugates. Analysis of this matrix reveals that the notion of format-defines-function applies not only to bispecific antibodies, but also to antibody-drug conjugates. Designing optimal antibody-drug conjugates (ADCs) involves screening many complex parameters. Here, the authors present a payload-coupled chain-exchange technology for efficient ADC matrix production, demonstrating its power in designing ADCs targeting HER2. [ABSTRACT FROM AUTHOR]

The synthesis of a hapten and antigen for the preparation of a monoclonal antibody (mAb) for buprofezin is described. The recognition mechanism of hapten and buprofezin by monoclonal antibodies (mAb-19F2) is described. The effectiveness of the mAb-19F2 immunoassay technique was assessed, and the effective detection of buprofezin in tea samples was achieved through the establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA). The mAb-19F2 subtype was IgG1, with an IC50 of 1.8 ng/mL and a linear range (IC20-IC80) of 0.6-5.4 μg/L, and had a cross-reaction rate of less than 0.18% with 29 other pesticides (neonicotinoids and insect growth regulators). The study identified π-π stacking interactions between hapten and TYR-61 at the mAb-19F2 site and alkyl/phosphate interactions with TRP-105 and ARG-103. The ic-ELISA had an IC50 of 12.9 ng/mL in green tea and 5.65 ng/mL in black tea, with a recovery rate of 92.4%-101.0% and RSD of 2.1%-4.8%. The GICA had a limit of detection (LOD) was 500 ng/mL, with the complete disappearance of the test lines visible to the naked eye. The limit of quantitation (LOQ, IC20) was determined to be 16.8 ng/mL. Additionally, the developed GICA showed no cross-reactivity with neonicotinoid pesticides. The recovery rate of tea spiked recovered samples was 83.6%-92.2%, with an RSD of 5.3%-12.6%, and the results were consistent with the LC/MS method. This study is important for the real-time detection of buprofezin residues to ensure food safety and human health.

In the past two to three decades, synthetic glycoconjugate vaccines have shown great potential in the prevention of severe infections and protection of high‐risk populations. Conjugation of synthetic oligosaccharide haptens to carrier proteins is the key step for the vaccine preparation. In this review, the conjugation methods currently used in the synthesis of glycoconjugate vaccines from synthetic/homogeneous oligosaccharide haptens are summarized with the focus on the reaction conditions (pH and sugar/protein ratio) and performance. This information can help researchers choose the appropriate conjugation methods. Further research directions toward site‐specific conjugations and fully homogeneous glycoconjugate vaccines are also discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

Introduction. The combination of atopic dermatitis (AD) with allergic contact dermatitis (ACD) or the occurrence of ACD on the background of atopic dermatitis is called the overlap syndrome. Studies have demonstrated several reasons why patients with AD have a similar or even increased risk of developing ACD compared to those without AD. Allergens and haptens are trigger factors in a group of patients with AD and ACD overlap syndrome. The aim of the study. To confirm the diagnosis of ACD in a group of patients with AD – diagnose the overlap syndrome and analyze which allergens and haptens were the trigger factors in this group. Materials and methods. To confirm IgE-dependent sensitization in atopic dermatitis, skin prick tests or determination of specific IgE in blood serum were performed. Skin patch tests (European series S-1000) were performed to determine the mechanisms of delayed-type hypersensitivity. Results. It was found that the highest specific weight of positive allergic reactions has been recorded in response to the following allergens: ticks, ticks/ambrosia, birch and mold. The absolute majority of patients demonstrated positive specific IgE-dependent sensitization to Dermatophagoides pteronyssinus and Dermatophagoides farinae – 24 (50%), in turn, on Ambrósia – 14 (29.2%), and on Alternaria alternata – 8 (16.7%). Also, the reaction was most often recorded to haptens: cobalt, nickel, formaldehyde, PPD, textile dyes. Deterioration of the clinical course and shortening of AD remission periods were observed due to the formation of ACD against the background of impaired skin barrier function and the presence of chronic immune inflammation. Conclusions. Patients with AD are more often diagnosed with ACD, which predictably worsens the course of AD. Patients with confirmed overlap syndrome "AD + ACD" most often show reactions to haptens: Cobalt, Nikel, Formaldehyde, PPD, Textile dye mix – and in the vast majority to 2 haptens or more in one patient.

In this study, a previously unreported 3-((4-(isopropylamino)-6-(methylthio)-1,3,5-triazin-2-yl) amino) butyric acid hapten was designed and synthesized. This maximized the exposure of the antigen-determinant isopropyl of prometryn to the immune system in animals to induce the production of anticipated highly specific anti-prometryn antibodies. The hapten has a molecular weight of 285.37 Da. The compound was confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H NMR), nuclear magnetic resonance carbon spectroscopy (13C NMR), and high-resolution mass spectrometry (HRMS). By using the active ester approach, immunogens and coated antigens were created. Bovine serum albumin (BSA) was used as an immunogen, along with the successfully produced hapten, to immunize mice. The IC50 value of mouse monoclonal anti-prometryn antibody (mAb) 7D4 (the quantity of analyte that generated 50% prometryn inhibition) was 3.9 ng/mL. The anti-prometryn mAb was of the IgG1 subclass. The IC20 (80% binding level (B/B0) of prometryn)-IC80 (20% binding level (B/B0) of prometryn) range of the anti-prometryn monoclonal antibody standard curve working range was 0.9-18.1 ng/mL. The prepared mAb has good characteristics because it can specifically recognize prometryn, and the cross-reaction rates for ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hapten structure proposed can serve as an additional tool for modulating selectivity in detecting triazines.

AbstractImmunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection. [ABSTRACT FROM AUTHOR]

Background: Tissue‐resident memory T (TRM) cells are detrimental in allergic contact dermatitis (ACD), in which they contribute to the chronicity and severity of the disease. Methods: We assessed the impact of a standard topical corticosteroid (TCS) treatment, triamcinolone acetonide (TA), on the formation, maintenance and reactivation of epidermal TRM cells in a preclinical model of ACD to 2,4‐dinitrofluorobenzene. TA 0.01% was applied at different time points of ACD response and we monitored skin inflammation and tracked CD8+ CD69+ CD103+ TRM by flow cytometry and RNA sequencing. Results: The impact of TA on TRM formation depended on treatment regimen: (i) in a preventive mode, that is, in sensitized mice before challenge, TA transiently inhibited the infiltration of effector T cells and the accumulation of TRM upon hapten challenge. In contrast, (ii) in a curative mode, that is, at the peak of the ACD response, TA blocked skin inflammation but failed to prevent the formation of TRM. Finally, (iii) in a proactive mode, that is, on previous eczema lesions, TA had no effect on the survival of skin TRM, but transiently inhibited their reactivation program upon allergen reexposure. Indeed, specific TRM progressively regained proliferative functions upon TA discontinuation and expanded in the tissue, leading to exaggerated iterative responses. Interestingly, TRM re‐expansion correlated with the decreased clearance of hapten moieties from the skin induced by repeated TA applications. Conclusions: Our results demonstrate that TCS successfully treat ACD inflammation, but are mostly ineffective in impeding the formation and expansion of allergen‐specific TRM, which certainly restricts the induction of lasting tolerance in patients with chronic dermatitis. [ABSTRACT FROM AUTHOR]

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Quenchbody (Q-body) is a unique, reagentless, fluorescent antibody whose fluorescent intensity increases in an antigen-concentration-dependent manner. Q-body-based homogeneous immunoassay is superior to conventional immunoassays as it does not require multiple immobilization, reaction, and washing steps. In fact, simply mixing the Q-body and the sample containing the antigen enables the detection of the target antigen. To date, various Q-bodies have been developed to detect biomarkers of interest, including haptens, peptides, proteins, and cells. This review sought to describe the principle of Q-body-based immunoassay and the use of Q-body for various immunoassays. In particular, the Q-bodies were classified from a structural perspective to provide useful information for designing Q-bodies with an appropriate objective. [ABSTRACT FROM AUTHOR]

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD. • FITC-induced contact hypersensitivity response was attenuated in atopic dermatitis. • Attenuated contact hypersensitivity in atopic dermatitis may be hapten-independent. • DC migration after hapten sensitization was not altered in atopic dermatitis. • Tolerogenic phenotype of hapten-bearing DC was induced in atopic dermatitis. [ABSTRACT FROM AUTHOR]

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins to threaten humans, animals and environment. Ustilaginoidins are produced by Villosiclava virens, the rice false smut pathogen. To prepare antibodies for quantitatively analyzing ustilaginoidins in rice samples, hemiustilaginoidins D and F from the laccase gene deficiency mutant of V. virens respectively reacted with diazonium 4-aminobenzoic acid to obtain haptens with a carboxyl group, which further reacted with bovine serum albumin or ovalbumin to get their complete antigens. Two monoclonal antibodies (mAbs) designated as 4A12C6 and 5F4F6 were developed by immunization. The relationships between mAb sensitivity and 20 ustilaginoidins were described. 4A12C6 was chosen for further analysis as it could recognize main ustilaginoidins and was more sensitive than 5F4F6. The achieved indirect competitive enzyme-linked immunosorbent assay (icELISA) based on 4A12C6 had a half maximal inhibitory concentration (IC50) of 0.76 ng/mL and working range of 0.2–2.8 ng/mL to ustilaginoidin A. The results of ustilaginoidins-contaminated rice samples by icELISA detection were consistent with those determined by HPLC‒DAD detection. Therefore, we developed a new strategy to get haptens from the biosynthetic precursors with half structures of ustilaginoidins. The achieved icELISA was demonstrated as a convenient method to monitor ustilaginoidin content in rice samples, and showed that the contents of total ustilaginoidins from the rice cultivars with low resistance to rice false smut were more than those of high resistance cultivars.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

In this study, we synthesized two haptens similar in structure to nitrofen (NIT), and screened out five monoclonal antibodies with the ability to recognize NIT and bifenox (BIF) by competitive ELISA, with the lowest IC50 values of 0.87 ng mL−1 and 0.86 ng mL−1, respectively. The antibody 5G7 was selected to be combined with colloidal gold to establish a lateral flow immunochromatographic assay strip. This method was shown to qualitatively and quantitatively detect the residues of NIT and BIF in fruit samples. The visual limits of detection for qualitative detection were 5 μg kg−1 and 10 μg kg−1 for NIT and BIF, respectively. The calculated limits of detection for quantitative detection were 0.75 μg kg−1, 1.77 μg kg−1 and 2.55 μg kg−1 respectively, for nitrofen in orange, apple and grapes, and 3.54 μg kg−1, 4.96 μg kg−1 and 5.26 μg kg−1, respectively, for bifenox. Thus the strip assay could be used for rapid analysis of fruit samples. [ABSTRACT FROM AUTHOR]

Diphencyprone (DPCP) is a hapten that causes a delayed‐type hypersensitivity reaction when applied topically. It has clinical uses in the treatment of various conditions such as melanoma metastases, warts, and alopecia areata, but the mechanisms are currently not well understood in humans. To further characterize the immunologic effects of DPCP, the authors performed a proteomic analysis of normal skin of eight healthy volunteers following a single application of DPCP and compared them with placebo‐treated skin from the same volunteers. A total of 96 proteins were examined using the Olink immuno‐oncology panel at 3 days (peak response), 14 days (partially resolved response), and 120 days (completely resolved response). Our analysis revealed significant upregulation of markers of immune cell activation (interleukin [IL] 8), vascular and tissue remodeling (matrix metallopeptidase 12 [MMP12], nitric oxide synthase 3 [NOS3]), antineoplastic markers (granzyme B [GZMB]), and the Th1 axis (interferon gamma [IFNG], chemokine (C‐X‐C motif) ligand [CXCL] 9, CXCL10, CXCL11) at days 3 and 14 compared with placebo (p < 0.05). In addition, several negative regulators of immune function such as programmed cell death 1 (PD1), programmed cell death ligand 1 (PDL1) (p < 0.001), and lymphocyte activation gene 3 (LAG3) (p < 0.05) were significantly upregulated at days 3 and 14. This induction of negative regulators may explain the seemingly paradoxical therapeutic benefits of DPCP in autoimmune conditions such as alopecia areata. The current analysis also indicated IL‐4 upregulation only at day 3, followed by IL‐12 upregulation only at day 14, suggesting a transient Th2 response followed by Th1 polarization. Overall, these data suggest a complex and evolving immunological delayed‐type hypersensitivity response to a single application of DPCP over time. Future proteomic studies of samples from patients with melanoma metastases, warts, and alopecia areata treated long term with DPCP are needed to further evaluate its pharmacologic mechanisms. [ABSTRACT FROM AUTHOR]

Background Patch testing is an important investigation when dermatitis is unresponsive to, or worsened by, topical corticosteroid treatment. There is a balance to be struck between testing too many allergens, which is expensive, time consuming and risks causing sensitization, and testing too few, which risks missing the diagnosis. The current British Society for Cutaneous Allergy (BSCA) corticosteroid series comprises eight allergens and was last updated in February 2007. Aim To review and update the BSCA corticosteroid series. Methods We retrospectively analysed data from 16 patch test centres in the UK and Ireland for all patients who were patch tested to a corticosteroid series between August 2017 and July 2019. We recorded the allergens tested, the number and percentage tested to a corticosteroid series and the number of positive results for each allergen. We identified the allergens that test positive in ≥ 0.1% of selectively tested patients. Results Overall, 3531 patients were tested to a corticosteroid series in the 16 centres. The number of allergens tested ranged from 7 to 18 (mean 10). The proportion of patch test patients who were tested to a corticosteroid series ranged from 1% to 99%. Six allergens in the 2017 BSCA series tested positive in ≥ 0.1% of patients. Nine allergens not in the BSCA corticosteroid series tested positive in ≥ 0.1% of patients. Conclusion This audit demonstrates the importance of regular review of recommended series and the significant variations in practice. The new BSCA corticosteroid series that we recommend contains 13 haptens, with the addition of the patient's own steroid creams as appropriate. [ABSTRACT FROM AUTHOR]

The aim of the current study was to develop an in silico model to predict the sensitizing potential of cosmetic ingredients based on their physicochemical characteristics and to compare the predictions with historical animal data and results from "omics"-based in vitro studies. An in silico model was developed with the use of WEKA machine learning software fed with physicochemical and structural descriptors of haptens and trained with data from published epidemiological studies compiled into estimated odds ratio (eOR) and estimated attributable risk (eAR) indices. The outcome classification was compared to the results of animal studies and in vitro tests. Of all the models tested, the best results were obtained for the Naive Bayes classifier trained with 24 physicochemical descriptors and eAR, which yielded an accuracy of 86%, sensitivity of 80%, and specificity of 90%. This model was subsequently used to predict the sensitizing potential of 15 emerging and less-studied haptens, of which 7 were classified as sensitizers: cyclamen aldehyde, N,N-dimethylacrylamide, dimethylthiocarbamyl benzothiazole sulphide, geraniol hydroperoxide, isobornyl acrylate, neral, and prenyl caffeate. The best-performing model (NaiveBayes eAR, 24 parameters), along with an alternative model based on eOR (Random Comittee eOR, 17 parameters), are available for further tests by interested readers. In conclusion, the proposed infotechnomics approach allows for a prediction of the sensitizing potential of cosmetic ingredients (and possibly also other haptens) with accuracy comparable to historical animal tests and in vitro tests used nowadays. In silico models consume little resources, are free of ethical concerns, and can provide results for multiple chemicals almost instantly; therefore, the proposed approach seems useful in the safety assessment of cosmetics. [ABSTRACT FROM AUTHOR]

Although dicamba has long been one of the most widely used selective herbicides, some U.S. states have banned the sale and use of dicamba because of farmers complaints of drift and damage to nonresistant crops. To prevent illegal use of dicamba and allow monitoring of nonresistant crops, a rapid and sensitive method for detection of dicamba is critical. In this paper, three novel dicamba haptens with an aldehyde group were synthesized, conjugated to the carrier protein via a reductive-amination procedure and an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for dicamba was developed. The assay showed an IC50 of 0.874 ng/mL which was over 15 times lower than that of the conventional enzyme immunoassay. The immunoassay was used to quantify dicamba concentrations in field samples of soil and soybean obtained from fields sprayed with dicamba. The developed CLEIA showed an excellent correlation with LC-MS analysis in spike-and-recovery studies, as well as in real samples. The recovery of dicamba ranged from 86 to 108% in plant samples and from 105 to 107% in soil samples. Thus, this assay is a rapid and simple analytical tool for detecting and quantifying dicamba levels in environmental samples and potentially a great tool for on-site crop and field monitoring.

This article discusses the prevalence of contact sensitizers in cosmetics marketed as safe for pregnant women. The study analyzed leave-on and rinse-off products available in online drugstores in Poland and found that only 2.5% of the products did not contain haptens, which are substances that can cause allergic reactions. The most common haptens found in the products were perfume, tocopherol, and panthenol. The authors suggest that pregnant and breastfeeding women should carefully examine the composition of these products to minimize the risk of skin sensitization. [Extracted from the article]

Phage display technology is commonly applied for high‐throughput screening of single‐domain antibodies (sdAbs), and the problem of non‐specific adsorption caused by carrier proteins seriously affects the biopanning of single‐domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)‐functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR‐specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre‐incubation were applied to the biopanning process through single‐factor experiments. Results of flat colony counting showed that the phage output of AG‐ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR‐specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single‐domain antibodies to haptens. [ABSTRACT FROM AUTHOR]

Objectives Benzimidazoles (BZs) are commonly used for the treatment of soil-transmitted helminth infections in veterinary clinics; however, misuse and overdosing of BZs will cause residual problems and have the potential to damage human health through the food chain. Thus, the existence of BZs in foods needs more attention. This study aims to establish a broad-spectrum immunoassay for rapid detection and to simultaneously monitor BZs in milk. Materials and Methods Based on structure analysis, a 'zero epitope loss' strategy, which introduced a spacer arm into the imino group of the imidazole ring of albendazole, was first adopted for hapten modification to obtain an ultra-sensitive and broad-spectrum antibody. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for the detection of 18 BZs in milk sample with a single-step pretreatment. A quantitative structure–activity relationship model was constructed to interpret and predict the recognition. Results The antibody could recognize 20 BZs and the half-inhibitory concentrations ranged from 0.054 to 417.58 ng/mL, the limits of detection of icELISA ranged from 0.4 to 89.4 ng/mL, and the mean recovery rates ranged from 76.49% to 120.40%, with a coefficient of variation <20%. Substituent R1 of BZs was considered to be the main influencing factor for recognition, and the comparative molecular field analysis model (q 2=0.724, r 2=0.998) was finally chosen for further prediction. Conclusions The results indicated that the established icELISA could simultaneously identify 18 BZs, with good accuracy and precision, which was suitable for rapid detection of BZs in milk. [ABSTRACT FROM AUTHOR]

Folic acid (FA) and its other forms known as folates are small molecules vital for humans. The high demand for increasingly sensitive methods of measuring folate concentrations is due to the fact that abnormal levels of FA cause severe health disorders. Besides, folates are used as recognition molecules in targeted drug delivery. The majority of FA measuring techniques are rather expensive, laborious, sometimes not sufficiently sensitive and specific, and often employ consumables that are too costly to be single-use for routine medical diagnostics. Here, we present a procedure for transformation of a simple microscope cover glass slip without deposition of any metal or dielectric films into a cost-efficient chemosensor chip interrogated by spectral correlation interferometry for highly sensitive measurements of the concentration of small molecules, as well as a feasibility study of long-term monitoring of such molecules in a flow mode. The obtained chips were tested for folate detection. The highly specific and sensitive measurements can be performed in real-time in a wide dynamic range of 0.9–220,000 pM. The developed method and single-use consumables are promising for concentration measurements of low molecular weight substances in pharmaceuticals and in vitro diagnostics. [ABSTRACT FROM AUTHOR]

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 %. [Display omitted] • A computational chemical-assisted hapten screening strategy was used. • Four different haptens of AA and seven complete antigens were synthesized. • A monoclonal antibody that can specifically recognize both MAA and AA was prepared. • An ic-ELISA method which enables the simultaneous detection of MAA and AA residues in animal tissues was developed. [ABSTRACT FROM AUTHOR]

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 (IC 50) 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 (CCβ) 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. [Display omitted] • Computer-chemical visualization strategy was proposed for precise hapten design. • Duplex-recognition antibody toward bisoxatin adulterants was first reported. • Molecular mechanism governing the antibody recognition spectrum was elucidated. • Visual immunoassay was developed for authenticating bisoxatin adulterants in food. [ABSTRACT FROM AUTHOR]

Phenol contamination in ambient air is a factor which creates health risks for children living in a zone influenced by emissions from a ferrous metallurgy enterprise. Our research goal was to assess specific hapten sensitization in children living under excessive aerogenic exposure to phenol. We performed hygienic assessment of ambient air pollution on territories of pre-school children facilities located at various distances from a zone influenced by the examined enterprise (1 km and 5 km were the test territories No. 1 and 2 accordingly) which emitted phenol thus creating elevated concentrations of the chemical in ambient air being higher than single maximum MPC. Ambient air on a selected reference territory was not polluted with any industrial emissions. The test group No. 1 was made of 99 children (the test territory No. 1); the test group No. 2, 92 children (the test territory No. 2); and the reference group, 95 children (the reference territory). We analyzed phenol contents and levels of IgG specific to phenol in blood of all the examined children. Phenol concentrations in ambient air were higher than its permissible levels on the test territory No. 1, 1.7 single maximum MPC, and the test territory No. 2, 1.1 single maximum MPC. We comparatively assessed phenol contents in blood of children from all three groups. The assessment revealed that children from the test group No. 1 had a hydroxybenzene concentration in their blood which was statistically significantly (р = 0.031) by 1.9 times higher than in blood of children from the reference group. Production of specific G class antibodies was higher than the upper limit of the physiological standard in 60 % and 36 % children living and attending a preschool children facility in zones located accordingly at the minimal and maximum distance an emission source. The research results indicate that a hapten-associated increase in the level of IgG specific to phenol in preschool children is associated with excessive phenol contamination creating a substantial burden on biological media (OR = 14.75; 95 % CI = 6.45–33.73; р < 0.05).

Active immunotherapy of cancer with therapeutic vaccines has been the subject of experimental and clinical studies for at least 50 years. Our approach has employed 1) autologous, human cancer cells because of extensive evidence that tumor rejection antigens may differ between multiple tumors of the same histology; 2) the immunopotentiating drug, cyclophosphamide; and 3) haptens, particularly dinitrophenyl. Multiple clinical trials in 455 patients with melanoma and ovarian cancer have shown that administration of haptenized vaccines at the proper dosage-schedule regularly induces T cell-mediated immunity to autologous tumor cells as measured by delayed-type hypersensitivity. Moreover, the vaccine causes changes in the tumor site suggestive of an immune reaction, including inflammation and infiltration with CD8+ T lymphocytes that are activated and produce cytokines. The T cell response is oligoclonal, and dominant Vβ families differ between patients. Studies of measurable metastases show clinically important tumor regression. Commercial development of this technology is clearly feasible.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant pathogens. Regulatory agencies cannot do the rigorous, expensive testing required to keep up with the volume of seafood shipments. Current rapid test kits for these drugs enable the increase in testing needed for adequate monitoring of food supply chains, but they lack a high degree of accuracy. To combat this, we set out to discover and engineer single‐domain antibodies (VHHs) that bind to small molecule antibiotics, and that can be used in rapid test kits. The small size, solubility, and stability of VHHs are useful properties that can improve the reliability and shelf‐life of test kits for these adulterants. Here, we report a novel anti‐chloramphenicol VHH (Chl‐VHH) with a disassociation constant of 57 nM. This was achieved by immunizing a llama against a chloramphenicol‐keyhole limpet hemocyanin (KLH) conjugate and screening for high affinity binders through phage display. The crystal structure of the bound‐VHH to chloramphenicol was key to identifying a mutation in the binding pocket that resulted in a 16‐fold improvement in binding affinity. In addition, the structure provides new insights into VHH‐hapten interactions that can guide future engineering of VHHs against additional targets. PDB Code(s): 7TJC; [ABSTRACT FROM AUTHOR]

The dysfunctionality of the protective skin barrier in psoriasis allows easier cutaneous penetration of various contact haptens; thus, such patients can develop allergic contact hypersensitivity as a comorbidity. Both skin conditions involve T-cell-mediated mechanisms. Dermatologists and allergists should consider assessing allergic contact cell-mediated hypersensitivity in selected psoriasis patients, especially those with palmoplantar psoriasis and who are refractory to topical treatments, and in patients with psoriasis, with or without arthritis, treated with biologics that present skin lesions clinically suggestive of contact dermatitis. [ABSTRACT FROM AUTHOR]

Exploring the proximity ligation-induced quasi-Y-DNA assembly (plq YDA) for creating label-free fluorescence biosensor might be intriguing based on DNA loop-hosted red Ag nanocluster (lr AgNC) as a signaling reporter. For proof-of-concept, a bivalent digoxigenin antibody (anti-Dig) with two identical binding sites was used as an analyte model, and it can specifically distinguish two Dig haptens that were separately modified in two modular split strands (SS1 and SS2) with complements each other and invading domains. The strong and robust anti-Dig-Dig binding guided the proximity ligation of SS1 and SS2 to construct geometric-structure plq YDA with a paired stem and two sticky linkers. After introducing a dsDNA duplex from a reporting strand (RS) with seven-polycytosine template (C 7) of lr AgNC and a blocking strand (BS), entropy-driven hybridization and cooperative displacement occurred. As a result, RS was rationally immobilized in plq YDA to crimp single-coil C 7 as a closed and extruded loop, which preferably favored the development of emissive lr AgNC when adding AgNO 3 and NaBH 4. By collecting the distinct fluorescence signal, the label- and enzyme-free assay of anti-Dig was readily achieved, exhibiting good specificity and high sensitivity. This would be the first example of our best understanding of integrating plq YDA and lr AgNC for applicable biosensing and bioanalysis. • An antibody-responsive fluorescence biosensor was created via specific ligand proximity ligation. • Sensitive, simplified and nonenzymatic detection of bivalent digoxigenin antibody was achieved. • Identical dual-site binding of two haptens modulated quasi-Y-DNA assembly for signal transduction. • Two co-localized splits were merged to initiate cooperative hybridization and strand displacement. • Red-emitting Ag nanocluster was stabilized in an extruded DNA loop as label-free signal reporter. [ABSTRACT FROM AUTHOR]

Tolperisone is a centrally acting muscle relaxant that has been used for the treatment of post-stroke spasticity and low back pain. Recently, the safety of tolperisone pharmaceutical products has been reassessed due to growing concerns over allergic adverse events. Reactive degradants of tolperisone may be responsible for these hypersensitivity reactions. By forming adducts with proteins, they may act as haptens that could evoke allergic reactions. The objective of this study was to examine the presence of these degradants in tolperisone pharmaceutical products and to assess their reactivity to elucidate their possible role in the pro-allergic effect of tolperisone. For this purpose, capillary electrophoresis UV detection (CE-UV) method was developed and validated for the quantification of degradants. A dual cyclodextrin system was applied to achieve the appropriate migration order enabling the analysis of 2-methyl-1-(4-methylphenyl)prop-2-en-1-one (MMP) and 1-(4-methylphenyl)propan-1-one (MMPO) in the presence of high concentrations of tolperisone. MMP was identified as the main degradant in forced degradation tests of the active pharmaceutical ingredient. Differences in MMP content of tolperisone products by different manufacturers have also been found, highlighting the role of formulation in their stability. High reactivity of MMP was demonstrated as rapid and almost complete adduct formation with cysteine was found. This degradant thus might be responsible for the allergic adverse effects of tolperisone even when it is present in trace amounts in tablets by readily reacting with proteins in vivo. • A CE-UV method was developed for the analysis of tolperisone degradants. • 2-Methyl-1-(4-methylphenyl)prop-2-en-1-one (MMP) was found as the main degradant. • High reactivity of MMP to cysteine was demonstrated. • MMP might be responsible for allergic adverse events associated with tolperisone. • Formulation of tolperisone products determines their stability and thus MMP content. [ABSTRACT FROM AUTHOR]

Objective: Unusual clinical course. Background: Leukocytoclastic vasculitis is a small-vessel vasculitis associated with infections, autoimmune disorders, and certain drugs, but it may also be idiopathic. Case Report: We report the case of a 37-year-old woman with no significant past medical history who presented with a chief concern of a full-body rash. Before the rash appeared, she had been treated for group A Streptococcus with amoxicillin and prednisone. An outpatient skin biopsy revealed findings concerning for early leukocytoclastic vasculitis. On admission, she had a diffuse palpable rash on the trunk and upper and lower extremities. Laboratory test results were notable for neutrophilic leukocytosis with a left shift, reticulocytosis with normal hemoglobin, thrombocytosis, and elevated ESR and CRP. An infectious diseases workup was negative, serum levels of complement C3 and C4 were normal, and no evidence of hemolysis was found on blood smear. Results of schistocytes review, LDH, and haptoglobin were not consistent with hemolysis, and IgG, IgA, and IgM were all within normal limits. The patient was initially started on antibiotics due to concern for bullous impetigo, but the treatment regimen was changed to steroids because IgA vasculitis and leukocytoclastic vasculitis were suspected. Biopsy results were received 1 week later and did not reveal definitive findings of acute leukocytoclastic vasculitis. Staining with antibodies to human IgG, IgA, IgM, C3, fibrinogen, and albumin was negative. Conclusions: Leukocytoclastic vasculitis can be triggered by penicillins, cephalosporins, sulfonamides, phenytoin, and allopurinol acting as haptens and stimulating an immune response, resulting in development of vasculitis. [ABSTRACT FROM AUTHOR]

There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABAA ) receptors. General synthetic approach toward two groups of analogues was developed. Screening of the resulting library of compounds by FLIPR or whole-cell voltage-clamp revealed that, despite the structural differences, some of the TETS analogues retained GABAA receptor inhibition; however, their potency was an order of magnitude lower. Antibodies raised in rabbits against some of the TETS analogues conjugated to protein recognized free TETS and will be used for the development of an immunoassay for TETS.

Tetramethylenedisulfotetramine (TETS, tetramine) is a formerly used and highly neurotoxic rodenticide. Its lethality, recent history of intentional use for mass poisoning, and the absence of a known antidote raise public health concerns. Therefore, rapid, high throughput, and sensitive methods for detection and quantification of TETS are critical. Instrumental analysis method such as GC/MS is sensitive but not rapid or high throughput. Therefore, an immunoassay selective to TETS was developed. The assay shows an IC50 of 4.5 ± 1.2 ng/mL, with a limit of detection of 0.2 ng/mL, comparable to GC/MS. Performance of the immunoassay was demonstrated by a recovery study using known concentrations of TETS spiked into buffer and human and mouse serum matrices giving recoveries in the range of 80-120%. The assay demonstrated good correlation in TETS recovery with established GC/MS analysis. The immunoassay was then used to quantify TETS concentration in the serum of mice exposed to 2× LD50 dose of TETS and to monitor kinetics of TETS clearance from blood over a short period of time. TETS concentration in the serum reached 150 ng/mL without significant change over 4 h post-treatment. Results obtained with the immunoassay had good correlation with GC/MS analysis. Overall, this immunoassay is an important tool to rapidly detect and quantify levels of TETS from biological samples with high sensitivity. The assay can be adapted to multiple formats including field or hospital use.

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. [Display omitted] • Qualitative and quantitative methods have been developed to monitor Pyr in actual samples. • Five different Pyr haptens were simulated computationally to calculate the functional similarity. • A Pyr CGIA was developed that could detect results by eyes within 10 min. • The CGIA detection results was consistent with that detected by LC-MS/MS. [ABSTRACT FROM AUTHOR]

To overcome limitations with respect to the low sensitivity of rapid immunoassay methods due to dilution of the extract in extraction solution, ultra-sensitive antibodies are needed. The linker-tethering site between the target and carrier protein, the length of the spacer arm, and the atomic charge of the linker-tethering site between the target and spacer arm in haptens design play a pivotal role in the preparation of ultra-sensitive antibodies. In this study, carbendazim (CBZ), a systemic broad-spectrum fungicidal pesticide, was used as model compound. 2-Aminobenzimidazole was chosen as the common skeletal structure and reacted with different groups, such as alkanes and urine groups, to form haptens H1 (previously reported), H2 (novel), and H3 (novel), respectively. Subsequently, eight monoclonal antibodies (mAbs) were generated using these three haptens, which exhibited notable variation in sensitivity. The half maximal inhibitory concentration (IC 50) value of mAb 4B11 based on hapten H3 against CBZ was 0.04 ng/mL, which was seven times better than that of mAb 5B10 (IC 50 value of 0.28 ng/mL) prepared using hapten H2 and 129 times better than that of mAb 6D5 (IC 50 value of 5.15 ng/mL) prepared using hapten H1. Finally, the optimal antigen–antibody combination was employed to establish a sensitive colloidal gold lateral flow immunoassay (CG-LFA) for CBZ detection in vegetables and fruits with convenient sample pretreatment. The developed CG-LFA shows promising practical utility and prospects for application due to its low limit of detection (0.10–0.18 ng/mL) and high recovery ratios (76.0%–96.1%) in six different samples. [Display omitted] • A novel hapten design strategy focuses on the link site and spacer arm length of the target compound. • An ultra-sensitive monoclonal antibody (mAb) 4B11 against carbendazim was prepared. • A sensitive colloidal gold lateral flow immunoassay in fruits and vegetables was developed. [ABSTRACT FROM AUTHOR]

We present the first synthesis of β-lactam-derived haptens, leveraging the principles of diversity-oriented synthesis to discover compounds for drug allergy in vitro testing. We designed, synthesised, and performed in vitro immunological evaluation on 18 structurally diverse haptens derived from β-lactam antibiotics. The antigens obtained with the synthesised haptens allow for the detection of specific anti-β-lactam immunoglobulins G and E. Excellent diagnostic sensitivity (83%) and specificity (100%) were achieved when the panel of antigens was tested against a cohort of 31 human serum samples using a multiplexed compact disc-based in vitro testing tool. We posit that adopting this strategy could aid β-lactam delabeling initiatives. [ABSTRACT FROM AUTHOR]