A multi-colorimetric immunosensor for visual detection of ochratoxin A by mimetic enzyme etching of gold nanobipyramids.

A multi-colorimetric immunosensor basing on the mimetic enzyme etching of gold nanobipyramids (Au NBPs) was established to detect ochratoxin A (OTA). Octahedral Cu2O nanoparticles were successfully synthesized through a selective surface stabilization strategy, which can exhibit a peroxidase-like ability to oxidize 3,3',5,5'-tetramethylbenzidine (TMB). Au NBPs can be etched by the product, TMB2+, to form a significant longitudinal peak blue shift of local surface plasmon resonance. During the construction of the immunosensor, the microplate was coated with dopamine to immobilized OTA antigens, followed by the immunoreaction of OTA antibody and the Cu2O-labled secondary antibody. A linear relationship can be found between the local surface plasmon resonance (LSPR) peak changes with the logarithm of OTA concentration in a wide range from 1 ng/L to 5 µg/L, while the detection limit was 0.47 ng/L. Meanwhile, the approximate OTA concentration can be conveniently and intuitively observed by the vivid color changes. Benefiting from the high specificity, the proposed multi-colorimetric immunoassay detection of OTA in millet samples was achieved, indicating the available potential of the immunoassay for the determination of OTA in real samples.

A novel magneto-gold nanohybrid-enhanced lateral flow immunoassay for ultrasensitive and rapid detection of ochratoxin A in grape juice.

Conventional gold nanoparticle-based lateral flow immunoassay (LFIA) usually suffers a huge challenge in measuring target concentration in food matrices with dark color because of its poor resistance to the background matrix and color interference. To address this issue, we first report a novel bifunctional magneto-gold nanohybrid (MGNH) for the simultaneous magnetic separation and colorimetric target sensing by integrating MGNHs into LFIA. Under optimum conditions, an ultrasensitive detection of ochratoxin A (OTA) in grape juice was achieved with a limit of detection at 0.094 ng mL-1. The average recoveries of this MGNH-LFIA ranged from 92.31% to 108.97% with a coefficient of variation of below 12%. The excellent selectivity of our MGNH-LFIA against OTA was demonstrated. Besides, our MGNH-LFIA is comparable to liquid chromatography coupled with mass spectrometry in terms of accuracy, reproducibility, and practicability. The designed MGNH-LFIA platform is readily extended for improving other small molecule detection in food samples.

Development of an Ultrasensitive and Rapid Fluorescence Polarization Immunoassay for Ochratoxin A in Rice.

Ochratoxin A (OTA) is a known food contaminant that affects a wide range of food and agricultural products. The presence of this fungal metabolite in foods poses a threat to human health. Therefore, various detection and quantification methods have been developed to determine its presence in foods. Herein, we describe a rapid and ultrasensitive tracer-based fluorescence polarization immunoassay (FPIA) for the detection of OTA in rice samples. Four fluorescent tracers OTA-fluorescein thiocarbamoyl ethylenediamine (EDF), OTA-fluorescein thiocarbamoyl butane diamine (BDF), OTA-amino-methyl fluorescein (AMF), and OTA-fluorescein thiocarbamoyl hexame (HDF) with fluorescence polarization values (δFP = FPbind-FPfree) of 5, 100, 207, and 80 mP, respectively, were synthesized. The tracer with the highest δFP value (OTA-AMF) was selected and further optimized for the development of an ultrasensitive FPIA with a detection range of 0.03-0.78 ng/mL. A mean recovery of 70.0% to 110.0% was obtained from spiked rice samples with a relative standard deviation of equal to or less than 20%. Good correlations (r2 = 0.9966) were observed between OTA levels in contaminated rice samples obtained by the FPIA method and high-performance liquid chromatography (HPLC) as a reference method. The rapidity of the method was confirmed by analyzing ten rice samples that were analyzed within 25 min, on average. The sensitivity, accuracy, and rapidity of the method show that it is suitable for screening and quantification of OTA in food samples without the cumbersome pre-analytical steps required in other mycotoxin detection methods.

Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals.

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody's primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I-L), two of them in CDR2 (G-D, E-K), and three of them in CDR3 (Y-H, Y-W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol-1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol-1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL-1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL-1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL-1 with a limit of detection (LOD) of 0.003 ng mL-1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1-12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

Development of ELISA and Lateral Flow Immunoassays for Ochratoxins (OTA and OTB) Detection Based on Monoclonal Antibody.

Ochratoxins were important secondary metabolites secreted by fungi, and OTA and OTB are mainly significant mycotoxin, having toxic effects on humans and animals. Therefore, it is important to establish a rapid, sensitive, and precise method for ochratoxins detection and quantification in real samples. In this study, a stable monoclonal antibody (mAb) that recognizing both OTA and OTB toxins was employed for the establishment of indirect competitive ELISA (ic-ELISA), colloidal gold nanoparticles (CGNs), and nanoflowers gold strips (AuNFs) for detection of ochratoxins in real samples. A 6E5 hybridoma cell line stable secreting mAb against both OTA and OTB toxins was obtained by fusion of splenocytes with myeloma SP2/0 cells. The 6E5 mAb had a high affinity (3.7 × 108 L/mol) to OTA, and also showed similar binding activity to OTB. The optimized ic-ELISA resulted in a linear range of 0.06-0.6 ng/mL for ochratoxins (OTA and OTB) detection. The IC50 was 0.2 ng/mL and the limit of detection (LOD) was 0.03 ng/mL. The mean recovery rate from the spiked samples was 89.315 ± 2.257%, with a coefficient variation of 2.182%. The result from lateral flow immunoassays indicated that the LOD of CGNs and AuNFs were 5 and 1 µg/mL, respectively. All these results indicated that the developed ic-ELISA, CGNs, and AuNFs in this study could be used for the analysis of the residual of ochratoxins (OTA and OTB) in food and agricultural products.

Nanobody affinity improvement: Directed evolution of the anti-ochratoxin A single domain antibody.

The characteristics of single domain and ease of gene manipulation of the single domain antibody (sdAb) make it suitable for affinity maturation in vitro. Since the affinity of antibodies can influence the immunoassays' sensitivity, a nanobody (Nb), the anti-ochratoxin A sdAb (AOA-sdAb), was herein selected as the model antibody to explore feasible approach for improving its affinity. Homology modeling and molecular docking were used to analyze the interaction between OTA and the AOA-sdAb. After alanine scanning verification, Gly53, Met79, Ser102, and Leu149 were determined as the key amino acids of the AOA-sdAb. Two site-directed saturated mutation libraries were constructed by two-site mutation against those four key amino acids. After biopanning and identification, a mutant Nb-G53Q&S102D was obtained with a half maximal inhibition concentration (IC50) of 0.29 ng/mL and a KD value of 52 nM, which is 1.4-fold and 1.36-fold lower than that of the original sdAb, respectively. The computer simulation analysis indicated that the hydrogen bond, hydrophobic interaction, and side chain steric hindrance of amino acid residues are critical for the binding affinity of the AOA-sdAb. Overall, the techniques shown in this study are effective ways at 'identifying residues involved in antigen binding' that can be altered by site-directed mutation.

Ultrasensitive and rapid detection of ochratoxin A in agro-products by a nanobody-mediated FRET-based immunosensor.

Ochratoxin A (OTA) is a major concern for public health and the rapid detection of trace OTA in food is always a challenge. To minimize OTA exposure to consumers, a nanobody (Nb)-mediated förster resonance energy transfer (FRET)-based immunosensor using quantum dots (Nb-FRET immunosensor) was proposed for ultrasensitive, single-step and competitive detection of OTA in agro-products at present work. QDs of two sizes were covalently labeled with OTA and Nb, acting as the energy donor and acceptor, respectively. The free OTA competed with the donor to bind to acceptor, thus the FRET efficiency increased with the decrease of OTA concentration. The single-step assay could be finished in 5 min with a limit of detection of 5 pg/mL, which was attributed to the small size of Nb for shortening the effective FRET distance and improving the FRET efficiency. The Nb-FRET immunosensor exhibited high selectivity for OTA. Moreover, acceptable accuracy and precision were obtained in the analysis of cereals and confirmed by the liquid chromatography-tandem mass spectrometry. Thus the developed Nb-FRET immunosensor was demonstrated to be an efficient tool for ultrasensitive and rapid detection of OTA in cereals and provides a detection model for other toxic small molecules in food and environment.

An immunoassay for ochratoxin A using tetramethylrhodamine-labeled ochratoxin A as a probe based on a binding-induced change in fluorescence intensity.

Ochratoxin A (OTA) is a mycotoxin that can cause health risks to human/animal health. Contamination by OTA can occur in various foods and agricultural products, so sensitive and rapid detection of OTA is crucial. We describe a simple and sensitive fluorescence immunoassay for OTA using tetramethylrhodamine (TMR)-labeled OTA as a fluorescent probe. We conjugated tetramethylrhodamine to OTA through a covalent reaction, and obtained three TMR-OTA isomer probes after purification by high-performance liquid chromatography. All of the fluorescent probes showed high binding affinity to the anti-OTA antibody. Binding of the TMR-OTA probe to the antibody induced strong fluorescence of TMR-OTA due to the possible change in the local environment of TMR caused by affinity binding. In the presence of OTA, the OTA target competitively displaced the bound TMR-OTA probe from the antibody, causing a decrease in fluorescence. Measuring the change in fluorescence enabled rapid detection of OTA. This method was selective and allowed the detection of 1 nM OTA, showing potential for rapid OTA analysis in applications.

Simultaneous detection of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol in corn and wheat using surface plasmon resonance.

Mycotoxins are toxic metabolites produced by fungi or molds, which may cause serious harm to human health through polluted cereal foods. In order to measure the typical mycotoxin contaminations in wheat and corn, a surface plasmon resonance (SPR) method was established using SPR sensor chip that was fabricated based on self-assembled monolayer. The minimum detection limit of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol were identified as 0.59 ng/mL, 1.27 ng/mL, 7.07 ng/mL and 3.26 ng/mL, respectively. The cross-reactivity for all four mycotoxins were demonstrated to be low. Moreover, the test data were compared with HPLC-MS/MS confirmatory analysis results and good agreement was found between them. In conclusion, the SPR method for simultaneously detecting four mycotoxins has been developed with high sensitivity, good linearity and specificity, which can meet the detection requirements of cereal foods.

Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals.

Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC50 value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027-0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products.

Development of a new format of competitive immunochromatographic assay using secondary antibody-europium nanoparticle conjugates for ultrasensitive and quantitative determination of ochratoxin A.

In this study, to enhance the sensitivity of conventional immunochromatographic assay (ICA) two strategies including using a high sensitive label and changing the assay format, were simultaneously applied to develop an ultrasensitive format of ICA. In new format, primary monoclonal antibody against ochratoxin A (OTA) was used without any labeling, and a secondary polycolonal antibody was labeled with europium fluorescent nanoparticles (EuNPs). Detection was performed in a single step by inserting the test strip into a microtube containing all reagents. The results were obtained within 12 min and read by a portable fluorescent strip reader. Salient features of the new format of ICA compared with conventional format include: (1) A 100-fold decrease in limit of detection (LOD) due to application of two amplification strategy; (2) Reduction in expensive monoclonal antibody consumption. The established method was evaluated for the quantitative determination of OTA with LOD as low as 0.4 pg mL-1.

Ochratoxin A detection in coffee by competitive inhibition assay using chitosan-based surface plasmon resonance compact system.

This study demonstrates the evaluation of ochratoxin A (OTA) in coffee on compact surface plasmon resonance (SPR) biosensors based on crosslinked chitosan and carboxymethyl chitosan nanomatrix substrates. Ochratoxin A is a toxic secondary metabolite widely produced by Aspergillus and Penicillium fungi and requires regular quantification and detection in food samples. The gold coated SPR chips were synthesized with chitosan and carboxymethyl chitosan through spin coating technique. The SPR nanomatrix chips were used for the immobilization of ochratoxin A-bovine serum albumin (OTA-BSA) conjugate to develop a competitive inhibition immunoassay. The monoclonal ochratoxin A antibodies (mAb-OTA) were used as biological receptors for the detection of OTA in buffer and coffee samples. The limit of detection (LOD) in coffee for chitosan (CS) and carboxymethyl chitosan (CMC) substrates was 5.7 ng/mL and 3.8 ng/mL, respectively. Compact surface plasmon resonance (SPR) system based on chitosan-based (CS-AU) nanomatrix substrates provides a platform for the detection of ochratoxin A with high sensitivity, accuracy, ease-of-use and cost-effectiveness. This compact SPR system can be used at farm and industrial levels for the detection of OTA in food matrices.

Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice.

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL-1 and a limit of detection of 0.059 ng mL-1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin-Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal.

Ochratoxin A (OTA) is a common food contaminant that threatens consumers' safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC50) of 0.14 ng mL-1 and the limit of detection (LOD = IC10) of 0.028 ng mL-1 for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 µg kg-1 in barley, 0.56 µg kg-1 in oats, and 0.84 µg kg-1 in rice for OTA. The average recovery percent was in a range of 84-137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system.

Development of a Magnetic Nanoparticles-Based Screen-Printed Electrodes (MNPs-SPEs) Biosensor for the Quantification of Ochratoxin A in Cereal and Feed Samples.

A rapid and sensitive electrochemical biosensor based on magnetic nanoparticles and screen-printed electrodes (MNPs-SPEs sensor) was developed for the detection of ochratoxin A (OTA) in cereal and feed samples. Different types of magnetic nanoparticles-based ELISA (MNPs-ELISA) were optimized, and the signal detection, as well as sensitivity, was enhanced by the combined use of screen-printed electrodes (SPEs). Under the optimized conditions, the calibration curve of the MNPs-SPEs sensor was y = 0.3372x + 0.8324 (R² = 0.9805). The linear range of detection and the detection limit were 0.01⁻0.82 ng/mL and 0.007 ng/mL, respectively. In addition, 50% inhibition (IC50) was detectable at 0.10 ng/mL. The limit of detection (LOD) of this MNPs-SPEs sensor in cereal and feed samples was 0.28 µg/kg. The recovery rates in spiked samples were between 78.7% and 113.5%, and the relative standard deviations (RSDs) were 3.6⁻9.8%, with the coefficient of variation lower than 15%. Parallel analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-SPEs sensor and liquid chromatography tandem mass spectrometry (LC/MS-MS). This new method provides a rapid, highly sensitive, and less time-consuming method to determine levels of ochratoxin A in cereal and feedstuff samples.

Mycotoxin Biosensor Based on Optical Planar Waveguide.

The research aim of this work is to develop a simple and highly sensitive optical biosensor for detection of mycotoxins. This sensor is built on a planar waveguide operating on the polarization interferometry principle, i.e., detecting a phase shift between p- and s-components of polarized light developed during the binding of analyte molecules. The operation of the proposed sensor is similar to that of a Mach⁻Zehnder interferometer, while its design is much simpler and it does not require splitting the waveguide into two arms. The refractive index sensitivity of the polarization interferometer sensor was in the range of 5200 radians per refractive index unit (RIU). Several tests were conducted to detect ochratoxin A (OTA) at different concentrations in direct immunoassay with specific antibodies immobilized in the sensing window. The lowest concentration of OTA of 0.01 ng/mL caused a phase shift of nearly one period. The results obtained prove high sensitivity of the sensors, which are capable of detecting even lower concentrations of mycotoxins at the ppt (part-per-trillion) level.

Determination of Ochratoxin A traces in foodstuffs: Comparison of an automated on-line two-dimensional high-performance liquid chromatography and off-line immunoaffinity-high-performance liquid chromatography system.

Automated on-line two-dimensional high-performance liquid chromatography method (2D-HPLC) is proposed to determine Ochratoxin A (OTA) in food samples as an alternative to OTA immunoaffinity column (IAC). An on-line 2D-HPLC system is designed for the analysis of OTA using an affinity-based monolithic column in the first dimension and reversed-phase C18 column in the second dimension. Initially, optimal OTA separation efficiency is determined through traditional HPLC system consisting of a P(HEMAPA) monolithic column coupled with HPLC system. Secondly, after providing optimum conditions, OTA determination was investigated through the 2D-HPLC system. According to results, 2D-HPLC system showed good linearity in the range 0.5 to 20 ng/mL with limit of detection (LOD) and limit of quantification (LOQ) values of 21.2 pg/mL and 64.3 pg/mL, respectively. The P(HEMAPA)-4 monolithic column displayed good recovery of OTA ranging from 104.34% to 107.33%. Relative standard deviations (RSD) varied in the range 0.21% to 1.31% thus indicating the efficiency of P(HEMAPA)-4 monolithic column developed for OTA.

An ITO-based point-of-care colorimetric immunosensor for ochratoxin A detection.

The accurate and rapid ochratoxin A (OTA) detection at early stage can prevent people from getting cancer to a great extent due to its strong liver toxicity and renal toxicity. The development of an efficient strategy to detect OTA without sophisticated instruments is highly desired, but still remains a severe challenge. In this communication, a sandwich type point-of-care testing (POCT) immunoassay is developed for OTA detection. The 3-aminopropyltrimethoxysilane-glutaraldehyde (APTMS-GA) is used as color matrix and Au-N-TiO2 as labels for loading more captured antibodies and glucose oxidases (GOD). When glucose is added, GOD can be catalyzed to produce H2O2 which can weaken the color strength to quantify OTA concentrations. The mean gray values of reacted ITO-based immunosensors can be calculated by the software of Image J. The linear range of OTA is 0.0001-1 ng mL-1 with a low detection limit of 28 fg mL-1 at a signal-to-noise ratio of 3 s. This strategy can achieve the excellent performance without sophisticated instruments. This POCT method may has a potential application in the undeveloped countries with only a camera-equipped cellphone.

Label-Free QCM Immunosensor for the Detection of Ochratoxin A.

Ochratoxin A (OTA) is a potent mycotoxin that poses a risk in food and feed moieties and subject to worldwide regulation. Laboratory-based analytical methods are traditionally employed for reliable OTA quantification, but these methods cannot provide rapid and on-site analysis, where biosensors fill this gap. In this study a label-free quartz crystal microbalance (QCM)-based immunosensor for the detection of OTA, which is one of the most important small molecule contaminants, was developed by direct immobilization of OTA to amine-bearing sensor surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) chemistry. The protein-free sensor surface enabled regeneration of sensor surface with 50 mM NaOH and 1% SDS up to 13 times without loss of performance, which would disrupt a protein-containing sensor surface. We developed a QCM immunosensor using the developed sensor surface with a 17.2-200 ng/mL detection range which can be used for on-site detection of feedstuffs.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.