Hapten-protein Conjugates Research Articles

Comparison of Conjugates Obtained Using DMSO and DMF as Solvents in the Production of Polyclonal Antibodies and ELISA Development: A Case Study on Bisphenol A

When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten–protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor—the conditions for obtaining the hapten–protein conjugate—is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSADMF–BVA, STIDMF–BVA, BSADMSO–BVA and STIDMSO–BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSADMF–BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STIDMF–BVA or STIDMSO–BVA conjugates. The use of the STIDMF–BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSADMF–BVA in an ELISA. A competitive interaction was observed only with the use of the STIDMSO–BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5–290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten–protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds.

Comparative Characteristics of Immunochromatographic Test Systems for Tylosin Antibiotic in Meat Products.

Tylosin (TYL) is a macrolide antibiotic widely used in animal husbandry. Due to associated health risks, there is a demand for sensitive methods for mass screening of TYL in products of animal origin. This article describes the development of lateral flow immunoassays (LFIAs) for TYL detection using direct (anti-TYL antibodies conjugated with nanoparticles) and indirect antibody labeling (anti-species antibodies conjugated with nanoparticles and combined with native anti-TYL antibodies). The choice of LFIA conditions, such as concentrations of hapten-protein conjugates, specific antibodies, and gold nanoparticle (GNP) conjugates with antibodies, as well as incubation time of reagents and the concentration of detergent in the sample buffer, is presented. The achieved limits of TYL detection using LFIAs with indirect labeling were 0.8 ng/mL (visual) and 0.07 ng/mL (instrumental), compared to 4 ng/mL (visual) and 0.4 ng/mL (instrumental) for the case of direct labeling. The sensitivity of the LFIA using the indirect format was up to seven times higher, allowing the determination of the target analyte at low concentrations. TYL detection in ground meat using LFIA with indirect antibody labeling ranged from 76-119%.

Development of Chemiluminescent ELISA for Detection of Diisobutyl Phthalate in Water, Lettuce and Aquatic Organisms

The use of plasticizers to improve the quality of plastics widely used for household purposes inevitably leads to an increase in their pollution of food and environmental objects. Diisobutyl phthalate (DiBP) is one of the ortho-substituted phthalic acid esters that negatively affect human health and ecosystems. This work is directed to the development of a chemiluminescent enzyme immunoassay (CL-ELISA) for the determination of diisobutyl phthalate in water and food. Luminol, which is oxidized with hydrogen peroxide in the presence of p-iodophenol as an enhancer, was chosen as the substrate for horseradish peroxidase used as a label in the analysis. For this development, rabbit anti-DiBP polyclonal antibodies were generated and tested with the synthesized hapten–protein conjugate. The developed chemiluminescent ELISA has a detection limit of 1.8 ng/mL; the operating range was 5.0–170.8 ng/mL at a content of 10% methanol in the assay medium. The assay was successfully applied to detect diisobutyl phthalate in lettuce leaves, seafood, and water. When using extraction with methanol and hexane, the recovery of DiBP in samples varies in the range of 76.9–134.2%; for assays in natural waters, the recovery rates are from 79.5 to 113.4%.

BPA Endocrine Disruptor Detection at the Cutting Edge: FPIA and ELISA Immunoassays.

BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20-30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten-protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 μg/L, working range from 2 to 155 μg/L. The validation of the methods was conducted using actual samples compared to LC-MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.

Development of a surface plasmon resonance based immunosensor for diclofenac quantification in water

A Surface Plasmon Resonance (SPR) biosensor based on an inhibition immunoassay was developed for the detection of diclofenac (DCF) in aqueous solution. Due to the small size of DCF, an hapten-protein conjugate was produced by coupling DCF to bovine serum albumin (BSA). DCF-BSA conjugate formation was confirmed via MALDI-TOF mass spectrometry. The resulting conjugate was immobilized onto the surface of a sensor fabricated via e-beam deposition of a 2 nm chromium adhesion layer followed by a 50 nm gold layer onto precleaned BK7 glass slides. Immobilization onto the nano thin gold surface was accomplished by covalent amide linkage through a self-assembled monolayer. Samples were composed of a mixture of antibody at a fixed concentration and DCF at different known concentrations in deionized water, causing the inhibition of anti-DCF on the sensor. The DCF-BSA was obtained with a ratio of 3 DCF molecules per BSA. A calibration curve was performed using concentrations between 2 and 32 μg L−1. The curve was fitted using the Boltzmann equation, reaching a limit of detection (LOD) of 3.15 μg L−1 and limit of quantification (LOQ) of 10.52 μg L−1, the inter-day precision was calculated and an RSD value of 1.96% was obtained; and analysis time of 10 min. The developed biosensor is a preliminary approach to the detection of DCF in environmental water samples, and the first SPR biosensor developed for DCF detection using a hapten-protein conjugate.

Universal and rapid detection of atrazine and bisphenol A using a reusable optical fiber chemiluminescent biosensor

Timely and accurately detection of small molecule pollutants is quite necessary to control environmental pollution and reduce harmfulness. Herein, a reusable optical fiber chemiluminescent biosensor (ROFC) was proposed for universal and rapid detection of two representative pollutants, pesticide atrazine (ATZ) and endocrine disruptor bisphenol A (BPA). The optical fiber modified with hapten-protein conjugates was regarded as both bio-probe and chemiluminescence signal transmission element, which effectively improved the light transmission efficiency and signal-to-noise ratio of the system. High-sensitive chemiluminescence signal detection is realized with a miniaturized ultrasensitive photodiode detector. Good regeneration performance of bio-probe can reduce detection cost and ensure detection reproducibility. Based on indirect competitive immunoassay principle, the chemiluminescence signal decreased with increasing pollutant concentration resulting from the less amount of antibody combined on the bio-probe surface. Under optimal conditions, the whole assay was achieved within 25 min with linear range of 1–100 μg/L and detection limits (LOD) for atrazine and BPA are 0.029 μg/L and 0.025 μg/L, respectively. The immunosensing optical fiber probe can be reused for 150 times at least without losing obvious bioactivity. The method was successfully applied to the detection of ATZ and BPA in three environmental samples, where recoveries between 93.4% and 116.6% were achieved. The ROFC biosensor provides a feasible platform for rapid detection of multiple small molecule pollutants in the environment.

Development of Lateral Flow Test-System for the Immunoassay of Dibutyl Phthalate in Natural Waters

The use of a large amount of toxic synthetic materials leads to an increase in the pollution of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger is conditioned by leaching into the environment and penetrating into living organisms with negative consequences and effects on various organs and tissues. This work presents the first development of lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of the phthalates group. To form a test zone, a hapten-protein conjugate was synthesized, and gold nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate. The work includes the preparation of immunoreagents, selectivity investigation, and the study of the characteristics of the medium providing a reliable optical signal. Under the selected conditions for the analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations was from 42.4 to 1500 ng/mL. Time of the assay-15 min. The developed technique was successfully applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

On-site rapid and simultaneous detection of acetamiprid and fipronil using a dual-fluorescence lab-on-fiber biosensor

Adual-fluorescence lab-on-fiber biosensor was developed for the rapid and simultaneous on-site determination of acetamiprid and fipronil, based on time-resolved effect and indirect competitive immunoassay principle. The optical fiber modified with two hapten-protein conjugates servesas a bifunctional bio-probe. The dual-color fluorescent reporters were prepared via labeling acetamiprid and fipronil antibodies with Cy5.5 and Alexa Fluor 555, which were excited at 635-nm and 520-nm laserwavelengths, respectively. In the presence of targets, the binding sites of corresponding antibodies were occupied and less antibodies were connected to the probe surface, resulting in the reduction of fluorescence signal. The concentration of acetamiprid and fipronil was determined by measuring the fluorescence signals at 568nm and 702nm (emission wavelengths), respectively. Under optimal conditions, the linear response range was 14.2-225.4ng/L for acetamiprid and 25.1-162.8ng/L for fipronil, and the limit of detection was 6.51ng/L and 17.8ng/L for acetamiprid and fipronil, respectively. The method was successfully applied to the simultaneous detection of acetamiprid and fipronil in three environmental samples, and the recoveries were between 90 and 128%. The dual-fluorescence lab-on-fiber biosensor provides a feasible platform for simultaneous and rapid detection of multiple pesticide residues. A dual-fluorescence lab-on-fiber biosensor was developed for the rapid and simultaneous on-site determination of acetamiprid and fipronil. A bifunctional bio-probe was prepared from the optical fiber modified with two hapten-protein conjugates. Acetamiprid and fipronil antibodies were labeled with different fluorophores and used as dual-color fluorescent reporters.

Double Competitive Immunodetection of Small Analyte: Realization for Highly Sensitive Lateral Flow Immunoassay of Chloramphenicol.

A new scheme of reagents interaction for lateral flow immunoassay (LFIA) is proposed, which combines the features of competitive and sandwich assay and provides highly sensitive detection of low-molecular-weight analytes. Namely, the antigen in the sample interferes with the formation of the antibody (on the membrane)–hapten-protein–antibody (on the nanoparticle-marker) complex, competing with hapten-protein conjugate in both reactions. The proposed scheme was modelled using COPASI software, with a prediction of limit of detection (LOD) decrease by one order of magnitude compared to the standard competitive LFIA. This feature was experimentally confirmed for the detection of chloramphenicol (CAP) in honey. When tested in spiked honey, the visual LOD was 50 ng/mL for the common scheme and 5 ng/mL for the proposed scheme. Instrumental LOD was 300 pg/mL (1.2 µg/kg in conversion per sample weight of honey) in the standard scheme and 20 pg/mL (80 ng/kg in conversion per sample weight of honey) in the proposed scheme.

Proteasome inhibition induces p53 driven apoptosis in early activated B-cells

Abstract Proteasome inhibition is a useful therapeutic strategy against plasma cell disorders in autoimmunity, neoplasias, and transplant rejection. However, how bortezomib induces cell death and the transcription factors that are involved in its execution are not well characterized. In this study, we aim to address the mechanism of action of proteasome inhibitors in plasma cells and in early precursor activated B-cells. We immunized mice with the hapten-protein conjugate nitrophenyl chicken-gamma-globulin (NP-CGG) and validated depletion of plasma cells and germinal center B-cells in vivo. At steady-state, we utilized B220 expression as a surface marker for newly-formed plasma cells and determined that bortezomib primarily depletes newly-formed plasma cells. To gain more mechanistic insight on how bortezomib affects newly-formed plasma cells, we employed an in vitro culture system of murine-derived primary follicular B-cells that were stimulated with CPG, IL-4, and IL5, to promote differentiation into the plasma cell fate. We find that deletion of the ER-stress induced transcription factor CHOP rescued in vitro generated plasma cells from low-dose bortezomib treatment. In addition, we identify the transcription factor p53 as a key inducer of apoptosis in in vitro generated early activated B-cells. Deletion of p53 rescued cells from apoptosis and reduced the number of cells expressing cleaved caspase-3 in response to bortezomib treatment. In conclusion, we have identified two transcription factors that are involved in bortezomib-induced cell death in B-lineage cells. Supported by NIAID Diversity Supplement: R01AI139123

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity.

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

LATERAL FLOW TECHNIQUE FOR THE DETERMINATION OF DIBUTYL PHTHALATE

An immunochromatographic method for the rapid determination of dibutyl phthalate has been developed. For this purpose, the competitive immunochemical interaction of monoclonal antibodies labeled with gold nanoparticles with a hapten-protein conjugate and a free analyte in various media was characterized. An immunochromatographic test system has been tested to detect and evaluate the content of dibutyl phthalate in natural waters.

Determination of the Fluoroquinolones Levofloxacin and Ciprofloxacin by a Piezoelectric Immunosensor Modified with Multiwalled Carbon Nanotubes (MWCNTs)

Methods for the high-sensitivity determination of fluoroquinolones using a piezoelectric immunosensor based on multi-walled carbon nanotubes (MWCNTs) are developed. The use of MWCNTs in the formation of a stable piezoelectric sensor detection layer increases the active specific surface area which is necessary for receptor molecule binding. The concentrations of hapten-protein conjugates (35/65 serial concentration) and polyclonal antibodies are determined in the direct (15/85 for levofloxacin, 18/82 for ciprofloxacin) and in the competitive immunoassay formats (14/86 for levofloxacin, 10/90 for ciprofloxacin). Conditions for the analysis in the flow-injection mode were studied. The carrier flow rate was 30 and 50 µl/min depending on the detection layer formation method. The characteristics of MWCNTs-based piezoelectric immunosensors were characterized for the determination of fluoroquinolones in the static and in the flow-injection modes. The direct and competitive immunoassay formats (detection limit, linear range of target concentrations, reproducibility) were determined. The detection limits were 9 and 8 ng/ml for levofloxacin and ciprofloxacin in the competitive format and 25 and 21 ng/ml in the direct assay. The piezoelectric immunosensors were employed for the analysis of milk.

A copper nanoparticle-based electrochemical immunosensor for carbaryl detection

A hapten-protein conjugate with copper nanoparticles (Hap-Car-BSA@CuNPs) was first synthesized in the present work for the determination of carbaryl. The copper nanoparticles (CuNPs) of the conjugate were used as electrochemical labels in the direct solid-phase competitive determination of carbaryl residues in flour from different crops. The signal was read by linear sweep anodic stripping voltammetry (LSASV) of copper (through the electrochemical stripping of accumulated elemental copper) on a gold-graphite electrode (GGE). To form a recognition receptor layer of monoclonal antibodies against the carbaryl on the surface of the GGE, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxy-2,5-pyrrolidinedione (NHS) were used as the best covalent cross-linkers. The concentrations of the antibodies and the Hap-Car-BSA@CuNPs conjugate were optimized for carbaryl detection by the electrochemical immunosensor. The electrochemical immunosensor can be used for highly sensitive determination of carbaryl residues in flour samples in the concentration range 0.8–32.3 μg·kg−1, with a limit of detection 0.08 μg·kg−1. The present work paves the path for a novel method for monitoring carbaryl in other food products, drinks, and soil samples.

COPPER-LABELED VOLTAMMETRIC IMMUNOSENSOR FOR CARBARYL DETECTION

A hapten-protein conjugate for determining carbaryl using copper nanoparticles was first synthesized in the present work. The copper nanoparticles were used as electrochemical labels in the direct solid-phase competitive analysis of carbaryl residues in flour from some crops. Copper nanoparticles stabilized by chitosan and a hapten-protein conjugate with copper nanoparticles stabilized by chitosan by UV/VIS - spectroscopy, transmission electron microscopy, and dynamic light scattering are characterized. To form a recognition receptor layer of monoclonal antibodies against the carbaryl on the surface of a gold-graphite electrode, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide and 1-hydroxy-2,5-pyrrolidinedione were found to be the best covalent cross-linkers. When selection the electrochemical immunosensor conditions, working dilutions of the antibodies and the hapten-protein conjugate with copper nanoparticles copper nanoparticles stabilized by chitosan were selected to detect the carbaryl. The effect of ethyl alcohol on the performance of the electrochemical immunosensor for the determination of carbaryl was evaluated. The specificity of the developed electrochemical immunosensor was investigated by testing structurally related carbaryl compounds; the results did not exceed 7%. The electrochemical immunosensor can be used for highly sensitive determination of carbaryl residues in flour samples with a detection limit of 0.08 μg /kg within a range of detectable contents of 0.8-32.3 μg/kg.

Synthesis of a deuterated 6-AmHap internal standard for the determination of hapten density in a heroin vaccine drug product.

A deuterated hapten was designed and synthesized that will be essential for a future study of residual hapten and stability of a hapten-protein conjugate. This hapten, 6-AmHap, was chosen for a heroin vaccine that is now slated for a Phase 1 clinical trial. A maleimide-thiol bioconjugation strategy was successfully applied to our heroin vaccine to connect the hapten 6-AmHap with an immunogenic carrier protein (tetanus toxoid, TT) through a trityl-protected 3-mercaptopropanamide linker. The antibodies induced by the vaccine have been found to have activity against several opioids, including heroin and its metabolites, and, importantly, leave alternate pain treatment medications such as methadone untouched. To the best of our knowledge, no other hapten for a heroin vaccine has been deuterated, yet this tool may prove to be of great importance in the study of residual hapten during product release and the long-term stability program of a hapten-protein conjugate as part of FDA regulatory requirements. Hydrocodone was the starting material for the synthesis of the deuterated 6-AmHap, with a stable amide at C6 and a 3-mercaptopropanamide linker attached at C3. The desired deuterated product was prepared as the disulfide, 3,3'-disulfanediylbis(N-((7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl)-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)propanamide), that could be easily reduced to form the needed hapten, N-((4aR,7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl]-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)-3-mercaptopropanamide.

ZBTB38 is dispensable for antibody responses.

Members of the broad complex, tram track, bric-a-brac and zinc finger (BTB-ZF) family of transcription factors, such as BCL-6, ZBTB20, and ZBTB32, regulate antigen-specific B cell differentiation, plasma cell longevity, and the duration of antibody production. We found that ZBTB38, a different member of the BTB-ZF family that binds methylated DNA at CpG motifs, is highly expressed by germinal center B cells and plasma cells. To define the functional role of ZBTB38 in B cell responses, we generated mice conditionally deficient in this transcription factor. Germinal center B cells lacking ZBTB38 dysregulated very few genes relative to wild-type and heterozygous littermate controls. Accordingly, mice with hematopoietic-specific deletion of Zbtb38 showed normal germinal center B cell numbers and antibody responses following immunization with hapten-protein conjugates. Memory B cells from these animals functioned normally in secondary recall responses. Despite expression of ZBTB38 in hematopoietic stem cells, progenitors and mature myeloid and lymphoid lineages were also present in normal numbers in mutant mice. These data demonstrate that ZBTB38 is dispensable for hematopoiesis and antibody responses. These conditional knockout mice may instead be useful in defining the functional importance of ZBTB38 in other cell types and contexts.

Characterizing Adduct Formation of Electrophilic Skin Allergens with Human Serum Albumin and Hemoglobin.

Skin (contact) allergy, the most predominant form of immunotoxicity in humans, is caused by small electrophilic compounds (haptens) that modify endogenous proteins. Approximately 20% of the general population in the Western world is affected by contact allergy. Although the importance of the hapten–protein conjugates is well established in the initiation of the immunological reaction, not much progress has been made regarding identification of these conjugates in vivo or exploration of their potential as diagnostic tools. In this study, the human serum albumin (HSA) and human hemoglobin (Hb) adductome for three representative contact allergens with different chemical properties, 1-chloro-2,4-dinitrobenzene (DNCB), 1,2-epoxy-3-phenoxypropane (PGE), and 2-bromo-2-(bromomethyl)glutaronitrile (MDBGN), were studied. Plasma and red blood cell lysate were used as a source for HSA and Hb, respectively. The Direct Peptide Reactivity Assay was used to investigate adduct formation of MDBGN with nucleophilic moieties and revealed that MDGBN is converted to 2-methylenepentanedinitrile in the presence of sulfhydryl groups prior to adduct formation. Following incubation of HSA and Hb with haptens, an Orbitrap Q Exactive high-resolution mass spectrometer was used to perform an initial untargeted analysis to screen for adduct formation, followed by confirmation by targeted Parallel Reaction Monitoring analysis. Although a subset of adducted sites was confirmed by targeted analysis, only some of the adducted peptides showed an increase in the relative amount of the adducted peptide with an increased concentration of hapten. In total, seven adduct sites for HSA and eight for Hb were confirmed for DNCB and PGE. These sites are believed to be the most reactive. Further, three of the HSA sites (Cys34, Cys62, and Lys190) and six of the Hb sites (subunit α: Val1, His45, His72; subunit β: Cys93, His97, and Cys112) were haptenated already at the lowest level of hapten to protein molar ratio (0.1:1), indicating that these sites are the most likely to be modified in vivo. To the best of our knowledge, this is the first time that the adductome of Hb has been studied in the context of contact allergens. Identification of the most reactive sites of abundant proteins, such as HSA and Hb, is the first step toward identification of contact allergy biomarkers that can be used for biomonitoring and to develop better diagnostic tools based on a blood sample.

The synthesis of antigens and the production of antibodies against patulin derivatives

Patulin hemisuccinate (P-HS) was synthesized and conjugated to bovine serum albumin (BSA) as the hapten-protein conjugation for immunization. Polyclonal antibodies were obtained from the antiascites of six immunized mice. Titers and specificity were determined using an indirect ELISA. The results indicated that three of the six mice were better immunized and that the antibodies acted strongly against patulin-hemisuccinate-BSA. Slight ELISA responses of the antibodies against patulin alone showed the potential for developing monoclonal antibodies.

Multiplex SERS-based lateral flow immunosensor for the detection of major mycotoxins in maize utilizing dual Raman labels and triple test lines

A multiplex surface-enhanced Raman scattering (SERS)-based lateral flow immunosensor was developed to determine six major mycotoxins in maize. Two characteristic Raman reporter molecules—5,5-dithiobis-2-nitrobenzoic acid (DTNB) and 4-mercaptobenzoic acid (MBA)—were used to label the synthesized Au@Ag core-shell nanoparticles for the preparation of SERS nanoprobes as detection reagents. Six corresponding hapten-protein conjugates were prepared and dispensed on three test lines of nitrocellulose membrane with two conjugates on each line as capture antigens. This design facilitates the simultaneous detection of the six mycotoxins in a single test. After optimizing the experimental parameters of immunosensor, the limits of detection were as low as 0.96 pg/mL for aflatoxin B1, 6.2 pg/mL for zearalenone, 0.26 ng/mL for fumonisin B1, 0.11 ng/mL for deoxynivalenol, 15.7 pg/mL for ochratoxin A, and 8.6 pg/mL for T-2 toxin, respectively. The spiking experiment showed high accuracy with recovery of 78.9–106.2 % and satisfactory assay precision with the coefficient of variations below 16 %. Moreover, this assay can be completed in less than 20 min, and its detection results were consistent with that of liquid chromatography-mass spectrometry. Therefore, the developed SERS-based lateral flow immunosensor is a promising approach for mycotoxin detection in the field.