Myasthenia gravis and myocarditis induced by chemoimmunotherapy in locally advanced gastric cancer: A case report.

The treatment strategy of patients with locally advanced gastric cancer has undergone notable changes since immune checkpoint inhibitors (ICIs) were developed. Although ICIs are generally well-tolerated, they can also cause serious adverse events, such as autoimmune diseases. In patients with gastric cancer and without a history of immune disease, the incidence of myasthenia gravis combined with myocarditis caused by ICI treatment is rare. Furthermore, cases of gastric cancer with ocular myasthenia gravis, without limb weakness or severe dyspnea, although with urination difficulties and symptoms of third-degree atrioventricular block have not been previously reported, to the best of our knowledge. The present study describes the case of a 72-year-old male patient with locally advanced gastric cancer that was treated with chemoimmunotherapy with oxaliplatin + tigio + sintilimab. At 19 days following only one cycle of therapy, the patient developed a left eyelid weakness and difficulty in urinating, as well as diplopia. At 5 days after the symptom of eyelid weakness, a third-degree atrioventricular block occurred. Hormone therapy, a temporary pacemaker and gamma-globulin therapy were administered, and the patient was discharged 1 month later with the resolution of myasthenia gravis and the atrioventricular block. At the final follow-up (1 month after discharge), the patient had a full recovery from myasthenia gravis and arrhythmias. Although some similar cases have been previously reported, the majority of patients with limb weakness and have eventually succumbed; moreover, clinical symptoms were identified at a late stage, and the disease evolution records were not detailed. Therefore, the present study describes the case of the patient and treatment strategy, also providing detailed laboratory indicators and clinical symptom evolution. This was performed with the aim to aid future research and the treatment of immune-related diseases.

Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

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.

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.

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.