Abstract：
:Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional manual methods. We have developed a novel approach to the identification and isolation of cells secreting phage encoding desirable antibodies that utilizes compartmentalization and Fluorescence Activated Cell Sorting (FACS). This method, termed Phage Emulsion, Secretion, and Capture (ESCape), allows us to individually query each phage against the antigen. Here, we demonstrate the ability of Phage ESCape to identify novel scFvs against a phosphopeptide epitope of the Her2 kinase from a phage display library containing approximately 10(8) synthetically diversified antibodies. Clones were analyzed by monoclonal phage ELISA against the Her2 phosphopeptide, and positive binders were identified as those showing a signal greater than 3-fold higher than the background signal against an irrelevant antigen. We isolated antibodies recognizing the phosphopeptide in a single round of selection by Phage ESCape, but the strength and specificity of the hits was substantially improved when the library was pre-enriched by a single round of biopanning. By minimizing the selection rounds required for phage display and using a FACS machine as a 'colony picker' equivalent, Phage ESCape has the potential to dramatically increase the throughput of in vitro screening methods.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Kiss MM,Babineau EG,Bonatsakis M,Buhr DL,Maksymiuk GM,Wang D,Alderman D,Gelperin DM,Weiner MPdoi
10.1016/j.jim.2010.09.034subject
Has Abstractpub_date
2011-03-31 00:00:00pages
17-26issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(10)00288-7journal_volume
367pub_type
杂志文章abstract：:An ultra-sensitive quantitative EPO (erythropoietin) lateral flow immunochromatographic test with a detection limit of 1.2 fM (10(-15) M), 0.035 ng EPO/L, which is 50-100 times more sensitive than a corresponding enzyme based immunoassay, is presented. In comparison with commercial lateral flow tests for other analyte...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2008.09.022
更新日期：2008-12-31 00:00:00
abstract：OBJECTIVE:Interleukin-13 (IL-13) has been difficult to quantify within human serum due to low abundance. Available assays have not been sensitive enough to detect IL-13 at the femtogram level. Thus, there are inconsistencies within the published literature as to IL-13 concentrations in normal or disease populations. To...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.08.006
更新日期：2015-11-01 00:00:00
abstract：:Counting of rat mast cells and assessment of degranulation were performed by flow-cytometry using a cytograph. It was shown that the number of mast cells in a total cell population may be obtained rapidly and reproducibly following toluidine blue staining. A dose-response for degranulation by compound 48/80 was perfor...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90085-x
更新日期：1980-01-01 00:00:00
abstract：:A rapid method for the simultaneous measurement of neutrophil superoxide generation and beta-glucuronidase release is described. Assay of beta-glucuronidase using a fluorescent substrate is shown to be valid in the presence of reduced or unreduced ferricytochrome C, a prerequisite for the simultaneous assessment of th...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90191-8
更新日期：1987-06-26 00:00:00
abstract：:Dot-immunobinding assay for anti-calmodulin (CaM), and immunoblot assay for CaM have become feasible by proper choice of a fixative, i.e., formaldehyde vapor. The protocol for these simple and convenient immunological procedures is presented. Dot-immunobinding assay could be accomplished in 3 h, and immunoblot assay i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90089-6
更新日期：1989-12-20 00:00:00
abstract：:A simple technique for raising specific antiserum to the native molecule of Epstein-Barr virus (EBV)-encoded nuclear antigen-2 (EBNA-2) from Raji cells is described. The procedure involves the use of immunoblotting to identify the EBNA-2 polypeptide followed by subsequent excision from an SDS-gel and immunization of e...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90543-0
更新日期：1986-03-13 00:00:00
abstract：:Procedures to reproducibly obtain pure preparations of murine Kupffer cells are described. Pure Kupffer cell preparations obtained following collagenase digestion, metrizamide separation and centrifugal elutriation remained viable and maintained their phagocytic functions for at least 4-5 days in vitro. Furthermore, w...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90281-b
更新日期：1993-08-26 00:00:00
abstract：:The length distributions of the third complementarity determining region (CDR3) of the T cell receptor (TCR) beta chain were determined quantitatively from peripheral blood lymphocytes of healthy donors. RT-PCR products of 26 V beta families and subfamilies were analyzed by an A.L.F. DNA sequencer and Fragment Manager...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00178-d
更新日期：1995-11-16 00:00:00
abstract：:This report describes the use of biotin-labeled antibodies in sensitive enzyme immunoassay systems for the measurement of bacterial antigens. Biotinylated immunoglobulins could be reproducibly formulated using N-hydroxysuccinimide biotin ester to link biotin to the immunoglobulin. Binding of the biotinylated antibody ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(83)80021-0
更新日期：1983-02-11 00:00:00
abstract：:Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diag...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.02.017
更新日期：2013-05-31 00:00:00
abstract：:Insulin was directly coupled and also indirectly coupled by a side chain to Sepharose 4B to form immunoabsorbents for affinity chromatography of insulin antibodies. The two direct derivatives were insulin lysyl and insulin phenylalanyl sepharose. The indirect derivative, insulin carboxyhexyl sepharose, was prepared in...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(77)90130-2
更新日期：1977-01-01 00:00:00
abstract：:The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response....
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2003.11.007
更新日期：2004-02-01 00:00:00
abstract：:A method is described for identification of the animal origin of mosquito bloodmeals (MBM). The immunoassay is named DOT-PAP, it makes use of nitrocellulose as solid phase and of peroxidase-anti-peroxidase soluble complexes as detectors. DOT-PAP includes a built-in absorbing system to remove possible cross-reactivitie...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90257-7
更新日期：1986-01-22 00:00:00
abstract：:We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90397-x
更新日期：1991-03-01 00:00:00
abstract：:Natural CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) control the activation of the immune system and therefore have become a major area of research in immunology. The generation of monoclonal antibodies against human Tregs offers the possibility to discover novel Treg-specific or Treg-associated surface markers an...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.01.002
更新日期：2010-02-28 00:00:00
abstract：:We describe an ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level. An exact number of newly excysted juveniles (NEJs) was injected into a gut segment with an intact blood supply and which was still attached to a live anaesthetized rat. NEJs that penetrated the gut wall ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(98)00026-x
更新日期：1998-04-15 00:00:00
abstract：:Malaria imposes a horrific public health burden - hundreds of millions of infections and millions of deaths - on large parts of the world. While this unacceptable health burden and its economic and social impact have made it a focal point of the international development agenda, it became consensual that malaria contr...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/j.jim.2014.05.001
更新日期：2014-08-01 00:00:00
abstract：:Rabbit immunoglobulin G (RIgG) was reduced with dithioerythritol and analyzed by high performance liquid chromatography. A quantitative method for determining the percentage of reduced half-molecules in the mixture was developed. An acetic acid concentration-dependent rate of dissociation of reduced half-molecules was...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90314-0
更新日期：1987-02-11 00:00:00
abstract：:There is an emerging trend in the pharmaceutical industry to evaluate a variety of surrogate biomarkers in Phase I/II clinical studies with the intention of determining potential activity of drugs early in clinical development. A number of cytokines expressed in pathological conditions are currently being considered a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00543-9
更新日期：2002-02-01 00:00:00
abstract：:The 7th International Workshop on Human Leucocyte Differentiation Antigens (HLDA7) studied a number of newly characterised molecules relevant to human leucocyte differentiation and function. The HLDA organisation, which devised and continues to maintain the CD nomenclature, is responsible, under the auspices of IUIS a...
journal_title：Journal of immunological methods
pub_type：
doi：10.1016/s0022-1759(03)00057-7
更新日期：2003-04-01 00:00:00
abstract：:A method of data analysis for flow cytometry is presented which enables up to eight-dimensional data to be handled by a microcomputer with 28K addressable plus a further 32K non-addressable memory. The multi-parameter coordinates are coded into single numbers using a minimal modification of the array vector mapping eq...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90333-x
更新日期：1988-10-26 00:00:00
abstract：:A dot-ELISA technique for the detection of G. lamblia specific antigen in stool eluates of clinical cases of giardiasis was developed and evaluated employing monospecific antibodies to a G. lamblia specific coproantigen with a molecular mass of 66 kDa. The assay detected 22 (91.7%) of the 24 microscopically confirmed ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90030-j
更新日期：1991-03-21 00:00:00
abstract：:By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 mu) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(77)90156-9
更新日期：1977-01-01 00:00:00
abstract：:A one-step immunospecific affinity chromatographic method for purification of mouse alpha-fetoprotein (AFP) directly from amniotic fluid is described. The procedure employed a rabbit anti-AFP immunoglobulin-G entrapped in a polyacrylamide gel matrix prepared as a gel slurry. The presence of impurities could not be dem...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90146-7
更新日期：1979-01-01 00:00:00
abstract：:Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosi...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00054-9
更新日期：1997-06-23 00:00:00
abstract：:The Raji cell assay for circulating immune complexes (CIC) is frequently the method of choice when the detection of large, complement fixing complexes is desired. We have developed an enzyme-linked immunosorbent assay modification (Raji-ELISA) of the Raji cell technique which is easy to perform, uses commercially avai...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90046-7
更新日期：1983-01-01 00:00:00
abstract：:Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at -221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5' nuclease (TaqMan) assay in combination with the use ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2004.01.025
更新日期：2004-04-01 00:00:00
abstract：:Human thyroxine binding globulin (TBG) was purified to near homogeneity by chromatography on thyroxine-substituted Sepharose 4B, DEAE-Sephadex A-50, Sephadex G-150 and n-decylamine-substituted Sepharose 4B. TBG was quantitated by homogeneous fluorescence excitation transfer immunoassay; the preparation of dye-protein ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90139-8
更新日期：1980-01-01 00:00:00
abstract：:Hybridoma cells producing monoclonal antibodies against tumor-shed antigens were generated by fusing mouse myeloma cells with spleen cells from immunosuppressed mice bearing human melanoma xenografts. Thirty-eight fusion experiments were performed at different stages of tumor growth. Hybridomas producing anti-melanoma...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90074-1
更新日期：1983-02-25 00:00:00
abstract：:Genetic mapping of differentiation specific surface antigens has been hampered by difficulty in preparation of interspecies hybrid cells which continue to express differentiated functions. A method has been developed for production of interspecies T cell hybrids which continue to express T cell specific cell surface m...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90108-4
更新日期：1983-08-12 00:00:00
