Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Protein chip-based microarray profiling of oxidized low density lipoprotein-treated cells

Commercially available high-content Ab380 and extensively validated DLM26 homemade protein microarrays were used to profile the effects of the pro-atherogenic molecule, oxidized low density lipoprotein (OxLDL), on human aortic smooth muscle cells. Protein microarrays detected 298 proteins in cell lysates and 54 of these were differentially regulated. Microarray data were validated by immunoblotting for a selected set of up- and down-regulated proteins. The protein microarray data sets were compared with our recent cDNA microarray-based gene expression results in order to characterize the global effect of OxLDL on smooth muscle cell functions. A group of cell-cell interaction molecules was classified as up-regulated by OxLDL, whereas nucleic acid/protein biosynthesis, structural and humoral response proteins/genes were under-expressed in cells treated by OxLDL. These findings reveal the major pattern of OxLDL-induced effects on the human aortic smooth muscle cells functions and also demonstrate that protein chip-based microarrays could be a useful proteomic tool to profile disease-related states of muscle cells.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV)

Protein microarrays for parallel detection of multiple viral antigens and antibodies have not yet been described in the field of human hepatitis virus infections. Here, we describe a simple, rapid and sensitive integrated protein microarray with three different reaction models. The integrated protein microarray could simultaneously determine in human sera two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses within 20 min. The results of the protein microarray were assessed directly by the naked eye but can also be analyzed by a quantitative detector. The detection limit of this protein microarray was 0.1 ng/ml for HBsAg. Overall, >85% concordance was observed between the integrated protein microarrays and an enzyme-linked immunosorbent assay for above hepatitis viral antigen and antibody detections in human sera. This integrated protein microarray can be easily optimized for clinical use and epidemiological screening for multiple hepatitis virus infections.     

流行性出血热循环免疫复合物组分分析

应用ＳＤＳ－聚丙烯酰胺凝胶电泳及免疫转印技术对流行性出血热患才血清中免疫合物组分进行了分析。流行性出血热循环免疫复合物经ＳＤＳ－ＰＡＧＥ分离,考马斯亮兰染色,显色主要有７条带,分子量分别为２３ｋＤ,５０ｋＤ,５２ｋＤ,６５ｋＤ,７２ｋＤ,８０ｋＤ及１００ｋＤ。采用该病毒特异性抗血清、单克隆抗体以及人免疫球蛋白、补体成分抗血清识别,在其特环免疫复合物中可检出特异性病毒抗在及相应的免疫球状蛋白和补体成     

反复呼吸道感染患儿血清微量元素及体液免疫水平测定及临床意义

目的:探讨反复呼吸道感染患儿血清微量元素及体液免疫水平测定及其临床意义。方法:选取2016年1月至2017年1月在我院接受治疗的反复呼吸道感染患儿64例作为观察组,另外选取同期来我院体检的健康儿童60例作为对照组,比较两组儿童血清微量元素钙(Ca)、铁(Fe)、铜(Cu)、锌(Zn)、镁(Mg)等的水平、体液免疫因子免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白G(IgG)水平及血清补体C3、C4、C5水平,并分析其相关性。结果:观察组患儿血清Ca、Fe、Zn水平显著低于对照组儿童(P0.05),两组儿童血清Cu、Mg水平比较差异无统计学意义(P0.05)。观察组患儿血清IgA、IgM、IgG水平低于对照组儿童(P0.05)。两组儿童血清补体C3、C4、C5水平比较差异无统计学意义(P0.05)。经Pearson相关性分析可得:反复呼吸道感染患儿血清Ca、Fe、Zn与血清IgA、IgM、IgG水平呈正相关(P0.05)。结论:反复呼吸道感染患儿存在血清Ca、Fe、Zn微量元素缺乏及血清IgA、IgM、IgG水平降低现象,且它们之间具有正相关关系,可能共同促进反复呼吸道感染的发生。     

基于夹心免疫分析的抗体微阵列的构建

目的：建立一种基于夹心免疫分析的抗体微阵列构建的优化方法。方法：将MCP-1的捕获抗体点样于修饰后的玻片,标准抗原加样覆盖所点阵列,生物素标记抗体和链酶亲和素-cy3依次加样孵育, 激光共聚焦扫描仪获取图象并进行数据分析。对捕获抗体浓度、封闭液种类、系统可重复性和定量检测能力、两种因子平行性检测对信号分析的影响及点样后玻片稳定性进行分析和评价。结果：随着捕获抗体浓度的升高,信号强度逐渐增加;2℅ BSA/PBS和5℅ 酪蛋白可作为本系统的封闭液;所构建系统具有较好的可重复性（组内变异 1.3%,组间变异8.7%）和定量分析能力（所建立的抗原浓度-相对信号强度标准曲线相关系数达0.9995）;并实现了两因子的平行性分析和点样后玻片的稳定性。结论：确立了基于夹心免疫分析的抗体微阵列构建的优化方法,为进一步构建多因子定量检测抗体微阵列奠定了基础。     

草鱼免疫应答的初步研究

研究了草鱼在不同水温条件下受抗原刺激后其中和抗体的变化。15℃培养条件下中和抗体上升缓慢,9周内滴度低于1:8;20℃时,3周后抗体可上升到1:256,最高达1:5270,而在25℃时,1周中和抗体即达到1:570,最高可达1:20000以上。并探索了从草鱼血清中提纯抗体的条件,研究其抗体的特性。草鱼血清中的抗体为大分子蛋白,容易解离为抗原性相同,分子量近似于人IgG的较小分子,含有较多的二硫键,具有类似IgM的某些特性。     

Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers

We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two-color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly-1-lysine coated glass with a photoreactive cross-linking layer. The hydrogel substrate yielded an average six-fold higher signal-to-noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from "reverse-labeled" experiment sets accurately predicted the agreement between the microarray measurements and enzyme-linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high-density antibody and protein microarrays in biomarker discovery studies.     

Further Evidence for Involvement of Both Cell Mediated and Humoral Immunity in Generalized Vitiligo

Immunohistochemical and immunoserological evidence supports the involvement of both cell-mediated and humoral mechanisms in the pathogenesis of melanocyte destruction in vitiligo. Punch biopsies from depigmented vitiliginous skin (VS), normal-looking pigmented skin (PS), and marginal skin (MS) from patients with generalized vitiligo (n = 15) were labeled with K 1.2.58, OKM1 (CD11b), Leu 11b (CD16), Leu 19 (CD56), IFN-γreceptor, IL-2 receptor (CD25), IgG, IgM, C3c, and C3d MoAbs. In addition, in vitro effects of vitiligo sera (n = 13) on human newborn melanocytes (HMel) under different culture conditions were studied. The immunohistochemical findings showed absence of K 1.2.58+ epidermal melanocytes in VS and abnormal morphology in MS. In these areas, a few CD11b + cells in the dermis and epidermis could be detected but no significant numbers of CD16+ or CD56+ cells were seen among the mononuclear cellular infiltrate. IL-2 and IFN-γ receptors were clearly expressed by the cellular infiltrate. No significant deposition of complement or immunoglobulin was seen. The addition of vitiligo sera to HMel cultures induced a significant cellular proliferation. The stimulation of cell proliferation occurred regardless whether the sera were added alone or when preheated (56°C for 1 hr) and then supplemented with a complement source (P < 0.01 at 2%, P < 0.001 at 10%, and P < 0.01 at 20% for sera alone) (P > 0.05 at 2%, P < 0.05 at 10%, and P < 0.01 at 20% for decomplemented sera plus complement). In contrast, incubation of vitiligo sera together with normal lymphocytes with HMel significantly decreased the number of living melanocytes in a dose dependent manner, suggesting an antibody-dependent cellular cytotoxicity (ADCC) reaction (P < 0.01 at 2% and 10%, P < 0.001 at 20%). The presence of lymphocytic infiltrate at marginal skin with evidence for IL-2- and IFN-γ-receptor expression and the decrease in the number of living cells by ADCC-like mechanisms provide further support for an autoimmune pathogenesis in vitiligo.     

Nucleotide Insertions and Deletions Complement Point Mutations to Massively Expand the Diversity Created by Somatic Hypermutation of Antibodies

During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.