Development of a Phage-Displayed Nanobody-Based Competitive Immunoassay for the Sensitive Detection of Soybean Agglutinin.

Soybean agglutinin (SBA) is a primary antinutritional factor in soybeans that can inhibit the growth of humans and mammals, disrupt the intestinal environment, and cause pathological changes. Therefore, detecting and monitoring SBA in foods is essential for safeguarding human health. In this paper, M13 phage-displayed nanobodies against SBA were isolated from a naive nanobody library. An M13 phage-displayed nanobody-based competitive enzyme-linked immunosorbent assay (P-cELISA) was then established for SBA analysis using biotinylated anti-M13 phage antibody (biotin-anti-M13) and streptavidin poly-HRP conjugate (SA-poly-HRP). The biotin-anti-M13@SA-poly-HRP probe can easily amplify the detection signal without the chemical modifications of phage-displayed nanobodies. The established P-cELISA presented a linear detection range of 0.56-250.23 ng/mL and a limit of detection (LOD) of 0.20 ng/mL, which was 12.6-fold more sensitive than the traditional phage-ELISA. Moreover, the developed method showed good specificity for SBA and acceptable recoveries (78.21-121.11%) in spiked wheat flour, albumen powder, and whole milk powder. This study proposes that P-cELISA based on biotin-anti-M13@SA-poly-HRP may provide a convenient and effective strategy for the sensitive detection of SBA.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products.

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen-antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

Dosage-sensitive and simultaneous detection of multiple small-molecule pollutants in environmental water and agriproducts using portable SERS-based lateral flow immunosensor.

The co-contamination of pesticide residues and mycotoxins in agricultural products is a global concern, with the potential for cumulative and synergistic damaging effects, imposing substantial health and economic burdens to the public. The dosage-sensitive and simultaneous detection of multiple pollutants, with a heightened sensitivity in real samples, poses a significant demand and challenge. Herein, we propose a portable detection method integrating surface-enhanced Raman scattering (SERS)-with lateral flow immunoassay (LFIA), offering high sensitivity and multiplex analysis capabilities. This approach enables the simultaneous detection of imidacloprid (IMI), pyraclostrobin (PYR) and aflatoxin B1 (AFB1) through a single test strip. Utilizing the immune-specific binding between antigen and antibodies, we immobilised antibody- conjugated SERS nanotags on three test lines of the strips to generate Raman signal amplification in the proposed biosensor. Accurate quantitative analysis was performed by measuring the SERS signal intensity on the test lines. The limits of detection were 8.6 pg/mL for IMI, 97.4 pg/mL for PYR and 8.9 pg/mL for AFB1, exhibiting sensitivities 12-fold, 102-fold and11-fold higher than the colorimetric signals, respectively. Importantly, the SERS-LFIA immunosensor demonstrated robust performance when applied to real samples, yielding recoveries ranging from 86.16 % to 115.0 %, with relative standard deviation values below 8.67 %. These results underscore the excellent stability, high selectivity and reliability the proposed SERS-LFIA immunosensor. Consequently, it holds promise for the detection of multiple pesticides and mycotoxins in both environmental and agricultural samples.

Protected Geographical Indication Discrimination of Zhejiang and Non-Zhejiang Ophiopogonis japonicus by Near-Infrared (NIR) Spectroscopy Combined with Chemometrics: The Influence of Different Stoichiometric and Spectrogram Pretreatment Methods.

This paper presents a method for the protected geographical indication discrimination of Ophiopogon japonicus from Zhejiang and elsewhere using near-infrared (NIR) spectroscopy combined with chemometrics. A total of 3657 Ophiopogon japonicus samples from five major production areas in China were analyzed by NIR spectroscopy, and divided into 2127 from Zhejiang and 1530 from other areas ('non-Zhejiang'). Principal component analysis (PCA) was selected to screen outliers and eliminate them. Monte Carlo cross validation (MCCV) was introduced to divide the training set and test set according to a ratio of 3:7. The raw spectra were preprocessed by nine single and partial combination methods such as the standard normal variable (SNV) and derivative, and then modeled by partial least squares regression (PLSR), a support vector machine (SVM), and soft independent modeling of class analogies (SIMCA). The effects of different pretreatment and chemometrics methods on the model are discussed. The results showed that the three pattern recognition methods were effective in geographical origin tracing, and selecting the appropriate preprocessing method could improve the traceability accuracy. The accuracy of PLSR after the standard normal variable was better, with R2 reaching 0.9979, while that of the second derivative was the lowest with an R2 of 0.9656. After the SNV pretreatment, the accuracy of the training set and test set of SVM reached the highest values, which were 99.73% and 98.40%, respectively. The accuracy of SIMCA pretreated with SNV and MSC was the highest for the origin traceability of Ophiopogon japonicus, which could reach 100%. The distance between the two classification models of SIMCA-SNV and SIMCA-MSC is greater than 3, indicating that the SIMCA model has good performance.

Quantum-Dot-Bead-Based Fluorescence-Linked Immunosorbent Assay for Sensitive Detection of Cry2A Toxin in Cereals Using Nanobodies.

In this study, a quantum-dot-bead (QB)-based fluorescence-linked immunosorbent assay (FLISA) using nanobodies was established for sensitive determination of the Cry2A toxin in cereal. QBs were used as the fluorescent probe and conjugated with a Cry2A polyclonal antibody. An anti-Cry2A nanobody P2 was expressed and used as the capture antibody. The results revealed that the low detection limit of the developed QB-FLISA was 0.41 ng/mL, which had a 19-times higher sensitivity than the traditional colorimetric ELISA. The proposed assay exhibited a high specificity for the Cry2A toxin, and it had no evident cross-reactions with other Cry toxins. The recoveries of Cry2A from the spiked cereal sample ranged from 86.6-117.3%, with a coefficient of variation lower than 9%. Moreover, sample analysis results of the QB-FLISA and commercial ELISA kit correlated well with each other. These results indicated that the developed QB-FLISA provides a potential approach for the sensitive determination of the Cry2A toxin in cereals.

A Smartphone Colorimetric Sensor Based on Pt@Au Nanozyme for Visual and Quantitative Detection of Omethoate.

A smartphone colorimetric sensor based on the Pt@Au nanozyme was successfully developed for the visual and quantitative detection of omethoate in fruit and vegetables. The anti-omethoate antibody was conjugated on the surface of the Pt@Au nanozyme as a catalytic functional signal probe, and coating antigen conjugated on the surface of magnetic polystyrene microspheres (MPMs) was used as a separation capture probe. In the sensing system, when the catalytic functional signal probe was combined with a separation capture probe containing no omethoate, the visible blue color appeared with the addition of tetramethylbenzidine (TMB) chromogenic solution, and the maximum B value of the sensing system was obtained via the smartphone. With increasing concentrations of omethoate, the visualization of the sensing system decreased, and the B-value obtained via the smartphone dropped. Under optimal detection conditions, the omethoate could be detected in a linear range of 0.5-50 µg/L (R2 = 0.9965), with a detection limit of 0.01 µg/L. The accuracy and reliability of the detection results of this colorimetric sensor were successfully confirmed by enzyme linked immunosorbent assay (ELISA) and gas chromatography. This colorimetric sensor provides a technical reference and potential strategy for the immunoassay of hazard factors in resource-scarce laboratories.

Low-Cost Detection of Methane Gas in Rice Cultivation by Gas Chromatography-Flame Ionization Detector Based on Manual Injection and Split Pattern.

Rice cultivation is one of the most significant human-created sources of methane gas. How to accurately measure the methane concentration produced by rice cultivation has become a major problem. The price of the automatic gas sampler used as a national standard for methane detection (HJ 38-2017) is higher than that of gas chromatography, which greatly increases the difficulty of methane detection in the laboratory. This study established a novel methane detection method based on manual injection and split pattern by changing the parameters of the national standard method without adding any additional automatic gas samplers. The standard curve and correlation coefficient obtained from the parallel determination of methane standard gas were y = 2.4192x + 0.1294 and 0.9998, respectively. Relative standard deviation (RSD, <2.82%), recycle rate (99.67−102.02%), limit of detection (LOD, 0.0567 ppm) and limit of quantification (LOQ, 0.189 ppm) of this manual injection method are satisfying, demonstrating that a gas chromatography-flame ionization detector (GC-FID), based on manual injection at a split ratio (SR) of 5:1, could be an effective and accurate method for methane detection. Methane gases produced by three kinds of low-methane rice treated with oxantel pamoate acid, fumaric acid and alcohol, were also collected and detected using the proposed manual injection approach Good peak shapes were obtained, indicating that this approach could also be used for quantification of methane concentration.

Highly Specific Monoclonal Antibody and Sensitive Quantum Dot Beads-Based Fluorescence Immunochromatographic Test Strip for Tebuconazole Assay in Agricultural Products.

A monoclonal antibody (mAb) was raised against tebuconazole (TEB) using a hapten where the p-chloro substituent of the TEB molecule was replaced with a long-chain carboxylic acid. The resulting mAb showed high sensitivity and specificity against TEB characterized by ELISA with a half-maximal inhibitory concentration (IC50) of 0.19 ng mL-1 and with cross-reactivity (CR) values below 0.01% to several analogues of triazole fungicides. On the basis of the mAb produced, a quantum dot beads-based fluorescence immunochromatographic test strip assay (QBs-FITSA) was developed for rapid and sensitive detection of TEB in agricultural product samples. The QBs-FITSA exhibited a linear detection range from 0.02 to 1.25 ng mL-1 with a limit of detection (LOD) of 0.02 ng mL-1. Furthermore, using produced mAb, multiple high-throughput rapid immunoassay formats could be achieved as a convenient monitoring tool for evaluation of human and environmental exposure to TEB.

Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody.

Cry toxins have been widely used in genetically modified organisms for pest control, raising public concern regarding their effects on the natural environment and food safety. In this work, a phage-mediated competitive chemiluminescent immunoassay (c-CLIA) was developed for determination of Cry1Ab toxin using anti-idiotypic camel nanobodies. By extracting RNA from camels' peripheral blood lymphocytes, a naive phage-displayed nanobody library was established. Using anti-Cry1Ab toxin monoclonal antibodies (mAbs) against the library for anti-idiotypic antibody screening, four anti-idiotypic nanobodies were selected and confirmed to be specific for anti-Cry1Ab mAb binding. Thereafter, a c-CLIA was developed for detection of Cry1Ab toxin based on anti-idiotypic camel nanobodies and employed for sample testing. The results revealed a half-inhibition concentration of developed assay to be 42.68 ± 2.54 ng/mL, in the linear range of 10.49-307.1 ng/mL. The established method is highly specific for Cry1Ab recognition, with negligible cross-reactivity for other Cry toxins. For spiked cereal samples, the recoveries of Cry1Ab toxin ranged from 77.4% to 127%, with coefficient of variation of less than 9%. This study demonstrated that the competitive format based on phage-displayed anti-idiotypic nanobodies can provide an alternative strategy for Cry toxin detection.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay.

In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL(-1) and 19.97 ± 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of "nanobody (N-28) - anti-DON MAb" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.

Anti-idiotypic nanobody: A strategy for development of sensitive and green immunoassay for Fumonisin B1.

Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2ß Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2ß Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27-5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1-BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2ß Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2ß Nb replacing the antigen-carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.

Use of cloneable peptide-MBP fusion protein as a mimetic coating antigen in the standardized immunoassay for mycotoxin ochratoxin A.

The quality of mycotoxin conjugates is essential to the development of reliability of immunoassays for mycotoxins. However, conventional mycotoxin conjugates are usually synthesized by chemical methods, which are harmful to the environment and yield unwanted cross-reactions. In this study, using ochratoxin A (OTA) as a model system, a selected OTA mimotope (phage-displayed peptide) that specifically binds to anti-OTA antibody was expressed as soluble and monovalent fusions to maltose binding protein (MBP). These prepared fusion proteins can serve as a mimetic coating antigen in both a quantitative chemiluminescent enzyme-linked immunoassay (CLEIA) and a qualitative dot immunoassay for OTA. One of the prepared mimetic coating antigen (L12-206-MBP)-based CLEIAs exhibited a half-inhibition concentration (IC50) of 0.82 ng/mL and a working range of 0.30-2.17 ng/mL, which resemble those of the conventional OTA-OVA conjugate-based immunoassay. The dot immunoassay developed with both the OTA-OVA conjugate and the mimetics showed identical visual cutoff values of 5 ng/mL. The mimetic coating antigen proposed here is an OTA-free product and can be prepared reproducibly as a homogeneous product and facilitates standardization of immunoassays for the mycotoxin OTA.

New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B(1) based on using peptide-MBP fusion protein as substitute for coating antigen.

Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are "clonable" homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 ± 0.13 ng/mL and 1.26 ± 0.08 ng/mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1-BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.

Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01-1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.