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, , 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.

Enzyme cascade-amplified immunoassay based on the nanobody-alkaline phosphatase fusion and MnO 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 MnO 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 MnO nanosheets mimicking the oxidase could be reduced by AA into Mn and catalyze the 3,3',5,5'-tetramethyl benzidine into blue oxidized product for quantification.

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.

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

A novel ultrasensitive electrochemiluminescence (ECL) immunoassay based on Au/CaCO was proposed for detecting ochratoxin A (OTA) in coffee. Au/CaCO 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) and conjugating a high-affinity nanobody (prepared by the phage display technique). Coupling of the Au/CaCO 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.

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.

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 MnO nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule.

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.

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 in rice and oat samples by eye.

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).

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.

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.