Hapten Conjugates Research Articles

A methamphetamine vaccine using short monoamine and diamine peptide linkers and poly-mannose

Methamphetamine (METH) substance use disorder is a long-standing and ever-growing public health concern. Efforts to develop successful immunotherapies are ongoing with vaccines that generate strong antibody responses are an area of significant research interest. Herein, we describe the development of a METH Hapten conjugate vaccine comprised of either two short-length peptides as linkers and mannan as an immunogenic delivery carrier. Initially, Hapten 1 (with a monoamine linker) and Hapten 2 (with a diamine linker) were synthesised. Each step of the Hapten synthesis were characterized by LC-MS and purified by Flash Chromatography and the identity of the purified Haptens were confirmed by 1H NMR. Haptens were conjugated with mannan (a polymannose), and conjugation efficiency was confirmed by LC-MS, TLC, 1H NMR, and 2,4 DNPH tests. The immunogenic potential of the two conjugated vaccines were assessed in mice with a 3-dose regimen. Concentrations of anti-METH antibodies were measured by enzyme-linked immunosorbent assay. All the analytical techniques confirmed the identity of Hapten 1 and 2 during the synthetic phase. Similarly, all the analytical approaches confirmed the conjugation between the Haptens and mannan. Mouse immunogenicity studies confirmed that both vaccine candidates were immunogenic and the vaccine with the monoamine linker plus adjuvants induced the highest antibody response after the second booster.

A two-dose regimen of Qβ virus-like particle-based vaccines elicit protective antibodies against heroin and fentanyl.

Opioid overdoses and the growing rate of opioid use disorder (OUD) are major public health concerns, particularly in the United States. Current treatment approaches for OUD have failed to slow the growth of the opioid crisis. Opioid vaccines have shown pre-clinical success in targeting multiple different opioid drugs. However, the need for many immunizations can limit their clinical implementation. In this study, we investigate the development of novel opioid vaccines by independently targeting fentanyl and the active metabolites of heroin using a bacteriophage virus-like particle (VLP) vaccine platform. We establish the successful conjugation of haptens to bacteriophage Qβ VLPs and demonstrate immunogenicity of Qβ-fentanyl, Qβ-morphine, and Qβ-6-acetylmorphine in animal models after one or two immunizations. We show that in independently or in combination, these vaccines elicit high-titer, high-avidity, and durable antibody responses. Moreover, we reveal their protective capacities against heroin or fentanyl challenge after two immunizations. Overall, these findings establish Qβ-VLP conjugated vaccines for heroin and fentanyl as very promising opioid vaccine candidates.

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.

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.

Immunotechniques for the Group Determination of Macrolide Antibiotics Traces in the Environment Using a Volume-Mediated Sensitivity Enhancement Strategy.

Macrolide antibiotics, which are effective antimicrobial agents, are intensively used in human and veterinary medicine, as well as in agriculture. Consequently, they are found all over the world as environmental pollutants, causing harm to sensitive ecological communities and provoking a selection of resistant forms. A novel azithromycin derivative, which was used as hapten conjugate, ensured the group immunorecognition of six major macrolide representatives (105-41%), namely erythromycin, erythromycin ethylsuccinate, clarithromycin, roxithromycin, azithromycin, and dirithromycin in a competitive immunoassay based on anti-clarithromycin antibodies. The heterologous hapten-based ELISA format resulted in a 5-fold increase in sensitivity, with an IC50 value of 0.04 ng/mL for erythromycin. In this study, we proposed an underexploited strategy in an immunoassay field to significantly improve the detectability of analytes in environmental samples. Unlike most approaches, it does not require special enhancers/amplifiers or additional concentration/extraction procedures; instead, it involves analyzing a larger volume of test samples. A gradual volume increase in the samples (from 0.025 to 10 mL) analyzed using a direct competitive ELISA, immunobeads, and immunofiltration assay formats based on the same reagents resulted in a significant improvement (more than 50-fold) in assay sensitivity and detection limit up to 5 and 1 pg/mL, respectively. The suitability of the test for detecting the macrolide contamination of natural water was confirmed by the recovery of macrolides from spiked blank samples (71.7-141.3%). During 2022-2023, a series of natural water samples from Lake Onega and its influents near Petrozavodsk were analyzed, using both the developed immunoassay and HPLC-MS/MS. The results revealed no contamination of macrolide antibiotic.

Development of ic-ELISA and colloidal cold-based immunochromatographic assay for red 2G detection in fruit drinks, red wine, and yoghurts

Red 2G (R2G), a cheap industrial colorant, cannot be added to food. An anti-R2G monoclonal antibody (mAb) was prepared by immunizing mice with the conjugate of R2G hapten and protein, which based on the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold-based immunochromatographic assay (CG-ICA) methods were used to determine R2G in fruit drinks, red wine, and yoghurts. A standard curve of the developed ic-ELISA showed that the IC50 of the anti-R2G mAb was 1.02 ng/mL, and limit of detection value (LOD) was 0.21 ng/mL. For the CG-ICA developed, the visual limit of detection values (vLOD) were 2 ng/mL and cut-off values of 100 ng/mL in samples. The results indicated that these two methods could be used to quickly detect R2G in fruit drinks, red wine, and yoghurts.

Development of an electrochemical immunosensor for the determination of molinate by using phages labeled with CdS nanocrystals as a novel strategy to signal amplification

Research on immunoassays and immunosensors is currently focused mainly on the development of strategies to amplify the resulting signal. In this sense, the development of new hapten tracers that allow simpler, more sensitive, and more reliable immunoassays is desirable. Therefore, we present the development of an electrochemical immunosensor using viral particles (phages) bound with dozens of CdS nanocrystals (CdS NCs) as an outperforming alternative to conventional competitive hapten conjugates. This allows us to obtain an multiplicator effect in signal obtained. Therefore, as proof of concept, an electrochemical immunosensor was developed for the detection of the molinate in river water samples, based on a noncompetitive immunoassay using recombinant M13 phage particles labeled with CdS NCs (CdS NCs-phage). The phage particles have on its surface peptides that recognize the molinate/antibody immune complex. Molinate determination was performed by square wave anodic stripping voltammetry of Cd2+ release from CdS NCs. The limit of detection and the midpoint sensitivity obtained were 34 pg mL-1 and 0.43 ng mL-1, respectively. In addition, other very good analytical parameters were obtained, where percent coefficient of variation (%CV) between 5 % and 25 %, and recovery percentages close to 99 % were found. Results obtained were compared with those determined by HPLC-UV visible, obtaining an excellent correlation between both methods. This electrochemical immunosensor was successfully used to analyze molinate in river water samples without pre-treatment, obtaining excellent recoveries.

Monoclonal antibody-based immunoassay for the specific quantification of licochalcone A: an active chalcone in licorice

ABSTRACT Licochalcone A (LicoA), which is found in the root of Chinese licorice (Glycyrrhiza inflata Batalin), has been reported as an effective anti-microbial and anti-inflammation and was approved for the treatment of rosacea and acne in clinical therapeutic. To develop a monoclonal antibody against LicoA, a mannich reaction hapten conjugate was used for immunization, followed by the hybridoma technique. Enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of LicoA was developed using a constructed antibody. The assay validation results were highly specific for the target compound, but minimally cross-reactive with the structure-related substances. After optimal conditions, the detection limit was 4.32 ng/mL and the quantification limit was 6.84–107.21 ng/mL. As a result, the developed ELISA was applied to determine the concentration of LicoA in raw licorice and marketed samples. This assay will aid in the quality control of licorice and derived products, as it can be used to identify plant species and determine bioactive markers.

Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines.

In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)–hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT–hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT–hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness.

Chemical Synthesis of Antibody-Hapten Conjugates Capable of Recruiting the Endogenous Antibody to Magnify the Fc Effector Immunity of Antibody for Cancer Immunotherapy.

Monoclonal antibodies (mAbs) with enhanced effector functions in cancer immunotherapy, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), could improve the clinical performance. Here, we develop an mAb-hapten conjugate strategy to augment the mAb effector functions with the engagement of endogenous antibodies. An "off-the-shelf" mAb, rituximab, is site-specifically conjugated with the rhamnose (Rha) hapten to generate rituximab-Rha conjugates. The octopus-like conjugates could recruit anti-Rha antibodies onto the cancer cell surface and further form an immune complex that is able to provide multivalent Fc domains to interact with immune cells or complement protein C1q, leading to magnified ADCC and CDC simultaneously. One optimal conjugate R2 with PEG2 as a linker exhibits the most potent in vitro cancer cell killing activity and significant in vivo antitumor efficacy in a xenograft model. This is a general and cost-effective approach to generate mAb with improved effector functions that may have broad applications.

Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library.

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development.

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.

Reprint of: Biotechnologies and the future of opioid addiction treatments

The future of treatment—including addiction treatment—is biotechnological. Depot injections, agonist/antagonist implants, deep brain stimulation, and hapten conjugate vaccines are hailed by researchers and pharmaceutical manufacturers as medicine's best hope to minimize illicit use, to decrease risk of overdose and painful withdrawal, and to prevent diversion of medicines to illicit markets. Marketing and use of new technologies reveal old tensions framing concepts of addiction and its treatment: between medical condition and disorder of the will, between criminal justice and health, and between patient choice and system control. Using the examples of depot naltrexone and implantable and injectable buprenorphine in the U.S., this essay considers the arc of long-acting opioid treatment and implications for the future. These include the rise of Vivitrol courts and “carceral prescription”—where criminal justice systems mandate medicine to lock up brain receptors much as they might lock up people themselves—as well as use of buprenorphine formulations positioned as increasing both patient benefit and provider control. We also consider lessons from debates on long-acting contraceptive technologies such as Norplant and Depo-Provera. While multiple new long-acting formulations are under development, success will be determined less by characteristics of particular formulations and more by whether or not the new technologies are accompanied by a new ethics of addiction treatment that emphasizes therapeutic alliance, concordance over compliance, and a genuine commitment to allowing patients the ability to narrate and be believed in their descriptions of their treatment experiences.

Development of Fluorescence Polarization Immunoassay for Imidacloprid in Environmental and Agricultural Samples.

A fluorescence polarization immunoassay (FPIA) for the determination of imidacloprid (IMI) was developed with advantages of simple operation and short assay time. The haptens of IMI, acetamiprid (ACE), and thiamethoxam (THI) were conjugated with fluorescein isothiocyanate ethylenediamine (EDF) and 4′-Aminomethyl fluorescein (AMF), respectively, to prepare six fluorescence tracers. The conjugation of IMI hapten and EDF (IMI-EDF) was selected to develop the FPIA due to the largest fluorescent polarization value increase in the presence of anti-IMI monoclonal antibody. Under the optimum condition, the limit of detection, 50% inhibition concentration and detection range of the FPIA were 1.7, 4.8, and 1.7–16.3 μg/L, respectively. The cross-reactivities (CRs) with the analogs of IMI were negligible except for imidaclothiz with CR of 79.13%. The average recovery of spiked paddy water, corn and cucumber samples were 82.4–118.5% with the RSDs of 7.0–15.9%, which indicated the FPIA had good accuracy. Thus, the developed FPIA was a potential tool for the rapid and accurate determination of IMI in agricultural and environmental samples.

Biotechnologies and the future of opioid addiction treatments

The future of treatment—including addiction treatment—is biotechnological. Depot injections, agonist/antagonist implants, deep brain stimulation, and hapten conjugate vaccines are hailed by researchers and pharmaceutical manufacturers as medicine's best hope to minimize illicit use, to decrease risk of overdose and painful withdrawal, and to prevent diversion of medicines to illicit markets. Marketing and use of new technologies reveal old tensions framing concepts of addiction and its treatment: between medical condition and disorder of the will, between criminal justice and health, and between patient choice and system control. Using the examples of depot naltrexone and implantable and injectable buprenorphine in the U.S., this essay considers the arc of long-acting opioid treatment and implications for the future. These include the rise of Vivitrol courts and “carceral prescription”—where criminal justice systems mandate medicine to lock up brain receptors much as they might lock up people themselves—as well as use of buprenorphine formulations positioned as increasing both patient benefit and provider control. We also consider lessons from debates on long-acting contraceptive technologies such as Norplant and Depo-Provera. While multiple new long-acting formulations are under development, success will be determined less by characteristics of particular formulations and more by whether or not the new technologies are accompanied by a new ethics of addiction treatment that emphasizes therapeutic alliance, concordance over compliance, and a genuine commitment to allowing patients the ability to narrate and be believed in their descriptions of their treatment experiences.

Determination of an organophosphate pesticide using antibody immobilised hybrid nanocomposites

ABSTRACT In this study, the malathion specific polyclonal antibodies (Abs) were raised for the fabrication of an electrochemical immunosensor using poly (3,4-ethylenedioxythiophene) (PEDOT) and carboxylated multiwalled carbon nanotubes (c-MWCNTs) nanocomposites. The conjugation of hapten (malathion diacid) with bovine serum albumin (BSA) through N-hydroxysuccinimide (NHS) ester method was optimised for the generation of Abs. New Zealand White (NZW) male rabbits were immunised subcutaneously for the development of Abs followed by characterisation and purification. The purified Abs were covalently immobilised on nanocomposites deposited onto fluorine tin oxide (FTO). Based on immunoreaction between malathion and anti-malathion Abs, the DPV peak current was found to be inversely proportional to increase in malathion concentration. The detection limit was 1.1 fM under all the optimised experimental parameters. The immunosensor was found to be specific as low current response was observed for malathion only in comparison to other interferents. Malathion recoveries from the spiked sample were found to be superior via immunosensor than HPLC.

Making Weak Antigens Strong: Cross-Linking Peptides to KLH with Maleimide

Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. KLH-hapten conjugates are commonly used in antibody generation in a variety of hosts such as mice, rats, and rabbits. Because KLH is harvested from mollusks, it is physiologically distant from mammalian species and less likely to produce antibodies that cross-react mammalian antigens. Maleimide activation of carrier proteins makes it possible to conjugate sulfhydryl-containing haptens, and hence this chemistry is widely used for conjugating KLH with haptens.

Hapten-mediated recruitment of polyclonal antibodies to tumors engenders antitumor immunity

Uptake of tumor antigens by tumor-infiltrating dendritic cells is limiting step in the induction of tumor immunity, which can be mediated through Fc receptor (FcR) triggering by antibody-coated tumor cells. Here we describe an approach to potentiate tumor immunity whereby hapten-specific polyclonal antibodies are recruited to tumors by coating tumor cells with the hapten. Vaccination of mice against dinitrophenol (DNP) followed by systemic administration of DNP targeted to tumors by conjugation to a VEGF or osteopontin aptamer elicits potent FcR dependent, T cell mediated, antitumor immunity. Recruitment of αGal-specific antibodies, the most abundant naturally occurring antibodies in human serum, inhibits tumor growth in mice treated with a VEGF aptamer–αGal hapten conjugate, and recruits antibodies from human serum to human tumor biopsies of distinct origin. Thus, treatment with αGal hapten conjugated to broad-spectrum tumor targeting ligands could enhance the susceptibility of a broad range of tumors to immune elimination.

Chemiluminescent detection integrated with microdialysis sampling for label-free measuring the affinity of ractopamine monoclonal antibody

A novel label-free protocol was developed for measuring the affinity between ractopamine and its monoclonal antibody (McAb) based on microdialysis (MD) on-line sampling integrated with flow injection chemiluminescent detection. In this study, unbound ractopamine was sampled by MD probe from homogeneous immunoreaction equilibrious systems, and then real-time quantified using flow injection chemiluminescent detection. The quantified concentrations of unbound ractopamine in the immunoreaction equilibrious systems were treated with Scatchard analysis and Klotz analysis to obtain the affinity constant. The mean recovery of MD probe for sampling ractopamine was found to be 24.2%. The affinity constants calculated by Scatchard analysis and Klotz analysis both were 1.0 × 106 M−1, indicating that the investigated ractopamine mouse McAb was a medium-affinity antibody. The result showed good agreement with that obtained from thiocyanate elution test. This protocol for measuring antibody affinity is free of protein conjugation of hapten and enzyme labeling of McAb. Therefore it avoids affinity decrease resulting from steric hindrance, occupancy of the antigenic determinants, and deactivation of antibody, which has been frequently encountered in the reported conventional approaches. It opens up a new pathway for direct measurement of antibody affinity with a facile, rapid, accurate and low-cost approach.

Broadening the Detection Spectrum of Small Analytes Using a Two-Antibody-Designed Hybrid Immunoassay.

The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb-hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.