Antibody complexes and methods for immunolabeling

an antibody complex and immunolabeling technology, applied in the field of immunolabeling complexes, can solve the problem that the time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, than 10 minutes

Inactive Publication Date: 2007-11-22
MOLECULAR PROBES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix. The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally

Problems solved by technology

The time required for the labeling reagent to selectively bind to the tar

Method used

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  • Antibody complexes and methods for immunolabeling
  • Antibody complexes and methods for immunolabeling
  • Antibody complexes and methods for immunolabeling

Examples

Experimental program
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Effect test

example 1

Preparation of Fc Antigen

[0245] Purified mouse and rabbit IgG was fragmented with the proteolytic enzyme papain (CURRENT PROTOCOLS IN CELL BIOLOGY, 16.4.1-16.4.10 (2000)). A 12 mL solution of mouse IgG was prepared at ˜2 mg / mL in phosphate-buffered saline (PBS). A solution containing 0.1 mg of papain in digestion buffer (PBS, 0.02 M EDTA, 0.02 M cysteine) was added to the antibody and allowed to react at 37° C. for 16 hours. The digestion was terminated by the addition 20 μL of 0.3 M iodoacetamide in PBS. The fragments were dialyzed against 2 L of PBS for 16 hours at 4° C. The Fc fragment was purified on a protein G-Sepharose CL-4B column. The bound fraction containing the Fc fragment was eluted from the column using 50-100 mM glycine / HCl buffer, pH 2.5-2.8. The eluate was collected in 1 mL fractions. The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction. The solution was then...

example 2

Production of Anti-Fc Antibodies

[0246] Polyclonal antibodies specific for the Fc region of an antibody were raised in goats against the purified FC region of an antibody from a different species (Example 1). Methods of immunizing animals are well known in the art, and suitable immunization protocols and immunogen concentrations can be readily determined by those skilled in the art (Current Protocols in Immunology 2.4.1-9 (1995); ILAR Journal 37, 93 (1995)). Briefly, individual goats were immunized with purified mouse Fc or purified rabbit Fc fragments. The initial immunization in 50% Freund's complete adjuvant (1000 μg conjugate (half subcutaneous, half intramuscularly)) was followed by 500 μg conjugate per goat in Freund's incomplete adjuvant two and four weeks later and at monthly intervals thereafter. Antibodies were purified from serum using protein A-Sepharose chromatography. Antibodies against mouse Fc isotypes can be prepared by starting with isotype-selected mouse Fc antige...

example 3

Preparation of Fab Fragments

[0247] Fragmentation of the goat anti-(mouse Fc) antibody to the monovalent Fab fragment was carried out using the proteolytic enzyme, papain, as described in Example 1. Following dialysis against PBS, the Fab fragment was purified on a protein A-Sepharose CL-4B column. The unbound fraction containing the Fab fragment and the papain was collected. This solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of ˜50 kDa were collected and analyzed by SDS-PAGE. The Fab fragments of goat anti-(rabbit Fc) can be prepared similarly.

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Abstract

The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling reagents have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody. The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Ser. No. 60 / 329,068, filed Oct. 12, 2001; U.S. Ser. No. 60 / 369,418 filed Apr. 1, 2002, U.S. Ser. No. 10 / 118,204 filed Apr. 5, 2002, and PCT / US02 / 31416 filed Oct. 2, 2002, which disclosures are herein incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to immuno-labeled complexes and methods for use in the detection and measurement of one or more targets in a biological sample. The invention has applications in the fields of molecular biology, cell biology, immunohistochemistry, diagnostics, and therapeutics. BACKGROUND OF THE INVENTION [0003] Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity. The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody. The ...

Claims

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Application Information

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IPC IPC(8): G01N33/00C12Q1/68G01N33/53G01N33/58G01N33/68
CPCB82Y5/00B82Y10/00G01N33/6857G01N33/53G01N33/58B82Y30/00
Inventor BEECHEM, JOSEPHHAGEN, DAVIDJOHNSON, IAIN
Owner MOLECULAR PROBES
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