Rapid and sensitive immunoassay for alpha-fetoprotein in serum by fabricating primary antibody-enzyme complexes using protein self-assembly.

Primary antibody-enzyme complexes (PAECs) are ideal immunosensing elements that simplify the immunoassay process and improve the uniformity of results due to their ability to both recognize antigens and catalyze substrates. However, the conventional fabrication methods of PAECs, such as direct gene fusion expression, chemical conjugation, enzymatic conjugation, etc., have low efficiency, poor reliability, and other defects, which limit the widespread application of PAECs. Therefore, we developed a convenient method for the fabrication of homogeneous multivalent PAECs using protein self-assembly and validated it using anti-alpha-fetoprotein nanobody (A1) and alkaline phosphatase (ALP) as models. Heptavalent PAECs showed a 4-fold enhancement in enzymatic catalytic activity compared to monovalent PAECs. Further, to verify the application of developed heptavalent PAECs in immunoassay, heptavalent PAECs were used as bifunctional probes to construct a double-antibody sandwich ELISA to detect AFP. The detection limit of the developed heptavalent PAEC-based ELISA is 0.69 ng mL-1, which is about 3 times higher than that of monovalent PAECs, and the whole detection process can be completed within 3 hours. In short, the proposed protein self-assembling method is a promising technology for developing high-performance heptavalent PACEs, which can simplify the detection process and improve detection sensitivity in various immunoassays.

Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum.

Alpha fetoprotein (AFP) is an important biomarker for diagnosis of hepatocellular carcinoma (HCC). Whereas, it is always a challenge to detect trace AFP in serum. In this work, a ratiometric fluorescence enzyme immunoassay (RFEIA) was developed using nanobody-alkaline phosphatase (Nb-AP) heptamer and MnFe layered double hydroxides nanoflakes (MnFe LDH) for ultrasensitive detection of AFP. The Nb-AP heptamer (Nb-C4bpα-AP) was constructed by fusion expression of Nb, AP, and α-chain of C4 binding protein (C4bpα), where the C4bpα contributed to multimerization through self-assembly. The dual functional Nb-C4bpα-AP can recognize AFP, dephosphorylate ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and thus tune the MnFe LDH-mediated ratiometric fluorescence, which was generated from the oxidization of MnFe LDH on o-phenylenediamine (OPD) and the catalyzation of MnFe LDH on the cyclization reaction between AA and OPD. By integration of Nb-C4bpα-AP, MnFe LDH, AAP, and OPD, the RFEIA showed a limit of detection of 0.013 ng/mL with good selectivity, accuracy and precision. Furthermore, results of clinical serum samples tested by the RFEIA were well confirmed by the automated chemiluminescence immunoassay analyzer. Thus, this work demonstrated that the Nb-C4bpα-AP is a robust immunoreagent and the developed RFEIA could be a very promising tool for diagnosis of HCC.

Fluonanobody-based nanosensor via fluorescence resonance energy transfer for ultrasensitive detection of ochratoxin A.

Ochratoxin A (OTA) contamination in food is a serious threat to public health. There is an urgent need for development of rapid and sensitive methods for OTA detection, to minimize consumer exposure to OTA. In this study, we constructed two OTA-specific fluonanobodies (FluoNbs), with a nanobody fused at the carboxyl-terminal (SGFP-Nb) or the amino-terminal (Nb-SGFP) of superfolder green fluorescence protein. SGFP-Nb, which displayed better fluorescence performance, was selected as the tracer for OTA, to develop a FluoNb-based nanosensor (FN-Nanosens) via the fluorescence resonance energy transfer, where the SGFP-Nb served as the donor and the chemical conjugates of OTA-quantum dots served as the acceptor. After optimization, FN-Nanosens showed a limit of detection of 5 pg/mL, with a linear detection range of 5-5000 pg/mL. FN-Nanosens was found to be highly selective for OTA and showed good accuracy and repeatability in recovery experiments using cereals with various complex matrix environments. Moreover, the contents of OTA in real samples measured using FN-Nanosens correlated well with those from the liquid chromatography with tandem mass spectrometry. Therefore, this work illustrated that the FluoNb is an ideal immunosensing tool and that FN-Nanosens is reliable for rapid detection of OTA in cereals with ultrahigh sensitivity.

Electrochemiluminescence Immunosensor Based on Nanobody and Au/CaCO3 Synthesized Using Waste Eggshells for Ultrasensitive Detection of Ochratoxin A.

A novel ultrasensitive electrochemiluminescence (ECL) immunoassay based on Au/CaCO3 was proposed for detecting ochratoxin A (OTA) in coffee. Au/CaCO3 nanocomposites synthesized using waste eggshells as the template with a large surface area and excellent electrochemical properties were applied for immobilizing a large amount of Ru(bpy)3 2+ and conjugating a high-affinity nanobody (prepared by the phage display technique). Coupling of the Au/CaCO3 nanocomposites and nanobody technologies provided an ultrasensitive and highly selective ECL immunosensor for OTA detection in the range of 10 pg/mL-100 ng/mL with a low detection limit of 5.7 pg/mL. Moreover, the as-prepared ECL immunosensor showed excellent performance and high stability. Finally, the proposed ECL sensor was applied to analyze OTA in coffee samples, confirming the desirable accuracy and practical applicability potential. Overall, this work presents a new nanomaterial for fabricating the sensing interface of immunosensors by harnessing natural waste as the source and a method for detecting toxic OTA in foods.

Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum.

Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO2 nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO2 nanoflakes mimic the oxidase for catalyzing the 3,3',5,5'-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO2 colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.

Enzyme cascade-amplified immunoassay based on the nanobody-alkaline phosphatase fusion and MnO2 nanosheets for the detection of ochratoxin A in coffee.

Ochratoxin A (OTA) is a common food contaminant with multiple toxicities and thus rapid and accurate detection of OTA is indispensable to minimize the threat of OTA to public health. Herein a novel enzyme cascade-amplified immunoassay (ECAIA) based on the mutated nanobody-alkaline phosphatase fusion (mNb-AP) and MnO2 nanosheets was established for detecting OTA in coffee. The detection principle is that the dual functional mNb-AP could specifically recognize OTA and dephosphorylate the ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and the MnO2 nanosheets mimicking the oxidase could be reduced by AA into Mn2+ and catalyze the 3,3',5,5'-tetramethyl benzidine into blue oxidized product for quantification. Using the optimal conditions, the ECAIA could be finished within 132.5 min and shows a limit of detection of 3.38 ng mL-1 (IC10) with an IC50 of 7.65 ng mL-1 and a linear range (IC20-IC80) of 4.55-12.85 ng mL-1. The ECAIA is highly selective for OTA. Good recovery rates (84.3-113%) with a relative standard deviation of 1.3-3% were obtained and confirmed by high performance liquid chromatography with a fluorescence detector. The developed ECAIA was demonstrated to be a useful tool for the detection of OTA in coffee which provides a reference for the analysis of other toxic small molecules.

Phage-mediated double-nanobody sandwich immunoassay for detecting alpha fetal protein in human serum.

Alpha fetal protein (AFP) is a significant biomarker of liver cancer. Herein we developed a novel phage-mediated double-nanobody sandwich immunoassay (P-ELISA) for sensitive detection of AFP in serum, where the phage displayed the nanobody for antigen recognition and multiple copies of major coat protein pVIII for signal amplification. The expressed nanobody Nb-A1 and the phage-displayed nanobody phage-A2 served as the capture antibody and detection antibody, respectively. Based on the optimal experimental conditions, the P-ELISA has a half maximal saturation concentration of 24.85 ng mL-1 and a limit of detection of 0.237 ng mL-1 for AFP. The P-ELISA is highly selective for AFP and ignorable cross-reactions were observed with other tested cancer biomarkers. After elimination of the matrix effect by 30-fold dilution with 0.5 × PBS, clinical serum samples were analyzed by the P-ELISA. The results correlated well with those of the AFP commercial ELISA kit and the Roche E601 automatic chemiluminescence analyzer. Thus, it showed the potential of the recombinant phage for highly sensitive and selective detection of AFP and provides a novel detection model for the other disease-related biomarkers.

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.

Development of a nanobody tagged with streptavidin-binding peptide and its application in a Luminex fluoroimmunoassay for alpha fetal protein in serum.

Sensitive and accurate detection of disease-related biomarkers can promote the early screening and diagnosis of cancers for improving the prognosis and survival of patients. Herein alpha fetal protein (AFP) was selected as the model macromolecule antigen and we developed AFP-specific alpaca nanobodies (Nbs) from an immunized phage-displayed Nb library. Then Nbs tagged with streptavidin-binding peptide (Nb-SBP) were constructed and used to develop an Nb-SBP-mediated fluoroimmunoassay based on the Luminex-200 system (NS-LFIA). Based on the optimal experimental conditions, the NS-LFIA has a limit of detection of 0.237 ng mL-1 with a linear detection range of 0.49-125 ng mL-1. The average recovery rate and relative standard derivation were in the range of 98.2-110% and 2.8-13.8%, respectively. The NS-LFIA is highly selective for AFP and ignorable cross-reaction was observed with the other biomarkers. The content of AFP in clinical serum samples was determined by both the developed NS-LFIA and the Roche E601 automatic chemiluminescence immunoassay analyzer and a good correlation was obtained between the two methods (R 2 = 0.9894). Moreover, the Nb-SBP can significantly improve the homogeneity of the fluorescent signals tested by the Luminex-200 system compared with the biotinylated conventional monoclonal antibodies, which could reduce the magnetic microsphere consumption and test cost by decreasing the repetitions of each sample. Thus the results demonstrated that the Nb-SBP was a very promising immunological diagnostic reagent and indicated the applicability and reliability of the NS-LFIA for sensitive detection of AFP and other disease-related biomarkers.

Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay.

Ochratoxin A (OTA) is a common cereal mycotoxin that seriously threatens food safety and public health. Herein a horseradish peroxidase-nanobody fusion protein (HRP-Nb) retaining antibody and enzyme activity was obtained after inclusion body denaturation and renaturation and enzyme reconstitution, which served both as the primary antibody and reporter enzyme and was applied to develop a membrane-based dot immunoassay (HN-DIA) for OTA visual detection. Based on the optimal experimental conditions, the HN-DIA could be finished in 10 min with a cut-off limit of 50 µg kg-1 in rice and oat samples by eye. The HN-DIA showed high selectivity for OTA and had good accuracy and reproducibility in the recovery experiments. Spiked sample analysis results of the HN-DIA and high performance liquid chromatography (HPLC) correlated well with each other. Therefore, the proposed HN-DIA has the potential for rapid screening of OTA and other small molecule pollutants in food and the environment by naked eye.

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.