Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.

Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.

Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

Nanobodies for Accurate Recognition of Iso-tenuazonic Acid and Development of Sensitive Immunoassay for Contaminant Detection in Foods.

The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.

Molecular Evolution of Antiparathion Nanobody with Enhanced Sensitivity and Specificity Based on Structural Analysis.

Nanobody (Nb) has gained significant attention in immunoassays owing to its numerous advantages, particularly its ease of molecular evolution. However, the limited understanding of how high sensitivity and specificity attained for antihapten Nbs hamper the development of high-performance Nbs. Herein, the antiparathion Nb (Nb9) we prepared previously was chosen as the model, and an approach based on X-ray crystallography, molecular docking, and rational site-directed saturation mutation for constructing a rapid and effective platform for nanobody evolution was described. Based on the structural analysis, two mutants, namely Nb-D5 (IC50 = 2.4 ± 0.2 ng/mL) and Nb-D12 (IC50 = 2.7 ± 0.1 ng/mL), were selected out from a six-sites directed saturation mutation library, 3.5-fold and 3.1-fold sensitivity enhancement over Nb9 to parathion, respectively. Besides, Nb-D12 exhibited improved sensitivity for quinalphos, triazophos, and coumaphos (5.4-35.4 ng/mL), indicating its broader detection potential. Overall, our study advances an effective strategy for the future rational evolution of Nbs with desirable performance.

Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

[Case control study on clinical effects of sacrococcygeal manipulation in the treatment of coccyx pain].

OBJECTIVE: To study the clinical efficacy of sacral manual therapy in the treatment of coccygodynia. METHODS: From November 2013 to July 2015, 184 patients with sacrococcygeal pain were divided into treatment group and control group. There were 26 males and 65 females in the treatment group, with an average age of (39.63±11.62) years old. In the control group, there were 31 males and 62 females, with an average age of (41.47±11.56) years old. The patients in the treatment group were treated with sacrococcygeal massage therapy, 3 times a week for 2 weeks. The patients in the control group were treated with Diclofenac Diethylamine Emulgel, 2 times a day for 2 weeks. The VAS pain score, score in rating scale of sacrococcygeal pain and degree of tenderness were obtained on the first day of treatment, 2, 7, 14 days and 3 months after treatment to evaluate clinical results. RESULTS: When comparing the VAS pain score of sacrococcygeal pain within the two groups, the differences began to reach statistical significance on the second day(P<0.001). The chagne of VAS pain scores, the change of scores in rating scale of sacrococcygeal pain and the degree of tenderness in the treatment group were all significontly larger that those in the contral group from the second day. CONCLUSIONS: The curative effect of sacral manipulation group is better than that of Diclofenac Diethylamine Emulgel group in the treatment of sacrococcygeal pain.

[Bacteriological study on adult chronic sinusitis operated after endoscopic sinus surgery].

OBJECTIVE: To explore the bacteria isolated from middle nasal meatus, maxillary sinus, ethmoid sinus and postoperative cavity of patients with chronic rhinosinusitis and their characteristics of antibiotic resistance. METHODS: Eighty-seven patients with chronic rhinosinusitis were operated on by ESS to obtain the pus specimen for bacterial culture and antibiotic susceptibility test, before and 1 month, 3 months and 6 months after operation. RESULTS: Totally 645 strains (26 species) of bacteria were detected in 464 specimens [total positive rate was 78.9% (366/464)], in which aerobic bacteria was 95.3% (615/645). Gram negative bacteria and gram positive bacteria were 51.2% (330/645) and 48.8% (315/645), respectively. There was supernumerary tendency in detectable rate of gram negative bacteria isolated from postoperative groups. The main pathogens of postoperative patients were gram negative bacteria, with Enterobacter aerogenes, Pseudomonas aeruginosa and Hemophilus influenza occupying the first 3 places. The detectable rate of multiple drug resistance bacteria in postoperative group was much higher than preoperative groups, in which gram negative bacteria was the most, especially for Pseudomonas aeruginosa. There was significant difference in beta-lactamase detectable rate of the bacteria isolated from the delayed recovery group and the preoperative group (chi2 = 4.85, P < 0.05), Enterobacteriaceae occupied the first place among the beta-lactamase detectable bacteria isolated from the delayed recovery group. There was no significant difference in detectable rate of kinds of bacteria isolated from recovery group and control group. CONCLUSIONS: The main pathogens of patients with chronic rhinosinusitis are multiple drug resistance gram negative bacteria after operation, in which Pseudomonas aeruginosa occupies the first place. Gram negative bacteria are becoming the main opportunity pathogenic bacteria, which shows antibiotic resistance. microbial population of postoperative cavity from recovery group are becoming balanced.