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    Immunochemical methods for the detection of antibody against HTLV-III
    1.
    发明授权
    Immunochemical methods for the detection of antibody against HTLV-III 失效
    用于检测针对HTLV-III的抗体的免疫化学方法

    公开(公告)号：US4774175A

    公开(公告)日：1988-09-27

    申请号：US707066

    申请日：1985-03-01

    申请人： Tse W. Chang ,  Ikunoshin Kato ,  Pranab Chanda ,  Nancy T. Chang

    发明人： Tse W. Chang ,  Ikunoshin Kato ,  Pranab Chanda ,  Nancy T. Chang

    IPC分类号： A61K38/00 ,  C07K14/16 ,  C12Q1/70 ,  G01N33/544

    CPC分类号： C07K14/005 ,  C12Q1/703 ,  A61K38/00 ,  C12N2740/16122 ,  Y10S435/805 ,  Y10S435/974 ,  Y10S436/811 ,  Y10S930/221

    摘要： Gene segments of human T cell lymphotropic virus type III (HTLV-III) were expressed in E. coli as peptides that are reactive with sera from patients with acquired immune deficiency syndrome (AIDS). Among recombinant peptides one designated HTLV-III polypeptide 121, contained 85 amino acid residues encoded by a gene segment in the env-lor region of the HTLV-III genome. The polypeptide is strongly reactive with AIDS patient sera. The peptide produced and purified as a fusion protein on a large scale. Solid phase immunoassays employing this recombinant peptide as an immunoabsorbent can reliably and reproducibly detect antibodies in sera of patients with HTLV-III infection. In two representative serum panels, the assay detected the presence of antibodies in 120 of 121 sera from patients with AIDS or AIDS-related complex (ARC), and only in 1 of 92 normal controls. Based upon HTLV-III polypeptide 121 as immunoreactive agent, sensitive and specific immunoassays for HTLV-III infection have been developed.

    摘要翻译： 人类T细胞淋巴细胞病毒III型（HTLV-III）的基因片段在大肠杆菌中表达为与获得性免疫缺陷综合征（AIDS）患者的血清反应的肽。 在一个称为HTLV-III多肽121的重组肽中，包含由HTLV-III基因组的环境区域中的基因区段编码的85个氨基酸残基。 多肽与艾滋病患者血清强烈反应。 该肽作为融合蛋白产生并纯化，大规模。 使用该重组肽作为免疫吸收剂的固相免疫测定法可以可靠且可重复地检测HTLV-III感染患者血清中的抗体。 在两个代表性的血清图中，该测定法检测了来自患有AIDS或AIDS相关复合物（ARC）的121个血清中的120个抗体中存在的抗体，并且仅在92个正常对照中的1个中存在抗体。 基于HTLV-III多肽121作为免疫反应剂，开发了HTLV-III感染的敏感和特异性免疫测定。

    Antibodies specific for isotype specific domains of human IgM and human IgG expressed or the B cell surface
    4.
    发明授权
    Antibodies specific for isotype specific domains of human IgM and human IgG expressed or the B cell surface 失效
    对人IgM和人IgG表达的同种型特异性结构域或B细胞表面特异的抗体

    公开(公告)号：US5298420A

    公开(公告)日：1994-03-29

    申请号：US902449

    申请日：1992-06-19

    申请人： Tse W. Chang

    发明人： Tse W. Chang

    IPC分类号： A61K38/00 ,  C07K16/42 ,  C12P21/08 ,  C07K15/28 ,  C12N1/21 ,  C12N5/12

    CPC分类号： C07K16/4283 ,  A61K38/00

    摘要： Membrane anchoring peptides are attached to the C terminal end of the heavy chain of the various immunoglobulin isotypes (IgM, IgD, IgA, IgE, or IgG). The membrane anchoring peptides span the cell membrane lipid bilayer of B cells thereby affixing the associated immunoglobulin to the cell membrane surface. The extracellular segments of these peptides are unique for different isotypes. Epitopes unique to the B cells which produce each isotype are formed, in whole or in part, by these extracellular segments. These membrane-bound immunoglobulin isotype-specific ("migis") extracellular epitopes are not present on the secreted, soluble form of the immunoglobulins, which are not bound to the cell surface by the membrane anchoring peptides. The antibodies of the invention (and other related products) specifically bind to the extracellular migis epitopes of human .mu. chain, human .delta. chain, or human .gamma. chain. The B cells which express the isotypes IgM, IgD or IgG are labeled for destruction when bound by such antibodies or products, and can be destroyed by the cytolytic or regulatory mechanisms of the immune system in order to cause immunosuppression.

    摘要翻译： 膜锚定肽连接到各种免疫球蛋白同种型（IgM，IgD，IgA，IgE或IgG）的重链的C末端。 膜锚定肽跨越B细胞的细胞膜脂质双层，从而将相关的免疫球蛋白附着于细胞膜表面。 这些肽的细胞外区段对于不同的同种型是独特的。 通过这些细胞外片段整体或部分形成产生每个同种型的B细胞特有的表位。 这些膜结合的免疫球蛋白同种型特异性（“migis”）细胞外表位不存在于分泌的可溶形式的免疫球蛋白，其通过膜锚定肽不结合细胞表面。 本发明的抗体（和其它相关产品）特异性结合人（my）链，人（delta）链或人（γ）链的细胞外迁移表位。 表达同种型IgM，IgD或IgG的B细胞在被这些抗体或产物结合时被标记为破坏，并且可以被免疫系统的细胞溶解或调节机制破坏以引起免疫抑制。

    Selecting low frequency antigen-specific single B lymphocytes
    5.
    发明授权
    Selecting low frequency antigen-specific single B lymphocytes 失效
    选择低频抗原特异性单一B淋巴细胞

    公开(公告)号：US5326696A

    公开(公告)日：1994-07-05

    申请号：US21619

    申请日：1993-02-17

    申请人： Tse W. Chang

    发明人： Tse W. Chang

    IPC分类号： G01N33/50 ,  G01N33/569 ,  G01N33/533 ,  G01N21/64 ,  G01N33/536

    CPC分类号： G01N33/5094 ,  G01N33/56972 ,  Y10S435/808 ,  Y10S435/962 ,  Y10S436/80

    摘要： Disclosed are immunofluorescence staining methods which increase the likelihood that antibodies expressed by a single B cell selected and sorted by fluorescence activated cell sorting are specific for the antigen of interest, and which also allow selection of B cells expressing antibodies of high affinity for the antigen of interest. The selection for B cells expressing antibodies to specific antigens is increased by labeling B cells with at least two antigen probes, where each antigen probe includes the antigen of interest and is labeled with a different fluorochrome. The positive selection is preferably combined with a negative selection step, in which autofluorescent cells and sticky cells are excluded out. The specificity of sorting of the desired B cells can be further enhanced by staining those antigen-specific B cells which produce the immunoglobulin isotype (typically IgG), with targeting molecules reactive with a B cell marker, such as .gamma. chain and CD19, that are conjugated with a different fluorochrome. Thus, the antigen-specific IgG-producing B cells of interest may be labeled with these unique reagents in four color FACS (including one for negative selection), which can sort the desired antigen-specific B cells at enhanced proportions. After sorting the B cells with FACS, those B cells with high affinity are preferred for analysis by the single cell-PCR procedure to amplify and clone the V.sub.H and V.sub.L segments of interest.

    摘要翻译： 公开了免疫荧光染色方法，其增加了通过荧光激活细胞分选选择和分选的单个B细胞表达的抗体对感兴趣的抗原特异的可能性，并且还允许选择表达对亲和素抗原具有高亲和力的抗体的B细胞 利益。 通过用至少两个抗原探针标记B细胞来增加表达针对特异性抗原的抗体的B细胞的选择，其中每个抗原探针包括感兴趣的抗原并用不同的荧光染料标记。 阳性选择优选与否定选择步骤组合，其中自发荧光细胞和粘性细胞被排除。 通过染色产生免疫球蛋白同种型（通常为IgG）的那些抗原特异性B细胞，可以进一步增强所需B细胞的分选的特异性，其中靶向分子与B细胞标记如γ链和CD19反应，其为 与不同的荧光染料缀合。 因此，可以用四色FACS（包括一种用于阴性选择）的这些独特的试剂来标记感兴趣的抗原特异性IgG产生B细胞，其可以以更高的比例对所需的抗原特异性B细胞进行分类。 在用FACS分选B细胞后，优选用亲和力高的B细胞通过单细胞PCR方法进行分析，以扩增和克隆感兴趣的VH和VL片段。

    Immunoassay device enclosing matrixes of antibody spots for cell determinations
    8.
    发明授权
    Immunoassay device enclosing matrixes of antibody spots for cell determinations 失效
    用于细胞测定的包含抗体斑点基质的免疫测定装置

    公开(公告)号：US4829010A

    公开(公告)日：1989-05-09

    申请号：US25501

    申请日：1987-03-13

    申请人： Tse W. Chang

    发明人： Tse W. Chang

    IPC分类号： G01N33/543 ,  B01L3/00 ,  G01N33/545 ,  G01N33/552

    CPC分类号： B01L3/508 ,  B01L3/5027 ,  G01N33/545 ,  G01N33/552 ,  B01J2219/00605 ,  B01J2219/0061 ,  B01J2219/00612 ,  B01J2219/00617 ,  B01J2219/0063 ,  B01J2219/00637 ,  B01J2219/00659 ,  B01L2200/0689 ,  B01L2300/0636 ,  B01L2300/0822 ,  B01L2400/0406 ,  Y10S436/807 ,  Y10S436/809

    摘要： An immunoassay device comprising a support which has a substantially planar surface 14 on which is located an array of small, closely-spaced, discrete, antibody coated areas (spots) 17. A cover 16 is spaced from the support surface 14 and is positioned over the array of antibody coated spots 17. The cover is attached to the support surface to provide an enclosed chamber 30. In the cover there is at least one aperture communicating with the interior of the chamber 30 for introducing a liquid sample into the chamber. In addition, there is at least one additional aperture 34 in the cover, which also communicates with the interior of the chamber to allow air to escape upon introduction of the sample into the chamber. When a cell sample is introduced into the chamber it immediately fills the chamber and cells will settle uniformly over the matrix. The antibody-coated spots function as tiny immunoadsorbents for cells bearing antigens recognized by the antibody. The device can be used to determine the proportion of cells in a sample cell population.

    摘要翻译： 一种免疫测定装置，其包括具有基本上平坦的表面14的支撑体，其上定位有小的，紧密间隔的离散的抗体涂覆区域（点）17.盖16与支撑表面14间隔开并且被定位在 抗体涂覆点阵列17.盖子附接到支撑表面以提供封闭的腔室30.在盖子中有至少一个与腔室30的内部连通的孔，用于将液体样品引入腔室。 此外，在盖中还有至少一个附加的孔34，其还与室的内部连通，以便在将样品引入室时允许空气逸出。 当细胞样品被引入腔室时，其立即填充腔室，细胞将均匀地沉降在基质上。 抗体包被的斑点作为抗体识别的携带抗原的细胞的微量免疫吸附剂。 该装置可用于确定样品细胞群体中细胞的比例。

    Methods for selecting low frequency antigen-specific single B lymphocytes
    10.
    发明授权
    Methods for selecting low frequency antigen-specific single B lymphocytes 失效
    选择低频抗原特异性单B淋巴细胞的方法

    公开(公告)号：US5213960A

    公开(公告)日：1993-05-25

    申请号：US848249

    申请日：1992-03-09

    申请人： Tse W. Chang

    发明人： Tse W. Chang

    IPC分类号： G01N33/569

    CPC分类号： G01N33/56972

    摘要： Disclosed are immunofluorescence staining methods which increase the likelihood that antibodies expressed by a single B cell selected and sorted by fluorescence activated cell sorting are specific for the antigen of interest, and which also allow selection of B cells expressing antibodies of high affinity for the antigen of interest. The selection for B cells expressing antibodies to specific antigens is increased by labeling B cells with at least two antigen probes, where each antigen probe includes the antigen of interest and the difference between the two probes is that each is labeled with a different fluorochrome. The specificity of sorting of the desired B cells can be further enhanced by staining those antigen-specific B cells which produce the immunoglobulin isotype (typically IgG), with targeting molecules reactive with B cell markers, such as .gamma. chain and CD19, that are conjugated with different fluorochromes. Thus, the antigen-specific IgG-producing B cells of interest may be labeled with these unique reagents in three or four color FACS, which can sort the desired antigen-specific B cells at enhanced proportions. The differences in relative intensities between the antigen labels and the isotype labels (e.g., IgG labels) among the different antibodies of the single cells selected can be used to determine the relative antigen binding affinity of those antibodies. For example, the ratio of antigen label to IgG label can be calculated for each labeled B cell. The higher the ratio, the higher the relative affinity of the antibodies on the B cells for the antigen. After sorting the B cells with FACS, those B cells with high affinity are preferred for analysis by the single cell-PCR procedure to amplify and clone the V.sub.H and V.sub.L segments of interest.

    摘要翻译： 公开了免疫荧光染色方法，其增加了通过荧光激活细胞分选选择和分选的单个B细胞表达的抗体对感兴趣的抗原特异的可能性，并且还允许选择表达对亲和素抗原具有高亲和力的抗体的B细胞 利益。 通过用至少两个抗原探针标记B细胞来增加表达针对特异性抗原的抗体的B细胞的选择，其中每个抗原探针包括感兴趣的抗原，并且两个探针之间的差异是每个用不同的荧光染料标记。 通过染色产生免疫球蛋白同种型（通常为IgG）的那些抗原特异性B细胞，可以进一步增强所需B细胞分选的特异性，其中靶向分子与缀合有B细胞标志物的γ细胞标记如γ链和CD19反应 与不同的荧光染料。 因此，感兴趣的抗原特异性IgG产生B细胞可以用三种或四种颜色的FACS用这些独特的试剂进行标记，这可以以增强的比例分类所需的抗原特异性B细胞。 可以使用选择的单个细胞的不同抗体之间的抗原标记与同种型标签（例如，IgG标记）之间的相对强度的差异来确定那些抗体的相对抗原结合亲和力。 例如，可以对每个标记的B细胞计算抗原标记与IgG标记的比例。 比例越高，抗体对抗原对B细胞的相对亲和力越高。 在用FACS分选B细胞后，优选用亲和力高的B细胞通过单细胞PCR方法进行分析，以扩增和克隆感兴趣的VH和VL片段。