Hapten-carrier Conjugates Research Articles

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.

Highly selective monoclonal antibody-based lateral flow immunoassay for visual and sensitive determination of conazole fungicides propiconazole in vegetables

ABSTRACT The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g−1 and a linear range from 0.5 to 80 ng g−1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g−1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry.

Research Status of Antigen Synthesis Methods

Most haptens are small molecules compounds. Because of its small molecular mass itself alone can not induce an immune response, it is necessary to form combined with the carrier hapten-carrier protein conjugate can stimulate the body's immune response. In recent years, in-depth research has been conducted at home and abroad on the synthesis of artificial antigens and the analysis of the results. Artificial antigen synthesis technology has been applied to many fields such as food and environment, which have more or less impact on our lives. This article reviews how to select carrier protein and the coupling method of hapten and carrier in the process of artificial antigen synthesis.

Research Status of Antigen Synthesis Methods

Most haptens are small molecules compounds. Because of its small molecular mass itself alone can not induce an immune response, it is necessary to form combined with the carrier hapten-carrier protein conjugate can stimulate the body's immune response. In recent years, in-depth research has been conducted at home and abroad on the synthesis of artificial antigens and the analysis of the results. Artificial antigen synthesis technology has been applied to many fields such as food and environment, which have more or less impact on our lives. This article reviews how to select carrier protein and the coupling method of hapten and carrier in the process of artificial antigen synthesis.

A new strategy for the preparation of antibody against natural glycoside: With astragaloside IV as an example

A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6”-CH2OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6”-COOH, which was subsequently condensed with -NH2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC50, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO4) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.

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

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

Generation of Monoclonal Antibodies Against Natural Products.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

Polysaccharide structure dictates mechanism of adaptive immune response to glycoconjugate vaccines

Glycoconjugate vaccines are among the most effective interventions for preventing several serious infectious diseases. Covalent linkage of the bacterial capsular polysaccharide to a carrier protein provides CD4+ T cells with epitopes that facilitate a memory response to the polysaccharide. Classically, the mechanism responsible for antigen processing was thought to be similar to what was known for hapten-carrier conjugates: protease digestion of the carrier protein in the endosome and presentation of a resulting peptide to the T cell receptor on classical peptide-recognizing CD4+ T cells. Recently, an alternative mechanism has been shown to be responsible for the memory response to some glycoconjugates. Processing of both the protein and the polysaccharide creates glycopeptides in the endosome of antigen-presenting cells. For presentation, the peptide portion of the glycopeptide is bound to MHCII, allowing the covalently linked glycan to activate carbohydrate-specific helper CD4+ T cells (Tcarbs). Herein, we assessed whether this same mechanism applies to conjugates prepared from other capsular polysaccharides. All of the glycoconjugates tested induced Tcarb-dependent responses except that made with group C Neisseria meningitidis; in the latter case, only peptides generated from the carrier protein were critical for helper T cell recognition. Digestion of this acid-sensitive polysaccharide, a linear homopolymer of α(2 → 9)-linked sialic acid, to the size of the monomeric unit resulted in a dominant CD4+ T cell response to peptides in the context of MHCII. Our results show that different mechanisms of presentation, based on the structure of the carbohydrate, are operative in response to different glycoconjugate vaccines.

Development of novel portable and reusable fiber optical chemiluminescent biosensor and its application for sensitive detection of microcystin-LR

A novel portable fiber optical chemiluminescent biosensor (FOCB) system was successfully constructed using fiber optic bio-probe as biorecognition element as well as transducer and a Si-based photodiode detector (PD-3000). The compact design of FOCB with free magnetic beads and photomultiplier allows it to be portable and suitable for on-site and automatic detection of targets, as well as to be cost-effective and small sample volume (only 40.0 µL). The sensitivity of 5 fW could be obtained for the FOCB due to the high performance of PD-3000 and no background light signal. Next, the FOCB was applied for the CL-based detection of microcystin-LR (MC-LR) in water using hapten-carrier protein conjugates modified fiber optic bio-probe as biorecognition element. Based on indirect competitive sandwich-like CL immunoassay principle, the highly sensitive detection of MC-LR was achieved using FOCB system. Under optimal conditions, the limit of detection for MC-LR is 0.03 µg/L, which is comparable with that of most reported MC-LR immunoassay methods. The robustness of the hapten–carrier protein-modified biosensor surface allowed multiple MC-LR immunoassays without any significant loss of performance, which ensured accurate and cost-effective detection results. The proposed strategy demonstrated good recovery, precision, and accuracy through the evaluation of the spiked water samples. The FOCB system can be readily extended toward the on-site real-time sensitive detection of other targets in the field of environment, food, and medical diagnosis.

Generation of Monoclonal Antibodies against S-Adenosylmethionine and Their Application in Diagnosing Liver Diseases

S-AdenosylMethionine (SAM) hapten-carrier conjugates were used to immunize BALB/c mice. Two strains of hybridoma cell lines that stably produced high-titer monoclonal antibodies against SAM were obtained. The biological properties of these two antibodies were characterized. A competitive ELISA (cELISA) was established using these antibodies. The sensitivity and specificity were analyzed, and human serum samples were measured using the SAM cELISA kit.Very little cross-reactivity (&lt; 1%) was observed between the monoclonal antibodies and SAM analogues. These two antibodies are IgG2b-type antibodies. The affinity measured using the cELISA is 5.75 x 109 L/mol for the 118-6 antibody and 7.29 x 1010L/mol for the 84-3 antibody. According to the Immunohistochemistry (IHC) and flow cytometry results, normal liver cells contain much higher levels of SAM than liver cancer cells (p &lt; 0.01). The results from 81 healthy subjects and 99 patients with liver disease showed the following sensitivity using the 240 nM cutoff: 95.65% (44/46) for hepatitis; 95% (19/20) for cirrhosis; 92.86% (13/14) for carcinoma; 100% (19/19) for liver failure; 30.86% (25/81) for healthy subjects; the detection specificity is 69.14% (56/81). Using the 120 nM cutoff, the sensitivitywas 80.43% for hepatitis; 85% for cirrhosis; 71.43% for carcinoma, 100% for liver failure; 3.70% for healthy subjects; the specificity is 96.30% (78/81). SAM, alanine transaminase and aspartate aminotransferase were compared in identifying liver diseases. Serum SAM allowed early detection of abnormal liver function and providing clinicians an important basis for a more accurate diagnosis of liver diseases than alanine transaminase and aspartate aminotransferase. Keywords: S-Adenosylmethionine (SAM); Monoclonal Antibody; Competitive ELISA; Liver Disease Diagnosis Abbreviations: Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); Chronic Hepatitis B (CHB), Competitive ELISA (CELISA); Hematoxylin and Eosin (HE); Hepatocellular Carcinoma (HCC); Homocysteine (HCY); Immunohistochemistry (IHC); Keyhole Limpet Hemocyanin (KLH); L-Methionine(L-Met); Methionine Adenosine Transferase (MAT); Methylthioadenosine (MTA); Optical Density (OD); S-Adenosylhomocysteine(SAH); S-Adenosylmethionine(SAM).

Ultrasensitive immunoassay for monocrotaline using monoclonal antibody produced by N, N’-carbonyldiimidazole mediated hapten-carrier protein conjugates

Monocrotaline (MCT) is a naturally occurring pyrrolizidine alkaloid mainly produced in plant genus Crotalaria (Fabaceae), in which various toxicities have been reported. Recently, demand of quantitative analysis for determination/detection of MCT has increased because MCT in plants can be transferred to human by food chain, leading to intoxication. In this study, monoclonal antibody (MAb) against MCT (MAb 4B8) was produced by unique one-pot conjugation between hydrolysate of MCT, retronecine (RN) and consecutive lysine residues of bovine-serum albumin (BSA) through N,N’-carbonyldiimidazole (CDI) mediated method. The cross-reactivity test evaluated by an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that MAb 4B8 is highly specific to MCT. In addition, the developed icELISA using MAb 4B8 exhibited ultrasensitive recognition to free MCT with the limit of detection of 48.8pgmL−1. Further validation analyses suggested that the developed icELISA is sensitive, accurate, and reliable enough to be used for determining/detecting MCT before ingestion.

New Advances in the Study of IgE Drug Recognition.

Allergic drug reactions are currently a major public health problem affecting patient health and increasing healthcare costs. They are caused by interactions between a drug and the human immune system and result in symptoms ranging from urticaria or angioedema to those more serious such as anaphylaxis or anaphylactic shock. The most commonly accepted mechanism for immunological activation is based on the hapten hypothesis. Drugs are low molecular weight substances that cannot cause an immune response on their own. However, they can act as haptens and form covalent adducts with proteins. The resulting hapten-carrier (drug-protein) conjugate can induce the production of IgE antibodies or T cells. An epitope, or antigenic determinant, is the part of the drug-protein antigen that is specifically recognized by the immune system. This may involve not only the drug derivative but also part of the carrier protein. Understanding the way in which drugs are metabolized after protein conjugation is vital in order to make progresses in the diagnosis of clinical allergy. In this review, recent advances in the identification of the chemical structures of antigenic determinants involved in immediate allergic reactions to drugs are presented. We have focused on drugs that most commonly elicit these reactions: betalactam and quinolone antibiotics and the non-steroidal anti-inflamatory drug pyrazolone. This will be discussed from a chemical point of view, relating our understanding of drug structure, chemical reactivity and immune recognition.

Novel Heat-Stable Enterotoxin (STa) Immunogen Based on Cationic Nanoliposomes: Preparation, Characterization and Immunization

The novelties encountered in the field of formulation have provided promising solutions for the problematic vaccine development of hapten molecules. In the current study, the novel preparation of a cationic nanoliposomal immunogen of the heat stable enterotoxin (STa) was reported. STa was produced from clinically ETEC isolate of diarrheic neonatal calves and purified using RP-HPLC. STa was loaded into the cationic vesicles which were characterized for their particle size, surface charge, morphology, STa loading, and stability. The STa loaded cationic nanoliposome was used for mice immunization and the generation of STa antibody was monitored using ELISA. Results displayed the spherical nature of the STa loaded vesicles, their suitable size and homogeneity represented by a particle size of 228.1 nm and a PDI of 0.202. The surface charge of the STa nanoliposomes was +29.9, demonstrating sufficient stability during refrigeration storage. The STa loaded cationic nanoliposome was able to elicit specific STa antibody response, and to confer effective protection against STa challenge in mice. The STa antibody binding titer and neutralization capacity were 105 and 104 mouse units/ml serum, respectively. The developed system is a one-step procedure, which overcomes the disadvantage of the complexity of generation of the hapten-carrier conjugate. In conclusion, the developed STa-cationic nanoliposomal immunogen is feasible and has the potential to improve effectiveness against ETEC, suggesting its applicability in preventing the harmful effects of ETEC infection in neonatal calves.

Synthesis of Hapten-Protein Conjugate Vaccines with Reproducible Hapten Densities.

The ability to prepare hapten-carrier conjugates reproducibly with consistent lot-to-lot hapten densities and protein yields is a critical component of hapten vaccine development. This entails the development of appropriate coupling chemistries that do not cause protein precipitation and the development of methods to quantify hapten density. Recently, extensive efforts have been devoted to design vaccines against drugs of abuse. We describe, herein, a method for conjugation of a morphine-like hapten (MorHap) to tetanus toxoid (TT), which involves conjugation of MorHap to the surface lysines of TT through the N-hydroxysuccinimide portion of a heterobifunctional linker and the subsequent attachment of the thiol on MorHap to the maleimide portion of the cross-linker. Methods are described for the analytical quantification of the hapten density of the conjugates using modified Ellman's test, trinitrobenzenesulfonic acid (TNBS) assay, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Design of generic haptens forO,O-diethyl organophosphorus pesticides and study of broad-specificity antibody recognition

Several generic haptens against O , O -diethyl organophosphorus pesticides (DOPs) were designed and used to produce broad-specificity antibodies. Molecular modeling, spectra analysis and genetic engineering technique were used to study the antibody recognition by structure-activity relationship, intermolecular interaction and three dimensional models. The conformation models of hapten-carrier protein conjugates were constructed based on quantum chemistry calculation and molecular modeling to study the correlation between hapten/antigen structures and antibody properties. The frontier-orbital energy (energy of the lowest unoccupied molecular orbital, E LUMO ) and hydrophobicity (log of the octanol/water partition coefficient, log P ) were mainly responsible for the antibody recognition. The key amino-acid residues for antibody recognition were also defined. The results of antibody recognition studies not only provide important theory on the interaction of antibody with small molecules, but also provide references for developing immunoassays for other small molecules contaminants.

Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR)

Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses.

Improved Detection Sensitivity and Selectivity Attained by Open-Sandwich Selection of an Anti-Estradiol Antibody

The development of a rapid and specific assay for 17β-estradiol (E2) will accelerate its in vitro diagnostics and/or environmental pollution control. Here, we employed an open-sandwich (OS) selection scheme to improve the sensitivity for E2 in an OS immunoassay, which is based on antigen-dependent stabilization of the antibody (Ab) variable region, Fv, where the two domains (V(H) and V(L)) dissociated in the absence of an antigen. The V(H) domain of a cloned anti-E2 antibody displayed on M13 phage was randomly mutated, and after three OS biopanning rounds, a mutant that showed higher sensitivity in OS-ELISA for E2 was identified. Interestingly, compared with the wild-type V(H), the cross-reactivity of the mutant was significantly decreased for the analogous steroid testosterone, both in OS and competitive ELISAs. This is the first report concerning selection for an anti-hapten Ab without using any hapten-carrier conjugates, and the method will be especially suitable for selecting Ab fragments that show better performance in hapten OS immunoassays.

Methodologies to anchor dendrimeric nanoconjugates to solid phase: toward an efficient in vitro detection of allergy to β-lactam antibiotics

β-lactam antibiotics are the most common cause of adverse drug reactions, and in vitro immunoassays are often employed to diagnose this allergy. Cellulose solid supports displaying hapten-carrier conjugates are traditionally used for quantifying specific IgE antibodies to determine whether a patient is allergic to the hapten. Our approach makes use of polyamidoamine dendrimers as carriers, and differs from traditional solid-phase activation methods by using two different haloalkanoyl halides as reagents instead of cyanogen bromide. Evaluation of the functionalization in the three different surfaces with benzylpenicilloyl-dendrimer conjugates showed a higher density of conjugates when using haloalkanoyl halides. The resulting surfaces were evaluated in vitro by radioimmunoassay using sera from patients allergic to penicillins. Selective and specific IgE recognition was observed in the different activated disks, though those activated with haloalkanoyl halides presented better sensitivity. From the Clinical EditorA novel and sensitive method of dendrimeric nanoconjugate-based in vitro detection of allergy to β-lactam antibiotics is presented in this basic science paper.

Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.