Abstract:
:Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Pini A,Spreafico A,Botti R,Neri D,Neri Pdoi
10.1016/s0022-1759(97)00107-5subject
Has Abstractpub_date
1997-08-07 00:00:00pages
171-82issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(97)00107-5journal_volume
206pub_type
杂志文章abstract::A simple and sensitive liposome immunoassay for gentamicin was developed using the cytolytic agent, phospholipase C, instead of complement. Liposomes entrapping a fluorescent marker, calcein, were prepared by the reverse-phase evaporation method from a mixture of dimyristoylphosphatidylcholine and cholesterol (molar r...
journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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abstract:OBJECTIVES:To report on a novel immunofluorescence pattern specifically associated with antibodies to SS-A/Ro. METHODS:A novel immunofluorescence pattern, herein designated SS-A/Ro pattern, was characterized as myriad discrete fine speckles throughout the nucleus. Eighty-six sequential samples presenting the SS-A/Ro p...
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journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/0022-1759(93)90063-d
更新日期:1993-01-04 00:00:00
abstract::A mild procedure for the purification of rabbit Fab' antibodies is described. A small column of normal goat IgG-Sepharose 4B (3 cm x 5mm) was saturated with F(ab')2 prepared from rabbit anti-goat IgG, and anti-goat IgG Fab was eluted from the column by splitting the disulfide bond in the hinge of the F(ab')2 molecule ...
journal_title:Journal of immunological methods
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doi:10.1016/0022-1759(80)90336-1
更新日期:1980-01-01 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::We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are ...
journal_title:Journal of immunological methods
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abstract::Immune analysis of polypeptides separated by SDS-PAGE provides a technique useful in the characterisation of complex mixtures. We describe a simple procedure involving immune analysis of fractionated PAGE gels. Polypeptides of purified herpesvirus simplex, HSV 'excreted antigen' and cell culture 'control' preparations...
journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90161-3
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1983-03-11 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90298-9
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1995-11-16 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90342-5
更新日期:1993-10-15 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90163-2
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