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An assay for measurement of antibodies binding to a therapeutic monoclonal antibody

A monoclonal antibody and therapeutic technology, which can be used in measurement devices, combinatorial chemistry, biological testing, etc., and can solve problems such as difficulties in partner modification of solid phases

Active Publication Date: 2013-05-15
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A buffer additive that has a positive effect in the case of one binding partner may even have an adverse effect o

Method used

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  • An assay for measurement of antibodies binding to a therapeutic monoclonal antibody
  • An assay for measurement of antibodies binding to a therapeutic monoclonal antibody
  • An assay for measurement of antibodies binding to a therapeutic monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0150] Preparation of biotin-conjugated Fab and F(ab') of specific therapeutic monoclonal antibodies 2 fragment

[0151] Fab Fragment: A full-length therapeutic monoclonal antibody of the immunoglobulin G class (IgG) in 100 mM phosphate, 2 mM EDTA buffer (pH 7.0) was synthesized with papain in the presence of 10-20 mM cysteine Incubate together (5-20 mU papain per mg IgG). Fragmentation was analyzed by analytical gel permeation chromatography and stopped after 60-120 min by addition of iodoacetamide solution (10 mM addition).

[0152] F(ab') 2 Fragments: Full-length therapeutic antibodies of the immunoglobulin G class (IgG) were incubated with pepsin (1-15 μg pepsin per mg IgG) in 100 mM sodium citrate buffer (pH 3.7). Fragmentation was analyzed by analytical gel permeation chromatography and stopped after 90 minutes by adjusting the pH to 6.5 with the addition of potassium phosphate.

[0153] Purification: Each of the two fragmented mixtures was dialyzed against...

Embodiment 2

[0158] Generation of monoclonal mouse IgM antibodies with rheumatoid factor-like specificity

[0159] Immunogen: H-IgG polymer

[0160] 10 mg of human IgG1 (Sigma Company) was dissolved in 0.6 ml of 25 mM bicarbonate buffer (pH 9.5). After adding 3.5 μl of 12.5% ​​glutaraldehyde solution, it was incubated at room temperature for 2 hours. Subsequently, it was cooled in an ice bath, adjusted to pH 8.3 with 50 mM triethanolamine solution (pH 8.0), and 0.15 ml of freshly prepared sodium borohydride solution (8 mg boron hydride / ml water) was added. After 2.5 hours at 0°C, the preparation was dialyzed against 10 mM potassium phosphate buffer / 0.2M NaCl (pH 7.5) at 4°C for 16 hours. Dialysate containing IgG multimers was stored in aliquots at -80°C or used for immunization and for specificity testing in hybridoma cell culture supernatants.

[0161] H-IgG3 multimers were generated in a similar manner starting from human IgG3 (Sigma Company).

[0162] Mouse immunization: ...

Embodiment 3

[0190] Fully automated ELISA assay on a multiparameter biochip platform

[0191] Multi-parameter biochip platforms are described in Hornauer, H. et al., BIOspectrum, Special Proteomics 10 (2004) 564-565 and Hornauer, H. et al., Laborwelt 4 (2004) 38-39.

[0192] A streptavidin coating is applied over the entire area of ​​the test zone of approximately 2.5 x 6 mm on the blackened polystyrene support (solid phase). Individual lines of approximately 10-20 identical spots per line consisting of biotinylated fragments of the therapeutic antibody were applied to the test area in an inkjet protocol; the diameter of each spot was approximately 150 μm.

[0193] Use the following test-specific reagents:

[0194] Sample dilution buffer:

[0195] 50mM Tris (pH7.6); 150mM NaCl; 0.1% detergent (polidocanol); 0.6% BSA; 0.2% preservatives (oxypyrion, oxypyrin and methylisothiazolone hydrochloride hydrochloride, MIT))

[0196] Wash buffer:

[0197] 10mM Tris, 0.01% polidoc...

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Abstract

The invention relates to an immunoassay method for determination of an anti- <therapeutic monoclonal antibody> antibody (anti-<TmAB>AB) in vitro in a sample from a patient treated with a therapeutic monoclonal antibody (TmAB). The method comprises the steps of (a) providing an F(ab) fragment of said TmAB bound to a solid phase, (b) incubating the solid phase provided in (a) with the sample, thereby binding the anti-<TmAB>AB to the solid phase via the F(ab) fragment, (c) incubating the solid phase obtained in (b) with a monoclonal antibody that binds to the anti-<TmAB>AB, (d) detecting the monoclonal antibody bound in (c) and thereby determining the anti- <TmAB>AB in the sample. The invention also concerns a method for the determination of antigen specific antibodies of a particular immunoglobulin class by means of an immunoassay in an array format in which the detection of an anti-<TmAB>AB to a TmAB in a sample provided from a patient treated with said TmAB is determined in vitro. Also disclosed is the use of such method for detection of an anti-<TmAB> antibody and in the identification of a patient who is at risk to develop an adverse drug reaction (ADR) during treatment with a TmAB.

Description

[0001] describe [0002] The present invention relates to a method for in vitro determination of anti-<therapeutic monoclonal antibody> antibody (anti- <tmab>AB) Immunoassay method from a patient treated with a therapeutic monoclonal antibody (TmAB). The method comprises the steps of: (a) providing the F(ab) fragment of said TmAB bound to a solid phase, (b) incubating the solid phase provided in (a) with the sample, whereby F(ab) ) fragments make anti <tmab>AB is combined with the solid phase, (c) the solid phase obtained in (b) is bound to the antibody <tmab>The monoclonal antibody of AB is incubated together, (d) detect the monoclonal antibody bound in (c), and thus determine the anti-antibody in the sample <tmab>AB. The present invention also contemplates a method for the determination of antigen-specific antibodies to a particular class of immunoglobulins by means of an immunoassay in the form of an array, wherein the antibody against TmAB i...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N2800/52G01N33/6854C40B30/04
Inventor V.P.格鲁纳特U.克劳斯P.库巴莱克M.罗思福斯B.厄普梅尔
Owner F HOFFMANN LA ROCHE & CO AG
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