WO1992020714A1 - Control of milk secretion - Google Patents
Control of milk secretion Download PDFInfo
- Publication number
- WO1992020714A1 WO1992020714A1 PCT/GB1991/000744 GB9100744W WO9220714A1 WO 1992020714 A1 WO1992020714 A1 WO 1992020714A1 GB 9100744 W GB9100744 W GB 9100744W WO 9220714 A1 WO9220714 A1 WO 9220714A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- milk
- protein
- fraction
- peak
- protein according
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to a newly isolated protein from cow's milk and the use of the protein or antibodies thereto for the control of milk secretion in lactating animals.
- the rate of milk secretion by a lactating animal is regulated by the frequency of milk removal.
- studies by workers at the Hannah Research Institute, Ayr, Scotland on lactating goats have shown that another factor is involved. This is an inhibitor which decreases milk secretion at a local level, i.e. at the individual gland of an udder.
- One currently preferred definition Is a protein which inhibits milk secretion by lactating cows and which is present in the eighth significant peak (6B) when a nominally 10-30 KDa fraction of the whey proteins of the milk is resolved on an anion exchange column using 10 mM imldazole buffer, pH 7.0 and a sodium chloride elution gradient.
- the column is composed of particles of mono-disperse hydrophilic polymer having pendant groups, the particle diameter being 10 ⁇ 0.5 ⁇ M.
- the protein of the invention can optionally be further defined as follows. It has a molecular weight, as determined by gel filtration chromatography of the product of the 6B peak, without further purification, is about 6.5. Its isoelectric point in a polyacrylamide gel is within the range 5.1 to 5.4.
- the protein of the invention can optionally further be defined by reference to its having a shared epitope with the milk secretion-inhibitory bovine 2A protein of our second above-mentioned prior application. This was demonstrated by raising a mouse monoclonal antibody by a conventional technique against the milk secretion-inhibitory goat peak 3 protein of our first above-mentioned prior application and testing its reactivity with the products of various bovine peaks. It was found that the anti-(goat 3 peak) monoclonal antibody reacted with the product of bovine peak 2 (equivalent to the combined product of peaks 2A and 2B), and also with peak 2A, but not with the products of at least the fifth and sixth peaks (4 and 5).
- any combination of one or more of the above features, together with the inhibitory action of the protein, might be sufficient to define the protein uniquely and accordingly applicant does not wish to be limited unnecessarily to specific combinations, in case one of them or some aspect of one of them might later be re-determined and found not sufficiently to approximate to their definition given above, while the remaining features are confirmed, and leave no doubt as to the identity of the protein.
- Precisely which features are the most meaningful and the most reliable are, in any case, a matter of judgement, the preferred definitions given above reflecting applicant's current judgement. It will be appreciated, therefore, that the protein defined by other combinations of features herein set forth is to be considered as encompassed by the invention.
- the protein can be in glycosylated or unglycosylated form.
- the protein of the invention exists in cow's milk, probably In glycosylated form. It is believed that the effect of glycosylation 1s simply for attachment of the protein to the appropriate cells within the mammary gland. It would be expected, therefore, that the protein could be administered locally to the gland in an unglycosylated form.
- Antibodies can be raised against the protein of the invention by any conventional methods, e.g. as polyclonal antisera, mouse monoclonal antibodies, cow-mouse hybrid monoclonal antibodies or as engineered antibodies, by any of the currently available methods. Passive Immunisation methods can then be used to generate a reduction in the effect of the natural inhibitor, when this is desired in order to increase milk yield. Frequently, however, there will be a need to reduce milk yield 1n order to meet milk quotas, in which event the inhibitor itself is administered. Conventional carriers and adjuvants known in vaccination can be used.
- the invention is applicable to any animal responsive to the inhibitor defined herein. Since the 10-30 KDa goat's milk fraction has been successfully found to reduce milk accumulation and relevant enzyme activities when injected into the mammary gland of rabbits, it is likely that the cow's milk inhibitor will be effective in some other lactating animals.
- a dose in the range of 1 to 50mg, especially 5 to 20mg of Inhibitor Is Hkely to be effective and should be repeated as required, e.g. daily, and possibly reduced when given over long periods.
- the protein of the invention can be obtained from cow's milk by the method described in the Example or some variant thereon or addition thereto, e.g. chromatofocussing as described in our second above-mentioned prior application. It can be recovered in pure form from an eluate by extensive dialysis against water
- Milk was obtained at the morning milking from Friesian cows, and was defatted by centrifugation (2500 g, 15°C, 20 min ) and filtered through glass wool. Defatted milk was centrifuged for 2 h at 15°C, yielding a pellet of casein micelles and a clear supernatant containing whey proteins. The whey fraction was dialysed against distilled water for 24 h.
- the whey fraction was subjected to ultrafiltration using a filter with a nominal cut-off value of molecular weight 30,000
- Fractions containing protein peaks eluted from the column were dialysed extensively against distilled water, freeze-dried and stored at -20°C, before use in the next stage.
- the sole Figure of the drawing shows the elution of protein from . the chromatography column. Protein concentration, as absorption of light at 280 nm, on the left-hand ordinate is plotted against cumulative volume of eluted material on the abscissa. The plot is shown as lines. The right-hand ordinate is calibrated to show the sodium chloride gradient, from 0 to 1.0M, used in the eluant. The broken line is a plot of the sodium chloride concentration.
- the numbering is 1, 2A, 2B 3, 4, 5, 6A, 6B, 6C, 7 and 8 (although the last two peaks do not correlate with those for the non-ultraflltered whey).
- Mammary tissue was cultured as explants, small pieces of parenchymal tissue approximately 1cm 3 and weighing 0.5-0.7 mg. Explants were prepared from mammary tissue of mid-pregnant New Zealand White rabbits as described by R. Dils & I.A. Forsyth in Methods in Enzymology 72, 724-742 (1981). The explants were cultured in a defined culture medium (Medium 199; Gibco Europe Ltd., Paisley, UK) on stainless steel grids each holding 30 explants, so that the explants were in contact with the medium but not completely submerged in it.
- a defined culture medium Medium 199; Gibco Europe Ltd., Paisley, UK
- the medium was supplemented throughout with insulin (5mg/ml), cortisol (lOOng/ml) and prolactin (lmg/ml).
- Explants were cultured in this medium under an atmosphere of air/C0 2 (19:1 v/v) for 42 h, with replenishment of medium after 24 h.
- groups of explants (3 or 4 groups per treatment) were transferred into fresh medium containing hormones and one of the fractions of cow milk under test.
- the milk fractions tested in this experiment were obtained from cow's milk whey which had no ⁇ been ultrafiltered (as distinct from the 10-30 KDa fraction referred to above), but which had been fractionated by anion exchange chromatography as described above.
- explants and culture medium were separated and stored frozen in liquid nitrogen.
- Explants were homogenized at 4°C in 1.0 ml of lOmM Tris/ HC1 , pH 7.0, containing 5mM ethyleneglycol-bis-(2-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and 2mM phenyl ethane- sulphonyl fluoride by 10 strokes with a glass/PTFE homogenizer, followed by sonlcation for 30 s. (Kontes ultrasonic cell disruptor, 307. maximum power), and a particle-free supernatant was prepared by centrifugation at 10,000g for 5 in.
- casein 3 H-labelled casein was prepared from the particle-free supernatant by precipitation at its isoelectric point, and the precipitate was subjected to SDS-polyacryl mide gel electrophoresis, as described by CO. Wilde e ⁇ al-, Exp. Cell Res. 151, 519-532 (1984). Bands corresponding to casein polypeptides were visualized by staining with Coomassie brilliant blue, and were excised and counted for [ 3 H] radioactivity as described by S.M. Russell ⁇ al., Blochim. Biophys. Acta 714, 34-45 (1982).
- the amount of radioactive material (casein and lactose) was expressed as a percentage of that produced by the explants to which no milk fraction had been added. The results are shown in Table 1. The figures in parenthesis are the numbers of experiments performed on the various peaks.
- Results are the mean ⁇ s.e. . for 3 experiments.
- Peak 6A contained a major peak of m.w 12,500 KDa plus other less well defined constituents, 6B eluted as one major peak with m.w 6,500 KDa and peak 6C as two components of m.w. 12,500 and 6,500 KDa. 5. Isoelectric focussing of peak 6B protein
- PhaseGel electrophoresis system
- the method used “PhastGel IEF 4-6.5".
- Phasegel IEF media are homogeneous polyacrylamide gels containing "Pharmalyte” carrier ampholytes.
- “Phar alyte” generates stable, linear pH gradients in the gels during electrophoresis, in this case in the pH range 4 to 6.5. Proteins migrate under an electric field, essentially unhindered by the porous gel, to a point in the pH gradient that corresponds to their pi (isoelectric point).
- mice Female balb/c mice were immunised with a goat milk protein fraction containing a feedback inhibitor of milk secretion (see the first above-mentioned prior application).
- the inhibitory milk protein fraction, designated peak 3 was prepared by anion-exchange chromatography of a 10-30 KDa fraction of goat whey proteins on a Pharmacia FPLC (Fast Protein Liquid Chromatography) HR 10/10 Mono-Q column.
- the elution buffer was 20 mM bis tris propane pH 7.0, and proteins were separated using a sodium acetate gradient. Peak 3 was dialysed extensively against water, freeze-dried and stored at -20°C until required.
- peak 3 was conjugated to bovine serum albumin (BSA) by glutaraldehyde treatment.
- BSA bovine serum albumin
- peak 3 ( lOO ⁇ g ) dissolved in 0.1M- sodium phosphate buffer pH 6.8 was mixed with lOO ⁇ g BSA and 0.001% (w/v) glutaraldehyde at room temperature for 1 h.
- lysine final concentration 25mM
- the conjugated protein was dialysed against water and freeze-dried.
- mice were immunised by subcutaneous injections of goat inhibitor-BSA conjugate (each containing 50-100 ⁇ g of inhibitor protein) until an antibody titre of greater than 1/1000 was obtained by ELISA. A final intraperitoneal booster of lOO ⁇ g was given 4 days prior to cell fusion.
- the methods for cell fusion and preparation of monoclonal antibody were as described by Campbell in "Monoclonal antibody technology” (Burton, R.H. & Van Knlppenberg, P.H., eds.), Elsevier Science Publishers, Amsterdam, 1984, Chapter 6 pp. 120-133.
- mouse spleen cells were fused with the mouse myeloma cell line Y63-Ag-653 at a ratio of 10:1 spleen:myeloma cells.
- Cells were fused with 50% polyethylene glycol 1500.
- Hybridomas were grown in RPMI medium and 20% (v/v) foetal calf serum containing hypoxanthine, aminopterin and thymidine (HAT medium).
- HAT medium foetal calf serum containing hypoxanthine, aminopterin and thymidine
- the fused cells were incubated at 37°C in a humidified C0 2 incubator. After one week, cells were fed with fresh HAT medium. After unfused myeloma cells and spleen cells had died, hybridomas were fed with HT medium i.e. HAT minus aminopterin, and the foetal calf serum concentration was gradually reduced to 10% (w/v).
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3509300A JPH06507600A (en) | 1991-05-10 | 1991-05-10 | Regulation of milk secretion |
AU78704/91A AU665743B2 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
CA002102800A CA2102800A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
EP91909536A EP0584061A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
PCT/GB1991/000744 WO1992020714A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
US08/140,183 US5496802A (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002102800A CA2102800A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
PCT/GB1991/000744 WO1992020714A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992020714A1 true WO1992020714A1 (en) | 1992-11-26 |
Family
ID=25676497
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1991/000744 WO1992020714A1 (en) | 1991-05-10 | 1991-05-10 | Control of milk secretion |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP0584061A1 (en) |
WO (1) | WO1992020714A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991007439A1 (en) * | 1989-11-13 | 1991-05-30 | British Technology Group Ltd | Control of secretion of milk |
WO1991007434A1 (en) * | 1989-11-13 | 1991-05-30 | British Technology Group Ltd | Control of milk secretion |
-
1991
- 1991-05-10 WO PCT/GB1991/000744 patent/WO1992020714A1/en not_active Application Discontinuation
- 1991-05-10 EP EP91909536A patent/EP0584061A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991007439A1 (en) * | 1989-11-13 | 1991-05-30 | British Technology Group Ltd | Control of secretion of milk |
WO1991007434A1 (en) * | 1989-11-13 | 1991-05-30 | British Technology Group Ltd | Control of milk secretion |
Also Published As
Publication number | Publication date |
---|---|
EP0584061A1 (en) | 1994-03-02 |
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