Abstract:
:Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and efficacy. For drugs administered intravitreally, systemic drug levels during the course of clinical studies are typically 100 to 1000-fold lower than those of similar therapeutics dosed intravenously, posing a significant bioanalytical challenge for PK measurements. Thus, the development of a highly-sensitive assay for measuring pg/mL levels of ranibizumab in patients' sera after intravitreal administration was needed to support clinical studies. In this report, we describe the development of a novel method that utilizes a high-affinity murine monoclonal anti-ranibizumab-VEGF-complexes antibody (MARA) reagent to measure ranibizumab in human serum. The assay format utilizes a semi-homogeneous solution phase step using a monoclonal antibody (the MARA) that binds specifically to the ranibizumab-VEGF complex, but not to either alone. This unique reagent exhibited low non-specific binding and high selectivity, increasing signal-to-noise readouts and maximizing assay sensitivity. The resulting MARA enzyme-linked immunosorbent assay (ELISA) has a lower limit of quantification of 15 pg/mL in human serum. In the assay, serum samples are incubated overnight with a mixture containing biotinylated-VEGF and MARA, which form a three-molecule complex with ranibizumab in the sample. These complexes are then captured onto streptavidin-coated wells, followed by enzymatic detection using a horseradish peroxidase-labeled-anti-murine antibody reagent and a colorimetric reaction. The assay conditions were optimized to allow for quantitative detection of "total" ranibizumab levels in serum. The assay was fully validated, establishing its high tolerance to sample matrix, as well as its suitable specificity, accuracy, dilution linearity, as well as intra- and inter-assay precision. The MARA ELISA's novel and unique approach has resulted in a considerably more sensitive ranibizumab PK assay compared to earlier versions of this assay. The MARA ELISA has been used for PK measurements in multiple ranibizumab studies, supporting this drug's life-cycle management and related preclinical and clinical-development studies.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Lowe J,Wakshull E,Shek T,Chuntharapai A,Elliott R,Rusit J,Maia Mdoi
10.1016/j.jim.2018.05.007subject
Has Abstractpub_date
2018-10-01 00:00:00pages
44-52eissn
0022-1759issn
1872-7905pii
S0022-1759(18)30039-5journal_volume
461pub_type
杂志文章abstract::Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secre...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90364-l
更新日期:1991-07-05 00:00:00
abstract::A sensitive direct assay has been developed to quantitate antigen specific helper factors (ThF). The assay depends on the ability of ThF to block the binding of free antibody to immobilized antigen. It has been used to follow the course of purification of ThF produced in short-term cultures of in vitro primed helper c...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90098-x
更新日期:1984-02-24 00:00:00
abstract::Recent developments in microarray technology have made it possible to perform immunoassays in a multiplexed format. This ability is highly desirable given the potential for greater throughput analysis. In spite of the obvious advantages, a number of issues arise as a result of multiplexing the reactions. In this study...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2005.04.007
更新日期:2005-07-01 00:00:00
abstract::We studied the binding of dog immunoglobulins G, A, M and E to protein A and protein G. Passive cutaneous anaphylaxis (PCA) testing was used for the measurement of dog IgE and enzyme-linked immunosorbent assays (ELISA) were used for the measurements of dog IgG, IgA and IgM. Protein A from lyophilized cells of Staphylo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90335-d
更新日期:1991-12-15 00:00:00
abstract::Immunochemical quantitative determination of a lipid antigen, ganglioside GM2, has been developed, based on the inhibition of the immune lysis of liposomes containing the antigen in their lipid bilayer. It has been shown that the full expression of the antigenicity of the competing lipid requires its dispersion in acc...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(77)90071-0
更新日期:1977-01-01 00:00:00
abstract::A method for separating IL-1 from plasma inhibitors by silica extraction has been developed and coupled to a highly sensitive bioassay using the LBRM TG6 cell line and the I1-2 dependent HT2A cell line. Using this assay we have detected IL-1 activity in plasma from patients undergoing elective surgery. ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90399-7
更新日期:1988-04-06 00:00:00
abstract::Monoclonal antibodies (MAbs) were selected for specific binding to spent media of cultured tumor cells. Out of more than 12,000 hybridomas screened, 19 were selected in preliminary inhibition assays for secretion of MAbs which detected antigens in cancer patients' sera. Antibody CO 29.11, which was studied in detail, ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90169-3
更新日期:1985-06-12 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::The sensitivity of crossed immunoelectrophoresis (CIE) and rocket immunoelectrophoresis (RIE) does not usually permit the study of diluted protein solutions or the detection of trace components. Antigen-antibody precipitates obtained after immunoelectrophoresis were therefore dissociated and transferred from the agaro...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90163-9
更新日期:1988-11-10 00:00:00
abstract::Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00287-1
更新日期:1996-05-10 00:00:00
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
doi:10.1016/0022-1759(80)90149-0
更新日期:1980-01-01 00:00:00
abstract::Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop co...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2003.07.011
更新日期:2003-10-01 00:00:00
abstract::Exosomes are nanometer-sized vesicles, secreted from most cell types, with documented immune-modulatory functions. Exosomes can be purified from cultured cells but to do so effectively, requires maintenance of cells at high density in order to obtain sufficient accumulation of exosomes in the culture medium, prior to ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2008.03.001
更新日期:2008-06-01 00:00:00
abstract::Interferons-alpha and -beta (IFN-alpha and -beta) are cytokines that are widely known to induce potent anti-viral activity. However, it has become increasingly apparent that IFN-alpha and -beta exert a variety of other biological effects, including anti-tumour and immunomodulatory activities and are increasingly used ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(96)00092-0
更新日期:1996-09-09 00:00:00
abstract::The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00159-2
更新日期:1997-12-15 00:00:00
abstract::The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents seve...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.02.020
更新日期:2016-06-01 00:00:00
abstract::Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to inf...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.09.004
更新日期:2016-12-01 00:00:00
abstract::Dispersed fluorocarbon oil droplets were used as the antigen-carrier particle in an immunological agglutination assay. The extent of agglutination was found to vary with the percentage of antigen to that of inert protein used in the solution to coat and stabilize the oil droplets. Maximum sensitivity was observed when...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90533-8
更新日期:1986-03-13 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::MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; howev...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2015.12.011
更新日期:2016-02-01 00:00:00
abstract::C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(01)00295-2
更新日期:2001-05-01 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
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90396-d
更新日期:1990-08-28 00:00:00
abstract::A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or IgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90488-0
更新日期:1985-01-21 00:00:00
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...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.02.001
更新日期:2016-04-01 00:00:00
abstract::Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of per...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90090-3
更新日期:1991-11-22 00:00:00
abstract::The identification of distinct T helper lymphocyte subsets (Th1/2) with polarised cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high ...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/s0022-1759(00)00230-1
更新日期:2000-09-21 00:00:00
abstract::The present study examined the use of replication-defective adenovirus for in vitro production of an immunoadhesin. A recombinant adenovirus, rendered replication defective by deletion of the E1 gene, was constructed to contain the murine interleukin-10 gene fused in frame with the hinge, CH2, and CH3 domains of the m...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(96)00233-5
更新日期:1997-03-10 00:00:00
abstract::A new and simple method for quantitation of IgM of low molecular weight in sera by radial immunodiffusion in 7% agar gel is described. The test is easy to perform and permits exact determination of low molecular weight IgM concentration down to very small quantities (1 mg%). Results obtained with the method described ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90094-7
更新日期:1978-01-01 00:00:00
abstract::The in vitro migration inhibition responses of peripheral blood leukocytes from tuberculin skin test positive and negative donors were tested to validate and determine optimal conditions for the agarose droplet method in the human. In vitro migration inhibition was observed in skin test positive donors in the presence...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90063-7
更新日期:1978-01-01 00:00:00
abstract::An enzyme-linked immunosorbent assay (ELISA) for determination of antibodies against the zona pellucida was developed and compared with the already available indirect immunofluorescence (IIF) technique. Sera from 100 women with explained and unexplained infertility were screened for the presence of autoantibodies to t...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90399-0
更新日期:1985-05-10 00:00:00