Abstract:
:Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Nath N,Godat B,Zimprich C,Dwight SJ,Corona C,McDougall M,Urh Mdoi
10.1016/j.jim.2016.02.001subject
Has Abstractpub_date
2016-04-01 00:00:00pages
11-21eissn
0022-1759issn
1872-7905pii
S0022-1759(16)30016-3journal_volume
431pub_type
杂志文章abstract::Mixtures of sheep erythrocytes and immune spleen cells from mice were incubated in shallow slide chambers coated with erythrocyte ghosts or anti-mouse Ig, with poly L-lysine as a coupling agent. Antigen-binding cells and erythrocytes surrounding antibody-releasing lymphocytes became bound to the reactive surfaces by i...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90180-1
更新日期:1978-01-01 00:00:00
abstract::We describe here a lateral flow-based assay (LFA) for the detection of antibodies against immunodominant antigen Pgp3 from Chlamydia trachomatis, the causative agent of urogenital chlamydia infection and ocular trachoma. Optimal signal detection was achieved when the gold-conjugate and test line contained Pgp3, creati...
journal_title:Journal of immunological methods
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更新日期:2016-08-01 00:00:00
abstract::Intracellular probes used for oxidative burst evaluation could be a substrate of the multidrug resistance proteins (MDR), which may cause misinterpretation of experimental data. We aimed to study the effect of the culture condition and of the MDR-interfering antioxidant quercetin on free radical measurement in serum d...
journal_title:Journal of immunological methods
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abstract::Liposomes have been widely used as carriers of protein or peptide antigens. Antigenic materials can be attached to the outer surface, encapsulated within the internal aqueous spaces, or reconstituted within the lipid bilayers of the liposomes. The natural tendency of liposomes to interact with macrophages has served a...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/0022-1759(91)90120-5
更新日期:1991-06-24 00:00:00
abstract::We have previously shown a polymorphism of human Fc receptors for mouse IgG1 using an EA rosette technique in which human erythrocytes sensitized with a murine IgG1 monoclonal antibody against glycophorin A acted as indicator cells. We now describe a method to quantitate this EA rosetting using the pseudoperoxidase ac...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90223-7
更新日期:1987-07-16 00:00:00
abstract::Fungi represent emerging infectious threats to human populations worldwide. Mice and other laboratory animals have proved invaluable in modeling clinical syndromes associated with superficial and life-threatening invasive mycoses. This review outlines salient features of common vertebrate animal model systems to study...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/j.jim.2014.03.022
更新日期:2014-08-01 00:00:00
abstract::A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence of multiple molecular ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90434-x
更新日期:1985-11-28 00:00:00
abstract::We have recently described a new colorimetric DNA enzyme immunoassay (DEIA) for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single stranded DNA and therefore reveals the hybridization event independently from the DNA seq...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(92)90050-4
更新日期:1992-01-21 00:00:00
abstract::To reduce the cost, time and waste in screening for HIV and HBV infections a combined assay for HIV-1 and -2 antibodies and HBsAg has been developed. Monoclonal anti-HBs antibodies were co-immobilized with synthetic peptides representing immunodominant regions of HIV-1 and -2. The presence of anti-HIV antibodies in th...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90262-6
更新日期:1993-01-14 00:00:00
abstract::Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera. Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS. ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90308-i
更新日期:1990-06-12 00:00:00
abstract::Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from m...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90160-0
更新日期:1994-12-28 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2017.04.005
更新日期:2017-08-01 00:00:00
abstract::The structural diversity of the immune response may be considerably restricted by the structure of the hapten used to elicit catalytic antibodies. The ligand-binding mode and the shapes of the binding pockets of hydrolytic antibodies induced to different transition-state analogs that contain an unsubstituted arylphosp...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/s0022-1759(02)00240-5
更新日期:2002-11-01 00:00:00
abstract::A microtechnique to study lymphocyte stimulation in solid cultures by autoradiography is described. Using this technique at a concentration of 2 x 10(6) lymphocytes/chamber, it was found that the percentage of proliferating cells was 8.4% with anti-lymphocyte serum, 3.9% with calcium ionophore A23187, 2.5% with phytoh...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(79)90251-5
更新日期:1979-01-01 00:00:00
abstract::Hepatitis B surface antigen (HBsAg)-specific T cells were analysed in 16 healthy individuals vaccinated against hepatitis B, using optimised protocols. HBsAg-specific IFN-gamma producing CD4(+) T cells were found at a similar low frequency (0.01%) within the naive T cell subset, the central and the effector memory T c...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2007.10.013
更新日期:2008-01-31 00:00:00
abstract::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 ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00262-6
更新日期:1996-04-19 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::Radioimmunoassays are usually developed empirically since there have been few established rules which cover general RIA behaviour. Evidence is presented which enables the delineation of 2 types of RIA with distinct group characteristics. (1) Simple RIA, conforming to the law of mass-action kinetics, is a result of uni...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90441-7
更新日期:1985-11-28 00:00:00
abstract::We report a simple procedure for the maintenance and retrieval of rodent peritoneal macrophages on dialysis membrane supports. Culture units are easily constructed from plastic petri dishes and dialysis membranes. Macrophages adhere readily but are detached easily from the dialysis membrane by mild mechanical agitatio...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90322-2
更新日期:1982-11-12 00:00:00
abstract::Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the beta-lactam ring giving a conjugate distinct from previously described immunogenic preparatio...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(12)80026-3
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90180-5
更新日期:1980-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(01)00505-1
更新日期:2002-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90176-4
更新日期:1982-09-30 00:00:00
abstract::A number of supravital fluorochromes are available to study leucocyte functions in vitro and in vivo. The fluorescein ester most widely used, fluorescein diacetate, has the disadvantage of rapid cellular efflux, whereas more recently developed fluorescent probes do not exhibit this inconvenient trait. However, their e...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90384-0
更新日期:1994-06-03 00:00:00
abstract::A microtitre plate enzyme immunoassay (EIA) for determining low concentrations of IgA is described and validated for serum and plasma products. The measuring range of the assay was 3.3-150 micrograms/l. The predilution requirement of samples was matrix dependent, ranging from 1/16 for serum to 1/2 for 4% albumin. Dilu...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90239-4
更新日期:1993-07-06 00:00:00
abstract::Studies in mice have advanced our understanding of the contribution of monocytes to inflammation and immunity. However, the lack of a straightforward and reliable method for the isolation of mouse blood monocytes remains a hurdle. Here we describe a fast and easy method for isolating monocytes from mouse blood based o...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2010.04.004
更新日期:2010-07-31 00:00:00
abstract::Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the cen...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90416-2
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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 ma...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2010.09.034
更新日期:2011-03-31 00:00:00
abstract::We describe a simple, rapid and reproducible in vitro culture system in which human peripheral blood lymphocytes (PBLs), donated 24 h prior to initiation of culture can be stimulated to produce antigen-specific antibodies. Peripheral blood lymphocytes purified by Ficoll-Hypaque centrifugation were passed over a G10 Se...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00256-1
更新日期:1996-03-28 00:00:00
abstract::Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(00)00318-5
更新日期:2001-01-01 00:00:00