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Anti-MIF antibody cell migration assay

A measurement method, a technology of cell migration, applied in chemical instruments and methods, specific peptides, measuring devices, etc.

Active Publication Date: 2015-09-02
BAXALTA INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although MIF proteins exhibited chemokine-like effects in earlier tests and these functions could be blocked by anti-MIF antibodies (e.g., Bernhagen et al., Nature Medicine, 2007), no method based on targeting Inhibition of e.g. autocrine-induced cell migration of monocytes qualified for stable MIF bioassays

Method used

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  • Anti-MIF antibody cell migration assay
  • Anti-MIF antibody cell migration assay
  • Anti-MIF antibody cell migration assay

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0112] The preparation of the anti-(ox)MIF antibody of the present invention includes any method using genetic engineering to produce recombinant DNA, for example, by reverse transcription of RNA and / or amplification of DNA and cloning it into an expression vector. In some embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in the host cell into which it is introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication). In other embodiments, the vector (eg, a non-episomal mammalian vector) can integrate into the genome of the host cell when introduced into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or sim...

Embodiment 1

[0170] Anti-MIF Antibody RAM9 Chemical Activation Assay; Cell-Based Assay

[0171] Intended purpose:

[0172] The assay was set up to test the function (= test item) of glycine buffered anti-MIF RAM9 preparations to inhibit the random migration (= chemical activation) of monocytes. The inventors have demonstrated that this assay, as well as the respective methods, can be used as a quality control test in the processing steps of the final drug product (FDP).

[0173] 1) Principle:

[0174] MIF is constitutively expressed on U397 (and other cancerous) monocytes and oxMIF is present on the surface of these cells (FACS data, see figure 2 ), which supports the migration function. It is an important feature of the invention to use cells expressing (ox)MIF endogenously or exogenously, eg cells from disease samples. This rationale is based on the inventors' previous findings that oxMIF is absent on healthy cells or tissues. The U397 cell line is a preferred embodiment for carryi...

Embodiment 2

[0242] In principle, this same assay can be used to determine whether antibodies can be provided to the upper chamber together with the cells.

[0243] summary:

[0244] Cell migration test: HTS Transwell plate (Coming): 5μm

[0245] *Cells: U937 (passage 10), starved overnight

[0246] *RAM9 Antibody: 30nM-0.04nM, pipet into culture chamber

[0247] * Glycine: 30nM; 10nM; 3,3nM Pipette into the culture chamber

[0248] *Negative Control: Glycine; Positive Control: FBS (Fetal Bovine Serum)

[0249] *Culture: 2 plates, 16 hours

[0250] * calculations for the results

[0251] result:

[0252] Tablet 1

[0253] Antibody concentration (nM)

Measurements

fit

0

1997,6

0,04

2452,7

2498,3

0,12

2490,8

2388,4

0,37

2019,2

2106,0

1,1

1642,0

1621,2

3,3

1179,6

1115,5

10

688,0

810,8

30

753,7

685,7

maximum value

＝A

2555

...

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Abstract

The present invention pertains to a robust, precise and easy-to-use assay for potency of anti-MIF antibodies. In particular, the invention discloses an anti-MIF antibody-cell migration assay and respective method and kit.

Description

[0001] The present invention relates to an assay suitable for testing the potency of anti-MIF antibodies. In particular, the present invention relates to an advantageous and easy-to-use assay for cell migration. Background technique [0002] Macrophage migration inhibitory factor (MIF) is a cytokine originally isolated on the basis of its ability to inhibit random migration in vitro of peritoneal exudate cells (containing macrophages) of tuberculin-allergic guinea pigs (Bloom et al., Science 1966 , 153, 80-2; David et al., PNAS 1966, 56, 72-7). Today, MIF is well known as a key upstream regulator of broad-spectrum activity in innate and adaptive immune responses. In 1989, the cDNA of human MIF was cloned (Weiser et al., PNAS 1989, 86, 7522-6), and its gene was located on chromosome 22. The human MIF gene product is a protein of 114 amino acids (after cleavage of the N-terminal methionine) and an apparent molecular weight of approximately 12.5 kDa. MIF has no significant seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/68
CPCG01N33/5029G01N33/6863C07K16/40C07K2317/76G01N33/5047G01N2333/4704G01N2333/99
Inventor M·蒂塞尔R·克西堡曼
Owner BAXALTA INC
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