食品安全免疫层析检测技术研究进展

免疫层析技术(immunochromatography assay, ICA)不仅有效结合了层析法卓越的分离能力以及常规免疫分析方法的高度特异性,而且简单、快速、灵敏,为现场即时筛查提供一种理想的技术手段。近年来,免疫层析技术在食品安全检测中不断更新、完善和发展,包括标记材料、信号增强、高通量和定量分析等的技术革新,显示出巨大的发展潜力和广阔的应用前景。本文总结归纳了ICA在食品安全检测中的最新应用进展,汇总分析了ICA在我国食品安全领域标准化的情况,并讨论了ICA现有的不足以及在未来应用中的发展方向,以期为我国食品安全快速检测的发展提供技术参考。     

脊髓灰质炎病毒Ⅲ型Sabin株C抗原和D抗原的分离及特性分析

目的分离脊髓灰质炎病毒Ⅲ型Sabin株(sPVⅢ)C抗原和D抗原,并分析其部分特性。方法采用CsCl密度梯度离心法分离sPVⅢ的C抗原和D抗原,电镜观察病毒形态;SDS-PAGE检测病毒结构蛋白组成;微量BCA法检测蛋白浓度,计算每种抗原所占比例;细胞病变法检测病毒滴度。同时检测上样浓度及超离次数对C抗原纯度的影响。结果sPVⅢC抗原和D抗原在透射电镜下均呈球形,C抗原病毒颗粒密度较小,外壳大多较为松散,D抗原病毒颗粒密度较大,结构更稳固;C抗原由VP0、VP1、VP3蛋白组成,D抗原由VP1、VP2、VP3和VP4蛋白组成;C抗原和D抗原分别约占总蛋白含量的10%和90%,部分C抗原在分离前及分离过程中易产生聚集;D抗原的病毒滴度高于C抗原。降低上样浓度及进行二次超离可提高C抗原纯度。结论采用CsCl密度梯度离心法成功分离了sPVⅢ的C抗原和D抗原,C抗原滴度远低于D抗原,且部分易产生聚集,较难达到较高纯度。     

The Impact of the ‘Mis-Peptidome’ on HLA Class I-Mediated Diseases: Contribution of ERAP1 and ERAP2 and Effects on the Immune Response

The strong association with the Major Histocompatibility Complex (MHC) class I genes represents a shared trait for a group of autoimmune/autoinflammatory disorders having in common immunopathogenetic basis as well as clinical features. Accordingly, the main risk factors for Ankylosing Spondylitis (AS), prototype of the Spondyloarthropathies (SpA), the Behçet’s disease (BD), the Psoriasis (Ps) and the Birdshot Chorioretinopathy (BSCR) are HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02, respectively. Despite the strength of the association, the HLA pathogenetic role in these diseases is far from being thoroughly understood. Furthermore, Genome-Wide Association Studies (GWAS) have highlighted other important susceptibility factors such as Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and, less frequently, ERAP2 that refine the peptidome presented by HLA class I molecules to CD8⁺ T cells. Mass spectrometry analysis provided considerable knowledge of HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02 immunopeptidome. However, the combined effect of several ERAP1 and ERAP2 allelic variants could generate an altered pool of peptides accounting for the “mis-immunopeptidome” that ranges from suboptimal to pathogenetic/harmful peptides able to induce non-canonical or autoreactive CD8⁺ T responses, activation of NK cells and/or garbling the classical functions of the HLA class I molecules. This review will focus on this class of epitopes as possible elicitors of atypical/harmful immune responses which can contribute to the pathogenesis of chronic inflammatory diseases.     

新型氨基甲酸乙酯人工抗原的制备与表征

目的合成新型氨基甲酸乙酯(ethylcarbamate,EC)人工抗原并对其效果进行表征分析。方法用4-(二苯基羟甲基)苯甲酸对EC衍生化合成半抗原,用核磁氢谱(proton nuclear magnetic resonance, NMR)、核磁碳谱和质谱法(massspectrometry,MS)对其结构进行表征;用活化酯法将EC半抗原与牛血清蛋白(bovine serum albumin, BSA)偶联得到EC人工抗原。用紫外光谱和荧光光谱法对人工抗原进行表征并计算偶联比,用考马斯亮蓝法测定蛋白含量,间接竞争酶联免疫法鉴定其免疫活性。结果 EC半抗原结构与理论结构一致,半抗原与BSA成功偶联,偶联比为13:1,冻干粉的蛋白含量为0.695 mg/mg,用该新型人工抗原免疫小鼠,得到的抗血清效价为1:10000,且特异性较好。结论该新型氨基甲酸乙酯半抗原具有较好的免疫活性。     

Optimizing PSMA Radioligand Therapy for Patients with Metastatic Castration-Resistant Prostate Cancer. A Systematic Review and Meta-Analysis

The aim of the review was to evaluate patient and treatment characteristics for patients with metastatic castration-resistant prostate cancer (mCRPC) treated with PSMA radioligand therapy (PRLT) associated with above-average outcome. The systematic review and meta-analysis followed recommendations by the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA). We searched for publications in PubMed, Embase, and ClinicalTrials.gov up to 31 September 2020. Thirty-six publications and four duplicates reported 2346 patients. Nearly two-thirds of the patients had bone metastases. Median overall survival (OS) was 16 months. Asymptomatic patients and patients with only lymph node metastases lived longer than symptomatic patients and patients with more extensive metastases. Patients treated with an intensified schedule of ¹⁷⁷Lu PRLT lived longer than those treated with a conventional schedule. Half of the patients obtained a PSA decline ≥ 50% and these patients lived longer than those with less PSA decline. Approximately 10% of the patients developed hematologic toxicity with anemia grade 3 as the most severe adverse effect. Characteristics for patients, cancer, restaging, and PRLT predict above average overall survival following treatment with PRLT.     

土霉素完全抗原的制备及ELISA检测方法的建立

为制备土霉素(Oxytetracycline,OTC)完全抗原,获得免疫学特性良好的土霉素鼠源多抗血清并建立OTC免疫学检测方法,采用重氮化法在OTC分子上引入羧基,进而利用改进碳二亚胺法将其与载体蛋白偶联制备完全抗原,通过红外扫描、紫外扫描和凝胶电泳鉴定其偶联效果,然后将完全抗原免疫BLAB/c小鼠获得多抗血清(pAb)并对其免疫学特性进行鉴定,通过优化条件建立OTC间接竞争ELISA检测方法。结果表明,偶联效果良好,免疫的4只小鼠多抗血清效价均达到1∶12 800;4只小鼠多抗血清均有抑制效果,其中2号小鼠产生的多抗血清效果最佳,基于该多抗血清建立的OTC间接竞争ELISA检测方法半数抑制浓度(IC_(50))为32.92ng/mL,检测限(LOD)为1.3ng/mL,牛奶样品中添加回收率在84.70%～87.45%,与金霉素、四环素的交叉反应率分别为5.27%,4.10%,与其他竞争物交叉反应率0.4%。该试验成功合成了OTC完全抗原,获得了免疫学特性良好的OTC多抗血清,并建立了OTC免疫学检测方法。     

Evaluation of the PSMA-Binding Ligand 212Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with ²¹²Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of ²¹²Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of ²¹²Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3–10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of ²¹²Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of ²¹²Pb-NG001.     

Dendrimer as nanocarrier for drug delivery

Dendrimers are novel three dimensional, hyperbranched globular nanopolymeric architectures. Attractive features like nanoscopic size, narrow polydispersity index, excellent control over molecular structure, availability of multiple functional groups at the periphery and cavities in the interior distinguish them amongst the available polymers. Applications of dendrimers in a large variety of fields have been explored. Drug delivery scientists are especially enthusiastic about possible utility of dendrimers as drug delivery tool. Terminal functionalities provide a platform for conjugation of the drug and targeting moieties. In addition, these peripheral functional groups can be employed to tailor-make the properties of dendrimers, enhancing their versatility. The present review highlights the contribution of dendrimers in the field of nanotechnology with intent to aid the researchers in exploring dendrimers in the field of drug delivery.     

Nipple Discharge of CA15-3, CA125, CEA and TSGF as a New Biomarker Panel for Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Serum biomarkers such as cancer antigen 15-3 (CA15-3), cancer antigen 125 (CA125), and carcinoembryonic antigen (CEA) can be used as diagnostic and prognostic factors and can also provide valuable information during follow-up. However, serum protein biomarkers show limited diagnostic sensitivity and specificity in stand-alone assays because their levels reflect tumor burden. To validate whether biomarkers in nipple discharge may serve as novel biomarkers for breast cancer, we composed a panel of potential cancer biomarkers, including CA15-3, CA125, CEA, and malignant tumor-specific growth factor (TSGF), and evaluated their expression in both serum and nipple discharge in order to explore the expression and significance of estrogen receptor (ER), progestrone receptor (PR), epidermal growth factor receptor type 2 (HER2/neu), CA15-3, CA125, CEA, and TSGF expression for their combined predictive value for breast cancer and in judging the prognosis of breast cancer. Univariate analysis revealed that combined detection of CA15-3, CA125, CEA, and TSGF in nipple discharge served as novel biomarkers for the diagnosis and prognosis of breast cancer, but in the multivariate analyses the adverse effects of the four biomarkers combination in nipple discharge positivity on overall survival were lost. Multivariate analysis revealed that the positivity of the combined detection of the four biomarkers in both nipple discharge and serum was significantly higher than that of other detection methods. Thus, the combined detection of these four biomarkers both in serum and nipple discharge was retained as an independent prognostic variable in breast cancer patients. Our results indicate that CA15-3, CA125, CEA, and TSGF in nipple discharge can serve as novel biomarkers in the diagnosis and prognosis of breast cancer.     

龙血素A和龙血素B的酶联免疫检测方法的建立

建立了龙血素A和龙血素B间接竞争酶联免疫吸附分析方法,并用于测定龙血素A和龙血素B的含量。分别合成龙血素A和龙血素B人工抗原制备其多克隆抗体,建立二者间接竞争酶联免疫吸附分析方法并评估其分析性能。结果表明:抗龙血素A血清和抗龙血素B血清的效价分别达到8 000和30 000;龙血素A和龙血素B的最低检测限分别为3.9ng·mL-1和26.1ng·mL-1,批内精密度分别为CV≤10.7%和CV≤9.9%,批间精密度分别为CV≤11.5%和CV≤12.3%,回收率分别为87.4%~110.8%和109.8%~113.5%,与各自的6种类似物的交叉反应率最大分别为9.20%和3.64%,在4种龙血竭药物中的最大含量和最小含量分别相差4.05倍和3.57倍。龙血素A和龙血素B间接竞争酶联免疫吸附分析方法的检测精密度、准确度和特异性均较好,可快捷有效地用于药物检测和分析。