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Native immunoglobulin binding reagents and methods for making and using same

A technology of immunoglobulin and natural immunity, which is applied in the field of binding reagents, can solve the problems of increased time for immunoprecipitation, no preferential identification of natural immunoglobulin reagents available, and increased complexity of other steps to achieve the effect of unique ability improvement

Inactive Publication Date: 2006-11-22
EBIOSCIENCE (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of the Seize X kit include the following factors: (i) time - using the kit adds additional hours required for immunoprecipitation, and (ii) complexity - additional steps are required in the process
[0009] For this purpose, prior to the present invention, no reagents that preferentially recognize native immunoglobulins were available

Method used

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  • Native immunoglobulin binding reagents and methods for making and using same
  • Native immunoglobulin binding reagents and methods for making and using same
  • Native immunoglobulin binding reagents and methods for making and using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Antibody production

[0133] Immunization and Fusion Methods

[0134] Lou / M rats were immunized with mouse serum immunoglobulin. Balb / c mice were immunized with rabbit serum immunoglobulin. Serum titers were tested after three immunizations. Spleens from animals with high serum titers were fused to the mouse myeloma fusion partner SP2 / O.

[0135] fusion method

[0136] A. Culture medium preparation

[0137] 1) IMDM basal medium:

[0138] 2) 20% FBS complete medium:

[0139] 500ml IMDM basal medium+100ml FBS+6.5ml P / S (stock solution concentration: 10,000 units / ml penicillin; 100,000 units / ml streptomycin)+6.5ml glutamine (stock solution concentration: 200μM).

[0140] 3) Supplements:

[0141] 100×HAT

[0142] 50 x HES (Hybridoma Enhanced Supplement)

[0143] 4) fusion medium:

[0144] 500ml complete medium

[0145] 5ml 100×HAT

[0146] 10ml 50×HES

[0147] 5) 50% PEG (Sigma, MW 1500)

[0148] B. Preparation of Myeloma Cells

[0149] 1) Myeloma cell l...

Embodiment 2

[0248] Preparation of Polyclonal Anti-NIgSAB Subtraction Immunization

[0249] method:

[0250] Day 1: 6 mice were injected (i.p.) with a tolerogen (denatured immunoglobulin) (25-50 mg) not required for final antibody production in complete adjuvant. Ten minutes later, cyclophosphamide (SJGMA) in sterile phosphate-buffered saline was injected at 100 mg / kg body weight. A 2 mg / ml solution of cyclophosphamide was prepared for this purpose.

[0251] Day 2: Another injection of cyclophosphamide (100 mg / kg body weight).

[0252] Day 3: Repeat injection of cyclophosphamide.

[0253] Day 7: Mice were bled and tested for antibody titers by ELISA.

[0254] Day 14: Tolerogen (25-50 mg) not required for final antibody production in incomplete adjuvant was used to inject 6 mice (i.p.). Ten minutes later, cyclophosphamide in sterile phosphate-buffered saline was injected at 100 mg / kg body weight.

[0255] Day 15: Another injection of cyclophosphamide (100 mg / kg body weight).

[025...

Embodiment 3

[0263] Cloning and Expression of Anti-NIGSAB in Mammalian Cells

[0264] A typical mammalian expression vector contains at least one promoter element that mediates the initiation of transcription of the mRNA, the antibody coding sequence and signals required for transcription termination and polyadenylation of the transcript. Other elements include enhancers, Kozak sequences, and intervening sequences flanked by donor and acceptor sites for RNA splicing. Efficient transcription can be achieved with the early and late promoters of SV40, the long terminal repeat (LTRS) of retroviruses (eg, RSV, HTLVI, HIVI), and the early promoter of cytomegalovirus (CMV). However, cellular elements (eg, the human actin promoter) can also be used. Suitable expression vectors for practicing the invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, California), pcDNA3.1(+ / -), pcDNA / Zeo (+ / -) or pcDNA3.1 / Hygro (+ / -) (Invitr...

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Abstract

Isolated native immunoglobulin binding reagents including antibodies are provided, along with articles of manufacture, compositions and kits that include the native immunoglobulin binding reagents. Labeled reagents and subtrates that comprise samples or the reagents are provided. Method of screening for, making and using the reagents are also provided.

Description

[0001] Cross References to Related Applications [0002] This application is non-provisional USSN 60 / 509,850, filed October 8, 2003, by Seed and Li, entitled "Natural Immunoglobulin Binding Reagents and Methods of Making and Using the Same." This application claims priority to and benefit of USSN 60 / 509,850, which is hereby incorporated by reference in its entirety for all purposes. field of invention [0003] The present invention generally relates to binding agents that preferentially bind native immunoglobulins over denatured immunoglobulins. The invention also relates generally to methods of producing these binding reagents and their use in various research, diagnostic and therapeutic adjustments. These methods and binding reagents substantially improve and enhance the analysis of various immunological interactions including, but not limited to, the effects of antibodies involving anti-antibodies, such as analysis of prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/42C12NC12N5/06G01N33/53
CPCA61K2039/505C07K16/00
Inventor B·锡德G·李
Owner EBIOSCIENCE (US)
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