Antibody-bound cell microarray for immunophenotyping: surface modification and lymphocyte subpopulations

Antibody-based cell microarrays may serve as a high-throughput diagnostic tool that requires precise surface design providing high biological specificity, high reliability, and high validity. A fundamental study on the quantitative evaluation of immunophenotyping using a cell microarray on which antibodies specific to cluster of differentiation (CD) antigens of blood cells are fixed was performed. The microarray, which was prepared by photomicropatterning self-assembled monolayers of alkanethiols, consisted of carboxyl group (3-mercaptopropionic acid)-packed domains regularly distributed in the methyl group (1-dodecanethiol)-packed matrix phase. This was verified by X-ray photoelectron spectroscopy and water wettability measurements. The patterned carboxylated domains of 1.0 mm or 0.2 mm diameter were covalently fixed with the anti-CD4 or anti-CD8 antibody by coupling reaction using a water-soluble carbodiimide and hydroxysuccinimide. Precision antibody fixation at almost complete conversion was verified by confocal laser scanning microscopy coupled with a specific dye staining technique. Peripheral blood mononuclear cells expressing CD4 or CD8 antigen, respectively, adhered on the anti-CD4 or anti-CD8 antibody-fixed domains of the microarray at a very high specificity, which was verified with flow cytometric analysis and an antibody-coated magnetic-bead-based cell isolation technique. The CD4/CD8 subset ratios determined using the antibody-fixed microarrays were very close to those obtained by flow cytometric analysis. These results indicate that microarrays, on which antibodies specific to cell surface antigens are covalently fixed, provide a solid basis of the high-throughput quantitative evaluation of immunophenotyping in medical diagnosis.     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

CD117 expression in diffuse large B‐cell lymphomas: Fact or fiction?

CD117 (KIT) is expressed in a variety of hematopoietic neoplasms but there are a paucity of data regarding its expression in diffuse large B-cell lymphomas (DLBCL). The purpose of the present paper was to describe the authors' experience of two CD117+ DLBCL (one of follicle center-cell origin and one nasal Epstein-Barr virus (EBV)- plasmablastic lymphoma associated with lytic bone lesions), as determined by tissue immunohistochemistry and flow cytometry. The CD117 expression in DLBCL was further evaluated using tissue microarrays and seven additional plasmablastic lymphomas, using two commercially available anti-CD117 antibodies (Ab-1, Oncogene and A4502, DakoCytomation). Membranous +/- cytoplasmic staining was seen with Ab-1 in 24/65 (37%) DLBCL, including 21/56 microarray DLBCL, two index cases, and 1/7 additional plasmablastic lymphomas, with persistent staining in 13% of microarray DLBCL despite preincubation with KIT peptide. However, A4502 had only membranous staining of the index cases and one additional EBV- plasmablastic lymphoma with medullary disease. The present study suggests that (i) CD117 expression can be detected sporadically in DLBCL of follicle center-cell origin and a subset of plasmablastic lymphomas; (ii) staining for CD117 might help in identifying EBV- plasmablastic lymphomas associated with bone marrow involvement; and (iii) CD117 antibodies should be carefully validated prior to use, because non-specific staining, as observed with Ab-1, could lead to false-positive results.     

Serum proteomic signature for cystic fibrosis using an antibody microarray platform

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.     

Autoantibodies to C-reactive protein is a common finding in SLE,but not in primary Sjögren's syndrome,rheumatoid arthritis or inflammatory bowel disease

The occurrence of antibodies to human C-reactive protein (CRP) was analysed by enzyme-linked immunosorbent assay (ELISA) in 56 patient sera known to contain antibodies to double-stranded DNA (dsDNA) and in 16 sera from patients with primary Sj?gren's syndrome (SS), 15 rheumatoid arthritis, 31 Crohn's disease, and 37 ulcerative colitis. Eighty-seven per cent of the patients with anti-dsDNA antibodies had systemic lupus erythematosus (SLE) and the remaining had autoimmune hepatitis. The cut-off for positive anti-CRP test was set at the 95th percentile of 100 healthy blood donors. Twenty of 56 anti-dsDNA sera (36%) and two of 16 SS sera (13%) had antibodies reactive with human CRP, whereas all other samples were negative. Thirteen of 27 SLE patients (48%) were positive on at least one occasion. The sera containing anti-CRP antibodies only reacted with surface-bound antigen, but not with native CRP in solution. In conclusion, we found that autoantibodies to CRP are common in sera from patients with anti-dsDNA antibodies. It is not likely that this explains the relative failure of CRP response in patients with active SLE. However, it cannot be excluded that anti-CRP autoantibodies have other biological potentials of pathophysiological interest in SLE, for instance by binding to CRP deposited on cell and tissue surfaces.     

应用蛋白微阵列同时检测人血清抗ToRCH抗体

目的：建立一种基于多元蛋白微阵列的免疫检测方法，用于同时检测人血清中识别弓形虫（TOXO）、风疹病毒（RV）、巨细胞病毒（CMV）、单纯疱疹Ⅰ型、Ⅱ型病毒（HSV-1、HSV-2）的特异性抗体。方法：将TOXO、RV、CMV、HSV-1和HSV-2抗原喷印到活化的玻片表面，抗原与活化基团共价结合后，制成蛋白微阵列。固定在玻片表面的抗原与病人血清中的特异性抗体反应、结合后，加入荧光标记的二抗，然后用高分辨率的激光共聚焦芯片扫描系统对蛋白微阵列进行扫描成像。所获得的图像用自行开发的软件进行分析，并自动判定并生成待测样本的定性结果。结果：使用此套蛋白微阵列系统检测抗TORCH抗体参考品，并与其它多种检测方法进行比较，各检测项目的符合率分别为92％～100％、92％～100％、96％、84％～96％、76％-96％。结论：多元蛋白微阵列法特异性强，灵敏度、准确度、精密度较高，具有应用和推广价值。     

C-reactive protein in acute viral infections

A sensitive solid-phase enzyme immunoassay procedure was used to determine the concentrations of C-reactive protein (CPR) in the acute and convalescent phase sera of patients with verified rubella, herpes simplex, cytomegalo, influenza A or B, enterovirus, or mycoplasma infection. In all infection groups about 90% (80% for influenza) elevated CRP values were observed in the acute phase sera (mean values in the different groups 16-57 micrograms/ml), the highest values exceeding or approaching 100 micrograms/ml. The serum CRP values were highest in all groups before the specific serum antibodies were detectable and decreased approaching the upper limit or normal controls (2 microgram/ml) within 2 weeks. Notable individual variation in the CRP production was seen. We conclude tha serum CRP determination should not be used as a reliable criterion to distinguish bacterial and viral infections.     

Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/mL range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.     

Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas

Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.     

Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2

Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.     

Development of a sensitive, colorometric microarray assay for allergen-responsive human IgE

With the existence of several thousand unique human allergens, a multiplex format, such as protein microarrays, is an attractive option for allergy screening. To determine the feasibility and sensitivity of using an enzyme-based, colorimetric protein microarray assay, three common allergens (mold, dustmite, grass) were arrayed and added sera assayed for responsive human IgE. Normal, low positive, and negative control samples were assayed to determine optimal reaction parameters. Sensitivity of the assay (in international units, IU) was determined by constructing a standard curve using World Health Organization (WHO) standards. The system described here can reliably detect allergen-specific IgE below 0.35 IU, the current WHO standard cutoff. By taking advantage of the sensitivity of enzyme-linked immunosorbent assays (ELISAs) and the multiplex format of microarrays, we have achieved a high throughput system, capable of screening patients for allergen-susceptibility with optimal sensitivity.     

Clinical validation of breast cancer biomarkers using tissue microarray technology.

The results of previous studies done in our laboratory on breast cancer gene expression profile, using DNA microarrays, led to the discovery of several genes associated with breast cancer progression. Further evaluation of these genes and their involvement at various stages of cancer progression required performance of immunohistochemistry on thousands of different tissue blocks. Tissue microarray (TMA) technology facilitates rapid translation of DNA microarrays results to clinical specimens by using immunohistochemical analysis of protein expression. DNA microarray analysis done in our laboratory showed a significantly higher expression of prostatic-specific antigen (PSA) in invasive ductal carcinomas as compared to ductal carcinoma in situ, a finding contrary to the previously published data for PSA immunoreactivity in breast carcinomas. To find out whether TMA strategy could be used to explore the expression of the candidate genes involved in the breast cancer progression, we constructed a breast cancer progression TMA. It consisted of 2 normal ductal epithelium, 8 ductal carcinoma in situ, 19 invasive ductal carcinomas, and 3 metastatic ductal carcinomas of breast in triplets. Two prostatic adenocarcinomas and 2 normal colons were used as positive and negative controls, respectively. We first used well-documented and well-tested markers, such as antibodies to estrogen receptor, progesterone receptor, and p53. Results of these 3 antibodies were according to the previously published data. To validate our result, we then used antibody to PSA and looked for the expression of this protein on breast cancer progression TMA. Except for the 2 positive controls all 98 cores were found to be negative for PSA expression highlighting the importance of validation studies for DNA microarray results.     

A protein multiplex microarray substrate with high sensitivity and specificity

The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three-dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen-specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specific signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laboratory environment.     

Assessing the potential of immunohistochemistry for systematic gene expression profiling

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.     

Elevated levels of soluble CD163 in sera and fluids from rheumatoid arthritis patients and inhibition of the shedding of CD163 by TIMP-3

The aim of the present study was to evaluate levels of soluble CD 163 in sera and fluids from rheumatoid arthritis (RA) patients and elucidate the mechanism that regulates the shedding of CD163. Levels of soluble CD163 in sera and fluids from RA patients were examined by a sandwich enzyme immunoassay and Western blotting. To determine the effects of tissue inhibitors of metalloproteinase (TIMPs) on the shedding of CD163 from monocytes/macrophages, levels of soluble CD163 in cultures of monocytes/macrophages and the expression of CD163 on monocytes/macrophages in the presence or absence of TIMPs were examined by a sandwich enzyme immunoassay and flow cytometry, respectively. The clinical marker that was most associated with serum levels of soluble CD163 was levels of CRP. TIMP-3, but not TIMP-1 or TIMP-2, inhibited the shedding of CD163 from monocytes/macrophages. It was shown that serum levels of soluble CD163 are a sensitive and reliable marker to monitor activated macrophages in synovitis from RA patients and the results imply that the responsible proteinase for the shedding of CD163 is not a member of the matrix metalloproteinases, but is likely to be a member of ADAMs.     

CD64的表达在成人麻疹并发细菌性肺炎患者中的诊断价值

目的 探讨中性粒细胞CD64的表达在麻疹并发细菌性肺炎患者的临床价值.方法 选取成人麻疹肺炎患者106例,按临床表现和细菌学检测结果 分为麻疹合并细菌性肺炎组及麻疹合并病毒肺炎组,应用流式细胞术测定中性粒细胞CD64,同时测外周血C反应蛋白（CRP）和白细胞.结果 麻疹合并细菌性肺炎组出疹期CD64水平为（32.15±11.07）MFI,明显高于恢复期（10.6±3.23）MFI（P＜0.01）和麻疹合并病毒性肺炎组（9.55±3.48）MFI（P＜0.01）,以CD64≥8.50MFI、CRP≥10.00 mg/L、WBC≥9.05×109/L阳性标准,三种指标的敏感度分别为78.12%、80.48%、59.37%,特异度分别为76.19、67.67%、64.28%,准确度为77.35%、74.52%、61.32%;CD64与CRP呈正相关.结论 与CRP比较,中性粒细胞CD64的表达可作为麻疹合并细菌性肺炎患者的早期诊断、并可用于判断病情程度的可靠指标之一.     

Simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.     

Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential

The differential expression of chemokine receptors may be an important mechanism for the regulation of T cell migration. To test this, we examined the expression and function of the monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 receptors on various populations of T cells. Using a simple and reliable transendothelial chemotaxis assay, both MCP-1 and IL-8 were shown to be chemotactic for subsets of blood T cells, although the relative response varied from donor to donor. To examine receptor expression and correlate it with chemotaxis of T cell subsets, monoclonal antibodies (mAb) to the receptors were produced by immunizing mice either with synthetic peptides (MCP-1 receptor), or with receptor transfectants (IL-8 receptors A and B). A flow cytometric analysis of blood T cells with an anti-MCP-1 receptor mAb revealed low expression on the CD26^hi subset and undetectable expression on other T cells. Staining of T cells with anti-IL-8RA and anti-IL-8RB showed much higher levels of expression, but only on a subset of CD3⁺ cells which were CD8⁺ and CD56^?. That IL-8 and MCP-1 attracted distinct subsets of T cells was best illustrated using the CD26 marker, since IL-8R⁺ T cells were CD26^?, whereas T cells expressing detectable MCP-1R or which responded to MCP-1 in chemotaxis assays were CD26^hi. T cells activated in vitro with anti-CD3 up-regulated expression of the MCP-1 receptor, but not the IL-8 receptors, and were attracted to MCP-1 much more efficiently than resting T cells. These results show that there is a clear distinction between the IL-8- and MCP-1-responsive T cell populations and that chemokine receptor expression on T cells may be regulated with respect to lineage as well as cellular activation.