Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1992-11-19
1994-06-28
Lilling, Herbert J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
536 20, 536 21, 536 33, 536 59, 536 61, 536100, 530395, 530412, 530413, 530417, 530425, C07K 300, C07K 1500
Patent
active
053248236
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a selective adsorbent for cellular fibronectin (cFN) and a technology for fractional purification of fibronectin (FN), particularly a method for fractionating an FN material containing both plasma fibronectin (pFN) and cFN into pFN and cFN and a method for eliminating cFN from blood by the first-mentioned method.
BACKGROUND ART
Fibronectin (FN) was first reported by Morrison et al. [Morrison, P. R. et al., J. Am. Chem. Soc., 70, 3103 (1948)] as one of plasma proteins in 1948. Being a multifunctional protein broadly distributed in various tissues and body fluids, this substance is known to be involved, as a cell adhesion factor, in a large variety of biological events such as the migration, differentiation, proliferation and canceration of cells [Sekiguchi, K.: Cell Engineering, 4 (6), 485-497 (1985)].
Meanwhile, FN as it is synthesized in the liver and occurring in the blood is known as plasma FN (pFN), and FN as it is detected on the cultured cell surface and culture medium is called cellular FN (cFN) [Sekiguchi et al., J. Biol. Chem., 260 (8) 5105-5114 (1985)]. It has been shown that these species of FN are subject to molecular diversity due to alternative splicing of the early gene transcription product. As the regions subject to such alternative splicing, there are three regions called EDA, EDB and IIIcs, and it is believed that a large number of molecular species occur according to varied combinations of expression of these regions. In pFN, the above-mentioned EDA and EDB regions have not been appreciably expressed. On the other hand, cFN is an FN with a high degree of expression of said EDA region. Peters J. H. et al. conjugated a peptide having 29 amino acids with keyhole limpet hemocyanin (KLH) to prepare an immunogen, constructed an anti-cFN polyclonal antibody specifically reactive to cFN having the EDA region, and using the antibody, demonstrated that the normal human blood contains traces of cFN and that the blood cFN level is markedly elevated in patients with collagen disease accompanied by vasculitis [Am. Rev. Respir. Dis., 138, 167-174 (1988); J. Lab. Clin. Med., 113 ( 5), 586-597 (1989)]. According to the above report of Peters et al., the physiological implications of increased blood cFN in patients with collagen disease accompanied by vasculitis are not definitely known but a correlation between the stage or severity of disease and the blood concentration of cFN is suggested. The investigation made by the inventors of the present invention using the anti-cFN monoclonal antibody which is to be described hereinafter revealed that whereas the plasma concentration of cFN in healthy humans is 1 to 2 .mu.g/ml, it is 4 to 8 .mu.g/ml in patients with various diseases and as high as 10 and odd .mu.g/ml in seriously ill patients. On the other hand, pFN is invariably within the normal range of 270 to 400 .mu.g/ml, irrespective of subjects.
Therefore, it is a subject of intriguing interest to investigate how the course of disease is modified by selective elimination of cFN from the blood of patients with collagen disease presenting with abnormal elevations of cFN. The recent study by the inventors of the present invention uncovered that when the plasma of a patient with rheumatoid arthritis is subjected to cryofiltration, cFN is partially removed from the plasma, resulting in relief of the symptoms. However, the cryofiltration procedure is disadvantageous in that it involves discarding of the useful plasma protein in large quantities and is not capable of selective elimination of cFN. Thus, both a technology for selective elimination of cFN from the patient's blood nor a selective adsorbent for such elimination remained to be developed. Furthermore, detailed research into the physiological role of cFN in patients with collagen disease and the functions and physiological implications of pFN and cFN require pFN and cFN in the pure form but since the difference between pFN and cFN, in structure, is of the minuscule order on a molecular scale, they ca
REFERENCES:
patent: 5120715 (1992-06-01), Kato et al.
JPOABS Japan 59-102436 (Oct. 4, 1984 Rb. Date ABS) Tani et al "Adsorbent Body".
Siri et al., Eur. J. Biochem., vol. 154, No. 3, (1986), pp. 533-538.
Borsi et al., FEBS Lett., vol. 192, No. 1, (1985), pp. 71-74.
Sekiguchi et al., J. Biol. Chem., vol. 260, No. 8, (1985), pp. 5105-5114.
Asakawa Kaneji
Hino Kazuo
Komai Takashi
Sakashita Eiji
Umemoto Ryoichi
Lilling Herbert J.
Otsuka Pharmaceutical Factory Inc.
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