Abstract:
:Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously adsorbed passively to the polystyrene. Data show that less than 3.0% of the binding sites of monoclonal CAbs and approximately 5-10% of those of polyclonal CAbs were capable of capturing antigen (FLU4.2-BSA) after passive adsorption. Immobilization of CAbs via an antiglobulin or a streptavidin bridge, resulted in the preservation of antibody binding sites to greater than 70% for some monoclonals although immobilization via the streptavidin bridge resulted in the highest number of functional sites/well. The data presented are consistent with studies on other adsorbed proteins which demonstrate that passive adsorption on polystyrene results in the loss of protein function. Furthermore, these data show that generally less than half of the binding sites of antibodies available in solution are available after solid-phase immobilization even when non-adsorptive methods are employed. Some polyclonal anti-FLU also have lower average avidity following passive adsorption compared with CAbs immobilization via a streptavidin bridge. Immunochemical studies revealed that adsorbed polyclonal-CAbs performed like monoclonals when tested with multivalent antigens (FLU10-IgA) but in an expected heterogeneous manner in Scatchard plots when tested using univalent FLU-insulin. This observation implied cross-linking of immobilized CAbs by the multivalent antigen. Because only 5-10% of adsorbed polyclonal CAbs are active, the survivors must be non-randomly distributed in clusters to explain the cross-linking. This was confirmed by scanning electron microscopy which gave rise to the hypothesis that antibodies which retain activity after adsorption, are those present in clusters, i.e., the functional adsorbed CAb is an antibody cluster. Data presented in this report on the behavior of adsorbed CAbs, and reviewed from the work of others for various adsorbed proteins, indicate that the method of passive adsorption at pH 9.6, which is widely used in popular microtiter ELISAs, and which has in many ways revolutionized immunoassay, is a method of protein denaturation. Assayists that utilize passive adsorption of proteins on hydrophobic supports as part of their research need to be cognizant of this phenomenon, while inventors of immunoassay should develop alternative methods of immobilization which do not destroy 90% of the functional activity of solid-phase reactant.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Butler JE,Ni L,Nessler R,Joshi KS,Suter M,Rosenberg B,Chang J,Brown WR,Cantarero LAdoi
10.1016/0022-1759(92)90066-3keywords:
subject
Has Abstractpub_date
1992-06-24 00:00:00pages
77-90issue
1-2eissn
0022-1759issn
1872-7905pii
0022-1759(92)90066-3journal_volume
150pub_type
杂志文章,评审abstract::A commercially available protein determination method (Pierce BCA protein determination kit) was used to assess the efficiency of coating of microtiter plates. Since the method is insensitive to detergents it permits the determination of adhered protein after washing the microtiter plate with a detergent-containing so...
journal_title:Journal of immunological methods
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doi:10.1016/0022-1759(86)90419-9
更新日期:1986-12-24 00:00:00
abstract::Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generat...
journal_title:Journal of immunological methods
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doi:10.1016/j.jim.2010.01.005
更新日期:2010-02-28 00:00:00
abstract::We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed b...
journal_title:Journal of immunological methods
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doi:10.1016/s0022-1759(01)00461-6
更新日期:2001-11-01 00:00:00
abstract::We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that o...
journal_title:Journal of immunological methods
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doi:10.1016/0022-1759(90)90396-d
更新日期:1990-08-28 00:00:00
abstract:BACKGROUND:Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) and monocyte subsets as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative inte...
journal_title:Journal of immunological methods
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doi:10.1016/j.jim.2012.04.003
更新日期:2012-07-31 00:00:00
abstract::Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transact...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2013.07.015
更新日期:2013-10-31 00:00:00
abstract::The effects of complement activation on the antigenic component of immune complexes have been studied, using 125I-BSA-rabbit anti-BSA-IgG complexes as models. Polyethylene glycol precipitates of 4 types of IC (those formed in native normal human serum, or NHS-containing EDTA, NHS-EDTA, and preformed soluble IC incubat...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90109-1
更新日期:1984-12-31 00:00:00
abstract::A modification of the COS cell transient expression system for isolating cDNAs encoding antibody defined molecules was used to clone the carcinoma antigen defined by monoclonal antibody CORC-6. Bacteria were transformed with cDNA prepared from colon carcinoma cells and immediately divided into aliquots containing appr...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00155-4
更新日期:1995-10-26 00:00:00
abstract::We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of app...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/s0022-1759(99)00143-x
更新日期:1999-12-10 00:00:00
abstract::This study describes a four-color flow cytometric assay that detects CD4+ T cell responses to the anthrax vaccine. Whole blood from seven volunteers who previously obtained the anthrax vaccine was inoculated in vitro with varying concentrations of the anthrax antigen. TNF-alpha and IFN-gamma production from memory CD4...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2003.07.012
更新日期:2003-11-01 00:00:00
abstract::The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of th...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(01)00395-7
更新日期:2001-08-01 00:00:00
abstract::Concanavalin A-activated murine suppressor T cells act via the mediator, soluble immune response suppressor (SIRS) which non-specifically suppresses IgM and IgG antibody responses to a variety of antigens, cytotoxic T lymphocyte responses and proliferative responses to alloantigens and mitogens in vitro. SIRS is a pro...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90235-6
更新日期:1982-08-27 00:00:00
abstract::A specific and sensitive enzyme immunoassay system for human secretory IgA was developed using anti-alpha-chain antibodies coupled to activated thiol-Sepharose, and anti-secretory component antibodies labeled with beta-D-galactosidase from Escherichia coli. The dose response of the enzyme activity in eluate was observ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(83)90053-4
更新日期:1983-01-01 00:00:00
abstract::The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2014.01.012
更新日期:2014-03-01 00:00:00
abstract::A new radioimmunoassay has been developed for measuring IgM and IgG rheumatoid factors. Diluted sera from donors and patients were incubated with immunoprecipitates prepared from sheep serum and rabbit anti-sheep IgG antiserum. The precipitates were washed, and radioiodinated rabbit F(ab')2 fragments specific from hum...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(81)90259-3
更新日期:1981-01-01 00:00:00
abstract::A new, 10-fold more sensitive method, based on an improved immunofixation technique, has been devised to detect oligoclonal IgM bands in unconcentrated cerebrospinal fluid (CSF). Using agarose gel electrophoresis, 5 microliters of an unconcentrated sample containing oligoclonal bands was separated and blotted on to a ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00023-9
更新日期:1997-04-25 00:00:00
abstract::A procedure is described making it possible to obtain quantitative data with the immunohistochemical peroxidase anti--peroxidase (PAP) technique. After applying PAP on fixed fibroblast preparations, peroxidase enzymic activity is measured on solubilized immune-complexes. The results show this technique to be sensitive...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90114-x
更新日期:1978-01-01 00:00:00
abstract::A rapid and simple immunostick immunosorbent ELISA has been developed for the diagnosis of myasthenia gravis. It is based on coating immunosticks with the major myasthenia gravis autoantigens acetylcholine receptor (AChR) or MuSK and it can be performed in <1 h, in a non-specialized examination place by non-skilled pe...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2018.06.016
更新日期:2018-09-01 00:00:00
abstract::A limiting dilution method for the efficient transformation by Epstein-Barr virus (EBV) of human B lymphocytes has been applied to the production of human monoclonal antibodies to ovarian cancer-associated antigens. Limited numbers (e.g., 2 X 10(5)) of EBV-infected B lymphocytes from ovarian cancer patient spleen, lym...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90274-2
更新日期:1987-12-24 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::To date, the applications of bispecific antibodies in immunoassay and immunocytochemical procedures have been directed at uniting two different biomolecules through the binding of epitopes on each respective substance. In this study, bispecific antibodies were constructed in which both binding sites were directed to t...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90042-6
更新日期:1994-05-16 00:00:00
abstract::An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(81)90162-9
更新日期:1981-01-01 00:00:00
abstract::Etanercept, a recombinant human tumor necrosis factor (TNF) receptor Fc fusion protein is an effective treatment option in adults with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or plaque psoriasis and paediatrics with juvenile idiotypic arthritis and plaque psoriasis. Patent expiration in Europ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:2017-08-01 00:00:00
abstract::IL-2 activity is commonly estimated in cell culture supernatants by an IL-2-dependent cell proliferation assay. This method is both reproducible and sensitive. However, it often appears from the literature that statistical analysis of the titration data either is disregarded or, when performed, is based on statistical...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90463-1
更新日期:1986-02-12 00:00:00
abstract::Whilst several centres have reported lymphocytotoxic antibody detection using single and dual fluorescent stains with analysis of the fluorescence emitted from the cell population present in a well of a multiwell plate, problems are encountered with cell concentration and light emission overlap. A method we have devel...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90042-1
更新日期:1987-05-04 00:00:00
abstract::A scheme for quantitation of serum and urinary paraproteins is described using isoelectric focusing and scanning densitometry. Paraproteins could be quantified using this system when present at concentrations ranging from 1 mg/ml to 26 mg/ml, depending on the immunoglobulin class. The relevance of these results to mon...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90082-7
更新日期:1986-06-24 00:00:00
abstract:OBJECTIVE:The objective of this study was the identification of the stain HIF-alpha using the Image Cytometry, and to help to count the positive cells (with HIF-alpha) and the negative cells (without HIF-alpha) from the same sample. METHOD:17 images of renal tissues from male rats of Winstar lineage; overall, there we...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2017.02.005
更新日期:2017-05-01 00:00:00
abstract::We have used a surface plasmon resonance biosensor (BIACORE 3000) to detect serum antibodies in chickens having current or recent infections. Three well-defined Salmonella flagellar recombinant DNA antigens reflecting Salmonella enteritidis (H:g,m flagellin) and Salmonella typhimurium (H:i and H:1,2 flagellins) expres...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(02)00102-3
更新日期:2002-08-01 00:00:00
abstract::A simple and highly sensitive analytical isoelectric focusing (IEF) technique using immunofixation with anti-C2 serum followed by silver staining has been developed in order to study simultaneously the structural polymorphism of both native C2 and C2 activation fragments (C2a MW 74,000 and C2b MW 34,000). Structural C...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90112-9
更新日期:1989-02-08 00:00:00
abstract::NIP-conjugated Fab' fragments from a rabbit hyperimmunized with sheep red blood cells (SRBC) were used to link the hapten NIP to target cells for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated lysis (ADCML). Target cells (SRBC) labelled with this NIP-Fab' complex were c...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90005-z
更新日期:1991-02-15 00:00:00