Application of experimental design techniques to optimize a competitive ELISA.


:This paper describes the application of experimental design techniques to optimize a sensitive ELISA for a hapten molecule with a calibration range of 0-1000 pg/ml. Ten factors that were expected to affect the assay performance were initially screened, followed by factorial experiments to delineate the effects of the critical factors identified at the screening stage. Assay performance was evaluated using a unique rating system based on standard curve reproducibility, assay detection limits and the use of desirability functions. This rating system allowed multiple responses to be evaluated simultaneously. It was found that the substrate incubation time and enzyme label lot played an important role, while dilutions of the enzyme label and the anti-hapten antibody showed significant interaction. These observations were in good agreement with optimal assay conditions based on historical data collected over a period of two to three years. Application of experimental design techniques enabled us to confirm the significance of the factors affecting the assay within a three month period, with a minimum number of experiments. In addition, information on interaction between factors were determined.


J Immunol Methods


Sittampalam GS,Smith WC,Miyakawa TW,Smith DR,McMorris C




Has Abstract


1996-04-19 00:00:00














  • 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

    abstract::2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingl...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Cayer MP,Drouin M,Proulx M,Jung D

    更新日期:2010-04-15 00:00:00

  • High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires.

    abstract::The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historica...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Gene RW,Kumaran J,Aroche C,van Faassen H,Hall JC,MacKenzie CR,Arbabi-Ghahroudi M

    更新日期:2015-01-01 00:00:00

  • U937 cells stimulated with opsonised zymozan particles provide a convenient laboratory source of tumour necrosis factor alpha.

    abstract::The U937 cell line has been shown to generate tumour necrosis factor alpha (TNF-alpha) in response to soluble stimuli such as PMA and LPS, but only after treatment with GM-CSF. We report here the generation of TNF-alpha from U937 cells following phagocytosis of opsonised zymozan particles without the need for pre-trea...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Jiang WG,Puntis MC,Hallett MB

    更新日期:1992-08-10 00:00:00

  • BlockLogo: visualization of peptide and sequence motif conservation.

    abstract::BlockLogo is a web-server application for the visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif posit...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Olsen LR,Kudahl UJ,Simon C,Sun J,Schönbach C,Reinherz EL,Zhang GL,Brusic V

    更新日期:2013-12-31 00:00:00

  • Quantitative measurement of T-lymphocyte activation by an enzyme-linked immunosorbent assay (ELISA) detecting interleukin-2 receptor expression.

    abstract::A monoclonal antibody prepared against the murine interleukin-2 receptor (IL-2R) was employed to develop an ELISA method for measuring the immunological activation of T-cells. The assay detects an increase in IL-2R expression on activated lymphocytes. Stimulated splenic lymphocytes displayed markedly higher IL-2R expr...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Igietseme JU,Herscowitz HB

    更新日期:1987-02-26 00:00:00

  • Biomarker analysis of fucosylated kininogen through depletion of lectin reactive heterophilic antibodies in hepatocellular carcinoma.

    abstract::Hepatocellular carcinoma (HCC) accounts for >700,000 deaths worldwide, largely related to poor rates of diagnosis. Our previous work identified glycoproteins with increased levels of fucosylation in HCC. Plate-based assays to measure this change were compromised by increased levels of heterophilic antibodies with glyc...

    journal_title:Journal of immunological methods

    pub_type: 临床试验,杂志文章


    authors: Wang M,Shen J,Herrera H,Singal A,Swindell C,Renquan L,Mehta A

    更新日期:2018-11-01 00:00:00

  • Eosinophils isolated with two different methods show different characteristics of activation.

    abstract::Eosinophils can be isolated from a mixed suspension of granulocytes by different procedures. We compared functional responses of human eosinophils purified according to two different principles: (1) an fMLP-induced difference in specific gravity between eosinophils and neutrophils and (2) selective removal of neutroph...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Blom M,Tool AT,Mul FP,Knol EF,Roos D,Verhoeven AJ

    更新日期:1995-01-27 00:00:00

  • Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells.

    abstract::ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesi...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Garceau V,Houle MG,Chouinard F,Gagnon S,Harbour D,Naccache PH,Bourgoin SG

    更新日期:2001-03-01 00:00:00

  • Presence of matrix-specific antibodies in affinity-purified polyclonal antibodies.

    abstract::In general, antigen affinity columns made with commercially prepared activated affinity supports bind antibody specific for the coupled antigen. Nonetheless, in some cases affinity purification may yield antibodies to molecules other than the molecule of interest. In this report, we demonstrate such an occurrence: an ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Sweet SC,Gnegy ME,Welsh MJ

    更新日期:1991-01-24 00:00:00

  • A flow cytometric rosetting assay for the analysis of IgG-Fc receptor interactions.

    abstract::We have developed a sensitive and flexible method for the qualitative evaluation of IgG-Fc receptor interactions in cell suspensions. The assay is based on the flow cytometric quantitation of antibody-coated erythrocyte (EA) rosetting using fluorescein-labelled indicator erythrocytes (E). The number of IgG molecules o...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Tuijnman WB,Van de Winkel JG,Capel PJ

    更新日期:1990-03-09 00:00:00

  • Selective binding of heat- and antigen-aggregated IgG to Blue-Sepharose. Radioimmunoassay of circulating immune complexes.

    abstract::In an attempt to find a direct method for the determination of circulating immune complexes in pathological sera we studied the ability of Cibacron Blue 3GA covalently bound to Sepharose 4B to bind heat- or antigen-aggregated human IgG. Blue-Sepharose was found to bind significant amounts of aggregated IgG (about 1.5 ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Dobre MA,Moţa G

    更新日期:1983-12-16 00:00:00

  • Quantitative studies of peroxidase labeled antibody. I. Indirect staining system analyzed by chessboard titraions.

    abstract::The sensitivity and specificity of immunohistological staining procedures employing horseadish peroxidase labeled antibody were evaluated with the aid of chessboard titrations. Two indirect staining systems, detecting antinuclear antibody and pemphigus intercellular antibody, were examined with reference to indirect i...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Shu S,Albini B

    更新日期:1976-01-01 00:00:00

  • Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology.

    abstract::Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10 ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Karsunke XY,Pschenitza M,Rieger M,Weber E,Niessner R,Knopp D

    更新日期:2011-08-31 00:00:00

  • Interaction of the octapeptide angiotensin II with a high-affinity single-chain Fv and with peptides derived from the antibody paratope.

    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


    authors: Cohen P,Laune D,Teulon I,Combes T,Pugnière M,Badouaille G,Granier C,Mani JC,Simon D

    更新日期:2001-08-01 00:00:00

  • Single-chain Fv antibody with specificity for Listeria monocytogenes.

    abstract::Single chain antibodies (scFv) exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFvs expressed on the surface of filamentous bacteriophages. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and n...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Paoli GC,Chen CY,Brewster JD

    更新日期:2004-06-01 00:00:00

  • Evaluation of antigen-specific responses using in vitro enriched T cells.

    abstract::Antigen-specific lymphocytes are important in the immune response to viral infection. Peripheral blood mononuclear cells (PBMC) are traditionally used as a source of effector cells in most immunological studies. We described here the use of the bispecific monoclonal antibodies (BSMAB) anti CD3:CD8 (CD3,8) and anti CD3...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Jones N,Agrawal D,Elrefaei M,Hanson A,Novitsky V,Wong JT,Cao H

    更新日期:2003-03-01 00:00:00

  • Production of monoclonal antibodies that recognize the extracellular domain of mouse langerin/CD207.

    abstract::Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monocl...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Cheong C,Idoyaga J,Do Y,Pack M,Park SH,Lee H,Kang YS,Choi JH,Kim JY,Bonito A,Inaba K,Yamazaki S,Steinman RM,Park CG

    更新日期:2007-07-31 00:00:00

  • Characteristics of chemically aggregated IgM in an immunoglobulin class-specific immune complex assay.

    abstract::Immune complexes have been implicated in the pathogenesis of a number of infectious diseases. The predominant immunoglobulin class associated with circulating immune complexes is IgG, although immune complexes containing IgM have been described. The role of IgM immune complexes in disease pathogenesis has been difficu...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Margolis HS,Nainan OV,Fields HA,Bradley DW

    更新日期:1987-08-24 00:00:00

  • Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets.

    abstract::Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage displa...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Spiliotopoulos A,Owen JP,Maddison BC,Dreveny I,Rees HC,Gough KC

    更新日期:2015-05-01 00:00:00

  • Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies.

    abstract::Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin. Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclon...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Thomas TE,Lansdorp PM

    更新日期:1988-09-13 00:00:00

  • Clonal expansion of antigen-specific CD8+ cytotoxic T lymphocytes is regulated by late exposure to serum to prevent apoptosis.

    abstract::Although serum-free media have been used to expand lymphokine-activated killer cells, antigen-specific CD8 T cell cytotoxicity does not develop in vitro in the absence of serum. The immunodominant Vbeta17 response to an influenza A matrix protein epitope restricted by HLA A2.1 was used to study the serum requirement f...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Trimble LA,Xu Z,Lieberman J

    更新日期:1999-05-27 00:00:00

  • Immunohistochemical application of monoclonal antibodies to reveal the structure and localization of carbohydrate antigens. N-acetyllactosamine-related carbohydrate antigens in human biliary epithelial cells.

    abstract::With the use of a panel of monoclonal antibodies against structurally related carbohydrate antigens, finely regulated cellular phenotype of the carbohydrate antigens can be characterized. As a model system, the expression of N-acetyllactosamine-related antigens in human biliary epithelial cells was investigated using ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Okada Y,Tsuji T

    更新日期:1988-09-13 00:00:00

  • Investigation of endogenous antigen processing by delivery of an intact protein into cells.

    abstract::Investigation of MHC class I endogenous antigen processing and presentation is important for understanding non-self immune recognition, vaccine design, immune evasion by pathogens and tumours, and elucidation of the mechanisms of certain auto-immune diseases. To study the MHC class I antigen-processing pathway, the pr...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Fu H,Ding J,Flutter B,Gao B

    更新日期:2008-06-01 00:00:00

  • Development of a quantitative lateral-flow assay for rapid detection of fatty acid-binding protein.

    abstract::Using human heart-type fatty acid-binding protein (H-FABP) as an early cardiac marker to confirm or exclude a diagnosis of acute myocardial infarction (AMI) soon after admission requires a rapid assay system. Due to the requirement of skillful technicians and complicated assay procedures, most immunochemical assays fo...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Chan CP,Sum KW,Cheung KY,Glatz JF,Sanderson JE,Hempel A,Lehmann M,Renneberg I,Renneberg R

    更新日期:2003-08-01 00:00:00

  • Rapid in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies to both viruses and toxins.

    abstract::A generally applicable method was developed for the rapid and quantitative detection of both toxin- and virus neutralizing antibodies. The method was optimized for three different biological agents, i.e., Shigella toxin, influenza viruses (A/Beying, A/Taiwan and B/Yamagata) and Chikungunya virus. The in vitro micro-cy...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: van de Water C,van Dura EA,van der Stap JG,Brands R,Boersma WJ

    更新日期:1993-12-03 00:00:00

  • Improved methods for immunohistochemical detection of BrdU in hard tissue.

    abstract::Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieva...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Shimada A,Shibata T,Komatsu K,Nifuji A

    更新日期:2008-11-30 00:00:00

  • An anti-human t-lymphocyte antiserum. Further characterization of the specificity for T-cell subpopulations and a comparison of methods for identification of T-lymphocytes in tissue sections.

    abstract::An anti-human T-lymphocyte antiserum has been further studied for specificity for T-cell subpopulations and application in tissue sections. Using a complement-dependent microcytotoxicity assay about 60% of normal peripheral blood T-cells were found to be sensitive to lysis, while 40% were resistant. When the T-cell su...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Braathen LR

    更新日期:1980-01-01 00:00:00

  • Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.

    abstract::Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Graber P,Jansen K,Pochon S,Shields J,Aubonney N,Turcatti G,Bonnefoy JY

    更新日期:1992-05-18 00:00:00

  • Hapten-specific naïve B cells are biomarkers of vaccine efficacy against drugs of abuse.

    abstract::Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address t...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Taylor JJ,Laudenbach M,Tucker AM,Jenkins MK,Pravetoni M

    更新日期:2014-03-01 00:00:00

  • Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies.

    abstract::In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that ...

    journal_title:Journal of immunological methods

    pub_type: 杂志文章


    authors: Kurosawa G,Sumitomo M,Akahori Y,Matsuda K,Muramatsu C,Takasaki A,Iba Y,Eguchi K,Tanaka M,Suzuki K,Morita M,Sato N,Sugiura M,Sugioka A,Hayashi N,Kurosawa Y

    更新日期:2009-12-31 00:00:00