Nanobody based immunoassay for alpha fetal protein detection using streptavidin-conjugated polymerized horseradish peroxidase for signal amplification.

The enzyme-linked immunosorbent assay (ELISA) offers several advantages, including simple operation, high throughput, and low cost, making it an ideal immunoassay method for efficient screening of disease-related biomarkers in clinical samples. However, the traditional colorimetric ELISA has relatively low sensitivity, which promotes the continuous emergence of various novel signal amplification technologies. In this work, we fused the AFP-specific nanobody (A1) with the streptavidin-binding peptide (SBP) to develop a fusion protein (A1-SBP) as biorecognition element in a colorimetric ELISA for detecting AFP.

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.

Nanobody-Nanoluciferase Fusion Protein-Enabled Immunoassay for Ochratoxin A in Coffee with Enhanced Specificity and Sensitivity.

Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase-nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples.

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).

Effect of EGCG-gelatin biofilm on the quality and microbial composition of tilapia fillets during chilled storage.

The effects of the (-)-Epigallocatechin gallate (EGCG)-gelatin biofilm treatment (EGT) on microbial composition and quality of tilapia fillets stored at low temperatures were evaluated. The changes in mechanical properties, microbial reproduction, as well as lipid and protein oxidation during fillets storage were determined. The results showed that EGT reduced the microbial count and the relative abundance of the fillets.