A quick change blow mold shell assembly is disclosed. The blow mold shell assembly includes a carrier plate and a blow mold half shell for being fastened to the carrier plate. A first slotted opening is defined in an edge of the carrier plate, and a corresponding second slotted opening is defined in an edge of the blow mold half shell. The first and second openings are placed in registry with one another as the shell is positioned on its carrier plate. A fastener clip is also provided, the clip being sized and shaped to be received in each one of the respective openings, and being constructed and arranged to align the first and second openings with respect to one another, to draw the shell snugly against the carrier plate, and to fasten the shell to its carrier plate as a fastener is passed through the clip and into the carrier plate.

This invention relates in general to blow mold machinery, and to the blow molding of containers. More particularly, this invention relates to an improved blow mold shell assembly having a blow mold shell and carrier plate in which the blow mold shell is adapted to be changed out for use in a quick and expedient fashion, while also aligning and fastening the blow mold shell to its carrier plate.
A large number of beverages, to include a wide variety of food products, are packaged in plastic bottles and/or containers. Plastic has proven to be a useful material as it can be readily adapted and formed into a variety of shapes and sizes, as well as designs. Moreover, a variety of plastics can be used to form bottles to package beverages, for example, as well as containers for semi-solid food products to include mayonnaise and peanut butter. Such plastics include polyethylene, polyvinylchloride, and polyethylene terephthalate also known as xe2x80x9cPETxe2x80x9d to those skilled in the industry.
The use of PET has grown due to the ease with which the material can be formed through a blow molding operation, either injection blow molding, stretch blow molding, or extrusion blow molding. Moreover, PET has the benefit of being lightweight, transparent, and has a superior resistance to impact, heat and pressure and lastly, it is 100% recyclable.
The shape, size and/or design of the molded PET container is a result of the design machined into a blow mold shell assembly. The shell assembly will typically include two opposed mold shell halves, each of which is machined, typically, from a ductile and durable metallic material, such as steel, to include stainless steel, and more recently aluminum. Aluminum offers the advantage of being lighter in weight, and easier to machine, although it can be more easily damaged. Once the mold shell halves are fabricated, they are then each affixed to a separate one of a pair of opposed support plates, also known as carrier plates or shell holders, which are themselves affixed to the blow mold machine, and more particularly to a shell opening and closing device.
In the blow mold process, a heated PET preform is positioned between the mold shell halves, whereupon the mold shell halves are then closed about the preform by the shell opening and closing device, at which point in time the preform is stretched, and compressed air or other suitable gases are passed into the preform such that it expands and takes on the shape of the mold cavity, and thus the container defined by the mold shell halves and an adjoining base portion of the mold which is moved into position as the shell halves close about the preform.
The known types of blow mold machines used for the process include linear or straight line blow mold machines which move the blow mold shells along a straight line toward and away from one another, as disclosed in U.S. Pat. No. 5,284,432 to Wurzer, and U.S. Pat. No. 5,551,861 to Baldi. The newer generation of blow molding machines are rotary blow mold machines, such as those shown in U.S. Pat. No. 3,854,855 to Pollock, et al.; U.S. Pat. No. 4,861,542 to Oles, et al.; U.S. Pat. Nos. 5,551,860 and 5,556,648, both to Budzynski, et al.; and U.S. Pat. No. 5,683,729 to Valles, which patent is assigned to Sidel S. A., the parent company of the assignee of this application.
In order to make a variety of containers in differing shapes, sizes and/or designs, it is necessary to xe2x80x9ccutxe2x80x9d molds for each such specific shape, size, and/or design. In order to produce a variety of shapes, sizes and designs among these blow mold containers, therefore, a container manufacturer or food packager can either have a machine dedicated to producing a certain type of container, or will be required to remove and replace differing blow mold shell sets, i.e. matching blow mold shell halves and the base assembly, from the blow mold machine when changing over from producing containers of a first size to a second size, for example. In a straight line blow mold machine such as that disclosed in the ""861 patent to Baldi or the ""432 patent to Wurzer, this is relatively easy to accomplish in that there typically is an adequate amount of room for a machine service technician to gain access to the molds, remove them from their carrier plates, and then install the new shell halves. However, on rotary blow mold machines, this becomes more of a problem.
U.S. Pat. No. 5,326,250 to Doudement, also assigned to the parent concern of the assignee of this invention, discloses an opening and closing mechanism for use in opening and closing a blow mold half shells on a rotary blow mold machine. As shown in Doudement, the mechanism is configured as a clam shell arrangement having a fixed pivot about which the mold shell halves will be pivoted for opening and closing. The carrier plates of the shell assembly will be affixed to the opening and closing mechanism, and in turn the blow mold shell halves will be removed from and replaced on their respective carrier plates based on the size, shape, or design of the container being produced.
The problem faced however, by machine service technicians is that as this is a clam shell type of opening and closing mechanism, there is a limited amount of space available for gaining access, to any threaded fasteners, or other fasteners which may be used for securing the blow mold shell halves to the holder or carrier plates. The changing over of the blow mold shell halves thus becomes a laborious and tedious process, made all the more difficult by the close confines within which these machines are typically housed. Thus, there is a need for an improved blow mold shell assembly in which the blow mold shells can be quickly and easily installed, removed, and replaced, as needed, to improve production efficiency and reduce the amount of machine downtime incurred when changing over the blow mold shell sets.
Another problem faced when changing over blow mold shell sets, more so on a clam shell type of holder arrangement on a rotary blow mold machine than with a straight line machine, is the alignment of the shell halves with their respective carrier plates. As known to those of skill in the art, a cooling or heat-treating fluid is oftentimes passed through conduits or channels defined in the carrier plate, the carrier plate in turn having ports which will be positioned in registry with inlet and discharge ports, respectively, defined in the blow mold shell halves. The precise alignment of the shell halves on the carrier plates is thus necessary in order to ensure that the cooling or heat-treating fluid does not leak out over the molds during the blow mold process, leading to undesirable or unacceptable containers, all of which tends to decrease machine efficiency. What is needed, therefore, is an improved blow mold shell assembly in which not only can the blow mold shells be quickly and easily removed and replaced, but which will also precisely align the blow mold shell with its respective carrier plate during the changeover process.
An additional problem faced by blow mold container manufacturers deals with the use of the newer generation of mold shell materials, for example aluminum. A great many blow mold shell halves are threadably affixed to their carrier plates by passing a threaded fastener through the carrier plate and into the mold shell, or by using clamps or straps which are threadably passed into both the carrier plate and the mold shell halve. The problem faced with using softer metals, for example aluminum, is that a machine service technician may over tighten a fastener such that the threads within the shell become stripped or destroyed, necessitating that the shell be removed from use and retapped, if possible, as the shell will no longer be securely fixed to its carrier plate, nor will it remain in a fixed position on its carrier plate during the high operating speeds employed by rotary blow mold machines. Moreover, due to the cost of machining blow mold shell halves, it is desirable that the service life of the shell half be extended for as long a time period as possible. What is needed, therefore, is an improved quick change blow mold shell assembly which will quickly and easily secure a blow mold shell half to its respective carrier plate without requiring the use of threaded fasteners passed into the blow mold shell.
The present invention provides an improved quick change blow mold shell assembly which overcomes some of the design deficiencies of the known blow mold shell assemblies. This invention provides a simple, efficient, and cost effective quick change blow mold shell assembly for use on the known types of blow mold machinery, and with which blow mold shell halves can be quickly, easily, and accurately changed out when it is desired to change the blow mold shells during a product size or design changeover, especially on rotary blow mold machines.
The improved quick change blow mold shell assembly of this invention thus includes a carrier plate adapted for being fastened to a blow mold shell assembly opening and closing device of the type known in the art. A first opening is defined in an edge of the carrier plate. The invention also includes a blow mold shell, or more preferably a half shell, having a second opening defined in an edge thereof. The first and second openings of the carrier plate and the half shell, respectively, are placed in registry with one another as the shell is positioned on its carrier plate for being mounted to a blow mold machine. A fastener clip sized and shaped to be received in each one of the respective openings defined in the carrier plate and the half shell, respectively, is provided. The fastener clip is constructed and arranged to align the first and second openings with respect to one another, thus aligning the shell with its carrier plate, while simultaneously drawing the shell snugly against its carrier plate as a fastener is passed through the clip and into the carrier plate for fastening the shell to its carrier plate.
The fastener clip is substantially U-shaped, and has a first leg and a spaced second leg. Each leg extends in a lengthwise direction away from a body portion of the clip, and each leg is tapered in its lengthwise direction. It is anticipated that the legs will be tapered at an angle of approximately 15xc2x0. In addition, each of the legs is beveled along the spaced parallel side edges of the length of the tapered surface.
In a preferred embodiment of the invention, each of the first and second openings defined in the carrier plate and the half shell, respectively, will also have a tapered surface sized complimentarily to the taper of the respective leg of the clip received therein, each such taper surface also being complimentarily beveled so that the clip progressively aligns the first and second openings with respect to one another, and progressively draws the shell and the carrier plate toward one another as the fastener, preferably an elongate threaded fastener, is passed through the fastener clip and into a threaded opening defined in the carrier plate.
It is anticipated that the half shell can be either a single or dual cavity blow mold shell. Moreover, in addition to the first and second openings defined in the top edges of the carrier plate and half shell, respectively, it is anticipated that additional openings, provided as corresponding pairs of openings, one each in the carrier plate and the half shell, may be provided along the side edges thereof, as desired, each such pair of openings being provided with one of the fastener clips as described above.
The openings defined in the edges of the carrier plate and half shell are formed as recessed slotted openings, sized and shaped such that as the fastener clip is received in such openings, it fits substantially flush with respect to the outwardly facing surfaces of the fastened half shell and carrier plate.
In a second embodiment, the invention includes a blow mold shell assembly for use with a known type of blow mold machine, the machine having a known type of clam shell style blow mold shell opening and closing mechanism to which the blow mold assembly is mounted. So provided, this embodiment of the invention will include first and second carrier plates adapted to be separately affixed to the blow mold shell opening and closing mechanism. First and second blow mold half shells are also provided for being separately and removably fastened to each respective carrier plate.
A first slotted opening is defined in a top edge of each carrier plate, and a corresponding second slotted opening is defined in the top edge of each half shell. The first and second openings are placed in registry with one another as the respective half shells are placed on their carrier plates, whereupon a fastener clip, as described above, one being provided for each pair of the first and second openings described, is received in each one of the openings of each pair of openings, and is constructed and arranged to align and draw each half shell snugly against its respective carrier plate as a fastener is passed through the clip and into its respective carrier plate.
It is, therefore, an object of the present invention to provide an improved quick change blow mold shell assembly.
Yet another object of the present invention is to provide an improved quick change blow mold shell assembly which is simple in design and construction, is rugged and durable in use, and is easy to use.
It is to these objects, as well as to the other objects, features, and advantages of the present invention, which will become apparent upon reading the specification, when taken in conjunction with the accompanying drawings, to which this invention is directed.


A dishwashing cleaning wipe comprising a fabric wherein the fabric is impregnated with a cleaning composition.

The present invention relates to a dishwashing cleaning wipe which is single or multi layer fabric substrate which has been impregnated with a liquid cleaning composition.
The patent literature describes numerous wipes for both body cleaning and cleaning of hard surfaces but none describe wipes for cleaning dishware, flatware, pots and pans. U.S. Pat. Nos. 5,980,931, 6,063,397and 6,074,655 teach a substantially dry disposable personal cleansing product useful for both cleansing and conditioning the skin and hair. U.S. Pat. No. 6,060,149 teaches a disposable wiping article having a substrate comprising multiple layers.
U.S. Patent Nos. 5,756,612; 5,763,332; 5,908,707; 5,914,177; 5,980,922 and 6,168,852 teach cleaning compositions which are inverse emulsions.
U.S. Pat. Nos. 6,183,315 and 6,183,763 teach cleaning compositions containing a proton donating agent and having an acidic pH. U.S. Pat. Nos. 5,863,663; 5,952,043; 6,063,746 and 6,121,165 teaches cleaning compositions which are oil in water emulsions.
A single use cleaning wipe for dishwashing application comprises a water insoluble substrate, with a cleaning composition containing at least one anionic surfactants and water coated onto and/or impregnated into the water insoluble substrate.
The liquid cleaning compositions of this invention are not an emulsion and do not contain potassium sorbate, a polysaccharide polymer, a polycarboxylate polymer, polyvinyl alcohol polymer, polyvinylpyrrolidone polymer or methyl vinyl ether polymer.
The present invention relates to a cleaning wipe for dishware, flatware, pots and pans which comprises approximately:
(a) 20 wt. % to 95 wt. % of a water insoluble substrate; and
(b) 5 wt. % to 85 wt. % of a liquid cleaning composition being coated onto and/or impregnated into said water insoluble substrate, wherein said liquid cleaning composition comprises:
(i) 0 wt. % to 60 wt. %, more preferably 8 wt. % to 40 wt. % of at least one anionic surfactant selected from the group of sulfate anionic surfactants and sulfonated anionic surfactants and mixtures thereof;
(ii) 0.25% to 30% of a surfactant selected from the group consisting of amine oxides, alkyl polyglucosides, zwitterionic surfactants, ethoxylated nonionic surfactants and alkanol amides and mixtures thereof; and
(iii) the balance being water, wherein the composition has a pH of 6 to 8 and does not contain ammonium hydroxide, an alkali metal hydroxide, potassium sorbate, a polysaccharide polymer, a polycarboxylate polymer, polyvinyl alcohol polymer, polyvinylpyrrolidone polymer or methyl vinyl ether polymer.
Suitable water-soluble non-soap, anionic surfactants used in the instant compositions include those surface-active or detergent compounds which contain an organic hydrophobic group containing generally 8 to 26 carbon atoms and preferably 10 to 18 carbon atoms in their molecular structure and at least one water-solubilizing group selected from the group of sulfonate, sulfate and carboxylate so as to form a water-soluble detergent. Usually, the hydrophobic group will include or comprise a C8-C22 alkyl, alkyl or acyl group. Such surfactants are employed in the form of water-soluble salts and the salt-forming cation usually is selected from the group consisting of sodium, potassium, ammonium, magnesium and mono-, di- or tri-C2-C3 alkanolammonium, with the sodium, magnesium and ammonium cations again being preferred.
Examples of suitable sulfonated anionic surfactants are the well known higher alkyl mononuclear aromatic sulfonates such as the higher alkyl benzene sulfonates containing from 10 to 16 carbon atoms in the higher alkyl group in a straight or branched chain, C8-C15 alkyl toluene sulfonates and C8-C15 alkyl phenol sulfonates.
A preferred sulfonate is a mixture of an alkali metal ammonium salt and an alkaline earth metal salt of a linear alkyl benzene sulfonate having a high content of 3- (or higher) phenyl isomers and a correspondingly low content (well below 50%) of 2- (or lower) phenyl isomers, that is, wherein the benzene ring is preferably attached in large part at the 3 or higher (for example, 4, 5, 6 or 7) position of the alkyl group and the content of the isomers in which the benzene ring is attached in the 2 or 1 position is correspondingly low.
Other suitable anionic surfactants are the olefin sulfonates, including long-chain alkene sulfonates, long-chain hydroxyalkane sulfonates or mixtures of alkene sulfonates and hydroxyalkane sulfonates. These olefin sulfonate detergents may be prepared in a known manner by the reaction of sulfur trioxide (SO3) with long-chain olefins containing 8 to 25, preferably 12 to 21 carbon atoms and having the formula RCHxe2x95x90CHR1 where R is a higher alkyl group of 6 to 23 carbons and R1 is an alkyl group of 1 to 17 carbons or hydrogen to form a mixture of sultones and alkene sulfonic acids which is then treated to convert the sultones to sulfonates. Preferred olefin sulfonates contain from 14 to 16 carbon atoms in the R alkyl group and are obtained by sulfonating an xcex1-olefin.
Other examples of suitable anionic sulfonate surfactants are the paraffin sulfonates containing 10 to 20, preferably 13 to 17, carbon atoms. Primary paraffin sulfonates are made by reacting long-chain alpha olefins and bisulfites and paraffin sulfonates having the sulfonate group distributed along the paraffin chain are shown in U.S. Pat. Nos. 2,503,280; 2,507,088; 3,260,744; 3,372,188; and German Patent 735,096.
Examples of satisfactory anionic sulfate surfactants are the alkali metal or ammonium salt C8-C18 alkyl sulfate salts the ethoxylated C8-C18 alkyl ether sulfate salts having the formula R(OC2H4)n OSO3M wherein n is 1 to 12, preferably 1 to 5, and M is a metal cation selected from the group consisting of sodium, potassium, ammonium, magnesium and mono-, di- and triethanol ammonium ions. The alkyl sulfates may be obtained by sulfating the alcohols obtained by reducing glycerides of coconut oil or tallow or mixtures thereof and neutralizing the resultant product.
On the other hand, the ethoxylated alkyl ether sulfates are obtained by sulfating the condensation product of ethylene oxide with a C8-C18 alkanol and neutralizing the resultant product. The alkyl sulfates may be obtained by sulfating the alcohols obtained by reducing glycerides of coconut oil or tallow or mixtures thereof and neutralizing the resultant product. The ethoxylated alkyl ether sulfates differ from one another in the number of moles of ethylene oxide reacted with one mole of alkanol. Preferred alkyl sulfates and preferred ethoxylated alkyl ether sulfates contain 10 to 16 carbon atoms in the alkyl group.
The ethoxylated C8-C12 alkylphenyl ether sulfates containing from 2 to 6 moles of ethylene oxide in the molecule also are suitable for use in the inventive compositions. These surfactants can be prepared by reacting an alkyl phenol with 2 to 6 moles of ethylene oxide and sulfating and neutralizing the resultant ethoxylated alkylphenol.
Other suitable anionic surfactants are the C9-C15 alkyl ether polyethenoxyl carboxylates having the structural formula R(OC2H4)nOX COOH wherein n is a number from 4 to 12, preferably 5 to 10 and X is selected from the group consisting of
CH2, (C(O)R1
and
xe2x80x83wherein R1 is a C1-C3 alkylene group. Preferred compounds include C9-C11 alkyl ether polyethenoxy (7-9) C(O) CH2CH2COOH, C13-C15 alkyl ether polyethenoxy (7-9)
xe2x80x83and C10-C12 alkyl ether polyethenoxy (5-7) CH2COOH. These compounds may be prepared by condensing ethylene oxide with appropriate alkanol and reacting this reaction product with chloracetic acid to make the ether carboxylic acids as shown in U.S. Pat. No. 3,741,911 or with succinic anhydride or phthalic anhydride. Obviously, these anionic surfactants will be present either in acid form or salt form depending upon the pH of the final composition, with salt forming cation being the same as for the other anionic surfactants.
The instant composition can optionally include 0.1 wt. % to 15 wt. % of a water-soluble zwitterionic surfactant, which can also be used provides good foaming properties and mildness to the present nonionic based liquid detergent. The zwitterionic surfactant is a water soluble betaine having the general formula:
wherein R1 is an alkyl group having 10 to 20 carbon atoms, preferably 12 to 16 carbon atoms, or the amido radical:
wherein R is an alkyl group having 9 to 19 carbon atoms and a is the integer 1 to 4; R2 and R3 are each alkyl groups having 1 to 3 carbons and preferably 1 carbon; R4 is an alkylene or hydroxyalkylene group having from 1 to 4 carbon atoms and, optionally, one hydroxyl group. Typical alkyldimethyl betaines include decyl dimethyl betaine or 2-(N-decyl-N, N-dimethyl-ammonia)acetate, coco dimethyl betaine or 2-(N-coco N, N-dimethylammonio)acetate, myristyl dimethyl betaine, palmityl dimethyl betaine, lauryl diemethyl betaine, cetyl dimethyl betaine, stearyl dimethyl betaine, etc. The amidobetaines similarly include cocoamidoethylbetaine, cocoamidopropyl betaine and the like. A preferred betaine is coco (C8-C18) amidopropyl dimethyl betaine.
The instant composition can include an amine oxide semi-polar nonionic surfactants comprise compounds and mixtures of compounds having the formula:
wherein R1 is an alkyl, 2-hydroxyalkyl, 3-hydroxyalkyl, or 3-alkoxy-2-hydroxypropyl radical in which the alkyl and alkoxy, respectively, contain from 8 to 18 carbon atoms, R2 and R3 are each methyl, ethyl, propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, or 3-hydroxypropyl, and n is from 0 to 10. Particularly preferred are amine oxides of the formula:
wherein R1 is a C12-16 alkyl and R2 and R3 are methyl or ethyl. The above ethylene oxide condensates, amides, and amine oxides are more fully described in U.S. Pat. No. 4,316,824 which is hereby incorporated herein by reference.
The instant composition can include a C12-14 alkyl monoalkanol amide such as lauryl monoalkanol amide or a C12-14 alkyl dialkanol amide such as lauryl diethanol amide or coco diethanol amide.
The water soluble nonionic surfactants utilized in this invention are commercially well known and include the primary aliphatic alcohol ethoxylates, secondary aliphatic alcohol ethoxylates, alkylphenol ethoxylates and ethylene-oxide-propylene oxide condensates on primary alkanols, such a Plurafacs (BASF) and condensates of ethylene oxide with sorbitan fatty acid esters such as the Tweens (ICI). The nonionic synthetic organic detergents generally are the condensation products of an organic aliphatic or alkyl aromatic hydrophobic compound and hydrophilic ethylene oxide groups. Practically any hydrophobic compound having a carboxy, hydroxy, amido, or amino group with a free hydrogen attached to the nitrogen can be condensed with ethylene oxide or with the polyhydration product thereof, polyethylene glycol, to form a water-soluble nonionic detergent. Further, the length of the polyethenoxy chain can be adjusted to achieve the desired balance between the hydrophobic and hydrophilic elements.
The nonionic detergent class includes the condensation products of a higher alcohol (e.g., an alkanol containing about 8 to 18 carbon atoms in a straight or branched chain configuration) condensed with about 5 to 30 moles of ethylene oxide, for example, lauryl or myristyl alcohol condensed with about 16 moles of ethylene oxide (EO), tridecanol condensed with about 6 to moles of EO, myristyl alcohol condensed with about 10 moles of EO per mole of myristyl alcohol, the condensation product of EO with a cut of coconut fatty alcohol containing a mixture of fatty alcohols with alkyl chains varying from 10 to about 14 carbon atoms in length and wherein the condensate contains either about 6 moles of EO per mole of total alcohol or about 9 moles of EO per mole of alcohol and tallow alcohol ethoxylates containing 6 EO to 11 EO per mole of alcohol.
A preferred group of the foregoing nonionic surfactants are the Neodol ethoxylates (Shell Co.), which are higher aliphatic, primary alcohol containing about 9-15 carbon atoms, such as C11 alkanol condensed with 9 moles of ethylene oxide (Neodol 1-9), C12-13 alkanol condensed with 6.5 moles ethylene oxide (Neodol 23-6.5), C12-15 alkanol condensed with 7 or 3 moles ethylene oxide (Neodol 25-7 or Neodol 25-3), C14-15 alkanol condensed with 13 moles ethylene oxide (Neodol 45-13), and the like. Such ethoxamers have an HLB (hydrophobic lipophilic balance) value of about 8 to 15 and give good O/W emulsification, whereas ethoxamers with HLB values below 8 contain less than 5 ethyleneoxide groups and tend to be poor emulsifiers and poor detergents.
Additional satisfactory water soluble alcohol ethylene oxide condensates are the condensation products of a secondary aliphatic alcohol containing 8 to 18 carbon atoms in a straight or branched chain configuration condensed with 5 to 30 moles of ethylene oxide. Examples of commercially available nonionic detergents of the foregoing type are C11-C15 secondary alkanol condensed with either 9 EO (Tergitol 15-S-9) or 12 EO (Tergitol 15-S-12) marketed by Union Carbide.
Other suitable nonionic detergents include the polyethylene oxide condensates of one mole of alkyl phenol containing from about 8 to 18 carbon atoms in a straight- or branched chain alkyl group with about 5 to 30 moles of ethylene oxide. Specific examples of alkyl phenol ethoxylates include nonyl condensed with about 9.5 moles of EO per mole of nonyl phenol, dinonyl phenol condensed with about 12 moles of EO per mole of phenol, dinonyl phenol condensed with about 15 moles of EO per mole of phenol and di-isoctylphenol condensed with about 15 moles of EO per mole of phenol. Commercially available nonionic surfactants of this type include Igepal CO-630 (nonyl phenol ethoxylate) marketed by GAF Corporation.
Also among the satisfactory nonionic detergents are the water-soluble condensation products of a C8-C20 alkanol with a heteric mixture of ethylene oxide and propylene oxide wherein the weight ratio of ethylene oxide to propylene oxide is from 2.5:1 to 4:1, preferably 2.8:1 to 3.3:1, with the total of the ethylene oxide and propylene oxide (including the terminal ethanol or propanol group) being from 60-85%, preferably 70-80%, by weight. Such detergents are commercially available from BASF-Wyandotte and a particularly preferred detergent is a C10-C16 alkanol condensate with ethylene oxide and propylene oxide, the weight ratio of ethylene oxide to propylene oxide being 3:1 and the total alkoxy content being about 75% by weight.
Condensates of 2 to 30 moles of ethylene oxide with sorbitan mono- and tri-C10-C20 alkanoic acid esters having a HLB of 8 to 15 also may be employed as the nonionic detergent ingredient in the described composition. These surfactants are well known and are available from Imperial Chemical Industries under the Tween trade name. Suitable surfactants include polyoxyethylene (4) sorbitan monolaurate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylene (20) sorbitan trioleate and polyoxyethylene (20) sorbitan tristearate.
Other suitable water-soluble nonionic detergents which are less preferred are marketed under the trade name xe2x80x9cPluronics.xe2x80x9d The compounds are formed by condensing ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The molecular weight of the hydrophobic portion of the molecule is of the order of 950 to 4000 and preferably 200 to 2,500. The addition of polyoxyethylene radicals to the hydrophobic portion tends to increase the solubility of the molecule as a whole so as to make the surfactant water-soluble. The molecular weight of the block polymers varies from 1,000 to 15,000 and the polyethylene oxide content may comprise 20% to 80% by weight. Preferably, these surfactants will be in liquid form and satisfactory surfactants are available as grades L 62 and L 64.
The alkyl polysaccharides surfactants, which can be used at a hydrophobic group containing from about 8 to about 20 carbon atoms, preferably from about 10 to about 16 carbon atoms, most preferably from about 12 to about 14 carbon atoms, and polysaccharide hydrophilic group containing from about 1.5 to about 10, preferably from about 1.5 to about 4, most preferably from about 1.6 to about 2.7 saccharide units (e.g., galactoside, glucoside, fructoside, glucosyl, fructosyl; and/or galactosyl units). Mixtures of saccharide moieties may be used in the alkyl polysaccharide surfactants. The number x indicates the number of saccharide units in a particular alkyl polysaccharide surfactant. For a particular alkyl polysaccharide molecule x can only assume integral values. In any physical sample of alkyl polysaccharide surfactants there will be in general molecules having different x values. The physical sample can be characterized by the average value of x and this average value can assume non-integral values. In this specification the values of x are to be understood to be average values. The hydrophobic group (R) can be attached at the 2-, 3-, or 4-positions rather than at the 1-position, (thus giving e.g. a glucosyl or galactosyl as opposed to a glucoside or galactoside). However, attachment through the 1-position, i.e., glucosides, galactoside, fructosides, etc., is preferred. In the preferred product the additional saccharide units are predominately attached to the previous saccharide unit""s 2-position. Attachment through the 3-, 4-, and 6-positions can also occur. Optionally and less desirably there can be a polyalkoxide chain joining the hydrophobic moiety (R) and the polysaccharide chain. The preferred alkoxide moiety is ethoxide.
Typical hydrophobic groups include alkyl groups, either saturated or unsaturated, branched or unbranched containing from about 8 to about 20, preferably from about 10 to about 18 carbon atoms. Preferably, the alkyl group is a straight chain saturated alkyl group. The alkyl group can contain up to 3 hydroxy groups and/or the polyalkoxide chain can contain up to about 30, preferably less than about 10, alkoxide moieties.
Suitable alkyl polysaccharides are decyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, and octadecyl, di-, tri-, tetra-, penta-, and hexaglucosides, galactosides, lactosides, fructosides, fructosyls, lactosyls, glucosyls and/or galactosyls and mixtures thereof.
The alkyl monosaccharides are relatively less soluble in water than the higher alkyl polysaccharides. When used in admixture with alkyl polysaccharides, the alkyl monosaccharides are solubilized to some extent. The use of alkyl monosaccharides in admixture with alkyl polysaccharides is a preferred mode of carrying out the invention. Suitable mixtures include coconut alkyl, di-, tri-, tetra-, and pentaglucosides and tallow alkyl tetra-, penta-, and hexaglucosides.
The preferred alkyl polysaccharides are alkyl polyglucosides having the formula
R2O(CnH2nO)r(Z)x
wherein Z is derived from glucose, R is a hydrophobic group selected from the group consisting of alkyl, alkylphenyl, hydroxyalkylphenyl, and mixtures thereof in which said alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14 carbon atoms; n is 2 or 3 preferably 2, r is from 0 to 10, preferable 0; and x is from 1.5 to 8, preferably from 1.5 to 4, most preferably from 1.6 to 2.7. To prepare these compounds a long chain alcohol (R2OH) can be reacted with glucose, in the presence of an acid catalyst to form the desired glucoside. Alternatively the alkyl polyglucosides can be prepared by a two step procedure in which a short chain alcohol (R1OH) can be reacted with glucose, in the presence of an acid catalyst to form the desired glucoside. Alternatively the alkyl polyglucosides can be prepared by a two step procedure in which a short chain alcohol (C1-6) is reacted with glucose or a polyglucoside (x=2 to 4) to yield a short chain alkyl glucoside (x=1 to 4) which can in turn be reacted with a longer chain alcohol (R2OH) to displace the short chain alcohol and obtain the desired alkyl polyglucoside. If this two step procedure is used, the short chain alkylglucosde content of the final alkyl polyglucoside material should be less than 50%, preferably less than 10%, more preferably less than about 5%, most preferably 0% of the alkyl polyglucoside.
The amount of unreacted alcohol (the free fatty alcohol content) in the desired alkyl polysaccharide surfactant is preferably less than about 2%, more preferably less than about 0.5% by weight of the total of the alkyl polysaccharide. For some uses it is desirable to have the alkyl monosaccharide content less than about 10%.
The used herein, xe2x80x9calkyl polysaccharide surfactantxe2x80x9d is intended to represent both the preferred glucose and galactose derived surfactants and the less preferred alkyl polysaccharide surfactants. Throughout this specification, xe2x80x9calkyl polyglucosidexe2x80x9d is used to include alkyl polyglycosides because the stereochemistry of the saccharide moiety is changed during the preparation reaction.
An especially preferred APG glycoside surfactant is APG 625 glycoside manufactured by the Henkel Corporation of Ambler, Pa. APG25 is a nonionic alkyl polyglycoside characterized by the formula:
CnH2n+1O(C6H10O5)xH
wherein n=10 (2%); n=122 (65%); n=14 (21-28%); n=16 (4-8%) and n=18 (0.5%) and x (degree of polymerization)=1.6. APG 625 has: a pH of 6 to 10 (10% of APG 625 in distilled water); a specific gravity at 25xc2x0 C. of 1.1 g/ml; a density at 25xc2x0 C. of 9.1 lbs/gallon; a calculated HLB of 12.1 and a Brookfield viscosity at 35C, 21 spindle, 5-10 RPM of 3,000 to 7,000 cps.
The instant composition can optionally contain 0.1 wt. % to 10 wt. % of a polyethylene glycol is depicted by the formula:
wherein n is about 8 to about 225, more preferably about 10 to about 100,000, wherein the polyethylene glycol has a molecular weight of about 200 to about 1,000. One preferred polyethylene glycerol is PEG1000 which is a polyethylene glycol having a molecular weight of about 1000.
The water is present in the composition at a concentration of about 5 wt. % to 70 wt. %.
The product of the present invention comprises a water insoluable substrate with one or more layers. Each layer may have different textures and abrasiveness. Differing textures can result from the use of different combinations of materials or from the use of different manufacturing processes or a combination thereof. A dual texture substrate can be made to provide the advantage of a more abrasive side for cleaning difficult to remove soils. A softer side can be used for fine dishware and flatware. The substrate should not dissolve or break apart in water. It is the vehicle for delivering the cleaning composition to dishware, flatware, pots and pans. Use of the substrate enhances lathering, cleaning and grease removal.
A wide variety of materials can be used as the substrate. It should have sufficient wet strength, abrasivity, loft and porosity. Examples include, non woven substrates, wovens substrates, hydroentangled substrates and sponges.
Examples of suitable non woven water insoluable substrates include, 100% cellulose Wadding Grade 1804 from Little Rapids Corporation, 100% polypropylene needlepunch material NB 701-2.8-W/R from American Non-wovens Corporation, a blend of cellulosic and synthetic fibres-Hydraspun 8579 from Ahlstrom Fibre Composites, and and0% Viscose/30% PES Code 9881 from PGI Nonwovens Polymer Corp.
Another useful substrate is manufactured by Jacob Holm-Lidro Rough. It is a composition material comprising a 65/35 viscose rayon/polyester hydroentangled spunlace layer with a hydroenlongated bonded polyeser scribbly layer.
Still another useful substrate is manufactured by Texel xe2x80x9cTIxe2x80x9d. It is a composite material manufactured from a layer of coarse fiber 100% polypropylene needlepunch, an absorbent cellulose core and a fine fiber polyester layer needlepunched together. The polypropylene layer can range from 1.5 to 3.5 oz/sq. yd. The cellulose core is a creped paper layer ranging from 0.5 to 2 oz./sq. yd. The fine fiber polyester layer can range from 0.5 to 2 oz./sq. yd.
Still another composite material manufactured by Texcel from a layer of coarse fiber 100% polypropylene needlepunch layer, an absorbent cellulose core and a fine fiber polyester layer needlepunched together. The polypropylene layer can range from 1.5 to 3.5 oz/sq. yd. The cellulose core is a creped paper layer ranging from 0.5 to 2 oz/sq. yd. The fine fiber polyester layer can range from 0.5 to 2 oz/sq. yd. The polypropylene layer is flame treated to further increase the level of abrasivity. The temperature of the flame and the length of time the material is exposed can be varied to create different levels of surface roughness.
The abrasiveness is tested by cutting one quarter inch thick Lucite boards to fit an abrader bed. The boards are marked to indicate the track of the wipes during abrasion (approximately 2xc2xc and 4xc2xd inches from one long edge) and three spots along the track (6, 9 and 12 inches from the short end). This gives unique and reproducible locations at which to do gloss measurements which are in the center of the abrasion track.
Using a BYK-Gardener Haze-Gloss glossmeter, the indicated spots are measured for starting gloss. An average and standard deviation is reported for each track using the three measurements.
Pieces of the wipes are cut approximately 3 inches by 2 inches. This piece is wrapped around a piece of sponge that fits in a holder for the abrader. (Indication should be made of whether the material is being used in the machine or cross direction). This wrapped sponge is placed in the holder, tucking all the edges of the wipe into the holder so that it is kept firmly in place. The dry sponge is wetted with approximately 20-25 g of water (either deionized, distilled or tap as the experiment desires). This is done so that there are two wipes in the trial.
The gloss measured Lucite board is placed in the abrader bed. Set the abrader for 500 cycles and start.
After the abrader cycles have ended, the Lucite board is removed. It is wiped dry with paper towel to remove any residual water. It is also inspected for any fingerprints incurred during handling and these are also wiped clean. Remeasure the gloss at the specified spots again and again report the average of these three spots and the standard deviation for each track.
The higher the abrasiveness of the wipe, the more that it roughens the surface of the Lucite and the more the gloss is reduced. The most abrasive of the wipes therefore give the greatest decrease in gloss. Results for this test are given below.
Ahlstrom Manufacturers:
A hydroentangled nonwoven created from a blend of cellulosic and polyester and/or polypropylene fibers with an abrasive side. The basis weight can range from 1.2 to 6 ounces per square yard.
A composite dual textured material manufactured by Kimberly Clark comprises a coarse meltblown polypropylene, polyethylene, or polyester and high loft spunbond polyester. The two materials can be laminated together using chemical adhesives or by coprocessing the two layers. The coarse meltblown layer can range from 1 to 3 ounces per square yard while the highloft spunbond layer can range from 1 to 3 ounces per square yard.
Another example of a composite is a dual textured material composed of coarse meltblown polypropylene, polyethylene, or polyester and polyester/cellulose coform. The two materials can be laminated together using chemical adhesives or by coprocessing the two layers. The coarse meltblown layer can range from 1 to 3 ounces per square yard. The coform layer can range in composition from 30% cellulose and 70% polyester to 70% cellulose and 30% polyester and the basis weight can range from 1.5 to 4.5 ounces per square yard.
The product of the present invention comprising mutliple layers may be ultrasonically bonded after applying the coating of one or more of the layers. Alternatively layers may be bonded together by needlepunch, thermal bonding, chemical bonding, or sonic bonding prior to applying the coating and/or impregnation.


The present invention relates to nucleic acid molecule compositions comprising MiniVectors™ encoding a nucleic acid sequence and methods of gene therapy using MiniVectors encoding a nucleic acid sequence.

Gene therapy involves the delivery of DNA or RNA to diseased organ or cells to correct defective genes implicated in disease. This may be achieved through a number of different approaches. If the condition is due to an absent or non-functional gene product, a functional copy of the gene may be delivered to the disease loci. Alternatively, gene expression may be controlled using RNA interference technologies such as small interfering RNA (siRNA), short hairpin RNA (snRNA) and microRNA (miRNA). RNA interference (RNAi), a natural cell process by which specific mRNAs are targeted for degradation by complementary small interfering RNAs (siRNAs), enables the specific silencing of a single gene at the cell level. A variety of biomedical1 and clinical research2-4 have showed that RNAi has a great potential as an efficient therapeutic approach. Typically, RNA interference is used to down-regulate expression of a pathogenic gene, however, up-regulation of genes is possible by targeting regulatory regions in gene promoters15.
Despite the tremendous therapeutic potential of gene therapy and the large number of disorders identified as good candidates, the field has so far been unsuccessful. These failures are largely due to complications associated with gene delivery. Delivery efficiency is high for viral vectors, the most common delivery method; however, these have limited therapeutic potential because of problems observed in clinical trials including toxicity, immune and inflammatory responses, difficulties in targeting and controlling dose. In addition, there is justifiable concern that the vectors will integrate into the genome, with unknown long-term effects, and the possibility that the virus may recover its ability to cause disease.
Much effort has therefore been directed towards non-viral vector systems, such as plasmid DNA. These vectors are attractive because they are simple to produce and store, and can stably persist in cells; however, they contain bacterial DNA sequences that may trigger immunotoxic responses. Some human cells, including dendritic cells and T-cells, cannot be efficiently transfected with current plasmid vectors. Short, linear DNA vectors and small RNAs such as short interfering RNA (siRNA) and micro-RNA (miRNA) are more easily introduced into cells than plasmids. Linear DNA and RNA ends, however, trigger rapid degradation by cells, therefore requiring continuous replenishment. Furthermore, the DNA ends can signal repair and recombination pathways to cause apoptosis. For these reasons, RNAi- and miRNA-based technologies have not yet been highly successful in the clinic There is clearly a desperate need for a new and efficient way to therapeutically deliver gene therapy vectors to cells.
Plasmid DNA vectors have some utility in basic research because they are straightforward to generate and isolate. They are propagated in bacterial strains and recovered from the bacterial cells. As mentioned above, however, this requires them to contain bacterial DNA sequences, notably a prokaryotic origin of replication and an antibiotic resistance marker for maintenance of the plasmid. The presence of these bacterial sequences has a number of very serious and deleterious consequences. Most notably, it limits how small the plasmids can be made. Large plasmids, of several kbp, are transfected at very low efficiency. Their large size also makes them to susceptible to hydrodynamic shearing forces associated with delivery (e.g., through aersolisation) or in the bloodstream. Shear-induced degradation leads to a loss of biological activity that is at least partially responsible for the current lack of success in using non-viral vectors for gene therapy. Various cationic and liposomal transfection reagents have been designed to try and alleviate these problems with transfection, but these suffer from problems with cytotoxicity. In addition, the bacterial sequences on plasmids can induce silencing of the gene carried on the plasmid14 leading to loss of efficacy even if the plasmid is successfully transfected. The CpG motifs that are more common in bacterial than eukaryotic DNA sequences also elicit immune responses in mammalian cells. Reducing the size of DNA vectors appears to be a reasonable approach to increase cell transfection efficiency. One may envision that the bacterial sequences on the plasmid could be physically removed and resultant short linear DNA fragments that contain only the therapeutic sequences more easily introduced into cells than conventional plasmid vectors. Unfortunately, the ends of linear DNA are highly bioreactive in vivo, triggering cellular DNA repair and recombination processes as well as apoptosis. Thus, there is a need for gene targeting therapies that are stable in biological environments and that allow for greater cell transfection and transgene expression.

Supercoiled minivectors for gene therapy applications


Sandwich assays for collagen degradation products are conducted using an antibody of the same specificity on both sides of the sandwich or using a first antibody reactive with an epitope contained in the sequence EKAHDGGR and a second antibody which may be the same or different.

The present invention relates to assays for collagen fragments in body fluids.
Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased susceptibility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15-20 million individuals in the USA alone. Its basis is an age-dependant imbalance in bone remodelling, i.e. in the rates of formation and resorption of bone tissue.
In the USA about 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $10 billion annually in the USA (Riggs, New England Journal of Medicine, 327:620-627 (1992)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited. Other disorders entailing (and correlated with) abnormalities in collagen metabolism include Paget""s disease, Marfan""s syndrome, osteogenesis imperfecta, neoplastic growth in collagenous tissue, dwarfism, rheumatoid arthritis, osteo-arthritis and vasculitis syndrome.
Three known classes of human collagen have been described to date. The Class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils. The amino-acid sequence of type I-III (to the extent it has been elucidated) is given in Appendix A of WO 95/08115.
Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteo-arthritis and collagen type III degradation in vasculitis syndrome.
Amino acid sequences of human type III collagen, human pro a1 (II) collagen, and the entire prepro a1 (III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers; see Loil et al., Nucleic Acid Research 12:9383-9394 (1984): Sangiorgi et al., Nucleic Acids Research, 13:2207-2225 (1985); Baldwin et al., Biochem J., 262:521-528 (1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989).
Type I, II, and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-terminal propeptide sequences, which are attached to the core collagen molecules. After removal of the pro-peptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecules consists largely of a triple-helical domain having terminal telopeptide sequences which are non-triple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extra-celluarly. The alpha-helical region also includes cross-linkable sites.
Intermolecular cross-links provide collagen fibrils with biomechanical stability. The formation of these cross-links is initiated by modification of lysine and hydroxylysine residues to the corresponding aldehydes. Several of these residues located on adjacent chains of collagen will spontaneously form different intermolecular cross-links. The exact position of the sites for cross-linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kxc3xchn, K., in Immunochemistry of the extracellular matrix, 1:1-29, CRC Press, Inc., Boca Raton, Fla. (1982), Eyre, D. R., Ann.Rev. Biochem., 53:717-48 (1984) or U.S. Pat. Nos. 5,140,103 and 5,455,179. Furthermore, the amino acid sequences of some potential sites for cross-linking in type I, II, and III collagen are given in Table 1 below.
The fibrous proteins, collagen and elastin, are cross-linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysine side chains. Four homologous loci of cross-linking are evident in molecules of type I, II and III collagens (for review see Kxc3xchn, K., in Immunochemistry of the extracellular matrix, 1:1-29 (1982)). Two are aldehyde sites, one in each telopeptide region. The other two sites are hydroxylysine symmetrically placed at about 90 residues from each end of the molecule. When collagen molecules pack into fibrils, these latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules. There is now strong evidence that 3-hydroxypyridinium residues are the mature cross-link coming from hydroxylysine-derived aldehydes. The mature cross-linking residues of the other pathway, i.e. from aldehyde formation of lysine residues, are however, still unknown.
As illustrated by formula in EP-0394296 discussed below, the two 3-hydroxypyridinium cross-links have been found to be hydroxylysyl pyridinoline (also known simply as xe2x80x9cpyridinolinexe2x80x9d) and lysyl pyridinoline (also known as xe2x80x9cdeoxypyridinolinexe2x80x9d). These cross-linking compounds are naturally fluorescent. Some hydroxylysyl pyridinoline cross-link are found to be glycosylated as discussed for instance in EP-A-0424428.
However, as described in Last et al, Int. J. Biochem. Vol. 22, No. 6, pp 559-564, 1990 other crosslinks occur naturally in collagen.
In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen.
For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget""s disease, a metabolic bone disorder in which bone turnover is greatly increased, as discussed further below.
For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation; Singer, F. R. et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.), 489-575 (1978), Academic Press, New York.
U.S. Pat. No. 3,600,132 discloses a process for the determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. The Patent states that hydroxyproline correlates with increased collagen anabolism or catabolism associated with patho-logical conditions such as Paget""s disease, Marfan""s syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism.
Bone resorption associated with Paget""s disease has also been monitored by measuring small peptides containing hydroxyproline, which are excreted in the urine following degradation of bone collagen; Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer, F. R., et al., supra.
In the case of Paget""s disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index for bone degradation. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline.
Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolised in the liver and never appears in the urine. Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good marker for osteoporosis as it is specific for collagen in bones even if it is not specific for bone resorption, but it is trouble-some to handle.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally non-specific markers of bone resorption; Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).
Other researchers have measured the cross-linking compound 3-hydroxypyridinium in urine as an index of collagen degradation in joint diseases. See, for back-ground and as examples, Wu and Eyre, Biochemistry, 23:1850 (1984): Black et al., Annals of the Rheumatic Diseases, 45:969-973 (1986); and Seibel et al., The Journal of Dermatology, 16:964 (1989). In contrast to the present invention, these prior researchers have hydrolysed peptides from body fluids and then looked for the presence of free 3-hydroxypyridinium residues.
Assays for determination of the degradation of type I, II, and III collagen are disclosed in EP-0394296 and U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103. However, these Patents are restricted to collagen fragments containing the cross-linker 3-hydroxypyridinium. Furthermore, the above mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.
Until recently very few clinical data using the approach described in U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above mentioned patents) of 3-hydroxypyridinium containing telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densitometry) had been published. Very recently however McClung et al (JBMR(1996)11:129) have concluded that results from the commercial NTx assay based on these Patents do not correlate to bone loss. More particularly, NTx did not correlate to bone loss in the normal population and also failed to predict bone changes in response to therapy. Gertz et al (JBMR (1994) 9(2): 135-142) have reported no significant correlation between baseline NTx measurements and bone loss and no significant correlation between change in NTx and change in bone loss during anti-resorptive therapy. Garnero et al (JBMR (1996) 11(10): 1531-1537) have reported that NTx was found not be predictive of hip fracture whilst other biochemical markers were associated with an approximately 100 percent increased risk of hip fracture.
The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and it would be desirable to avoid this dependence of the correct formation of the cross-linking structure.
GB Patent Application No. 2205643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
Schrxc3x6ter-Kermani et al., Immunol. Invest. 19:475-491 (1990) describe immunological measurement systems based on CNBr fragments of collagen type I and II. Use is made of pepsin-solubilised collagen, leaving the telopeptides in the tissue (cf. the above mentioned GB Patent Application No. 2205643). There is therefore no conformity between the fragments and the antibodies raised therefrom. Further, the reference only describes measurements on extracted tissue samples.
The development of a monoclonal antibody raised against pepsin-solubilised type I collagen is described in Werkmeister et al., Eur. J. Biochem. 1987:439-443 (1990). The antibody is used for immunohistochemical staining of tissue segments and for measuring the collagen content in cell cultures. The measurements are not carried out on body fluids.
EP Patent Application No. 0505210 describes the development of antibody reagents by immunisation with purified cross-linked C-terminal telopeptides from type I collagen. The immunogen is prepared by solubilising human bone collagen with bacterial collagenase. The antibodies thus prepared are able to react with both cross-linked and non-cross-linked telopeptides, and cross-linkers other than pyridinoline.
There are a number of reports indicating that collagen degradation can be measured by quantitating certain procollagen peptides. Propeptides are distinguished from telopeptides and alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see U.S. Pat. No. 4,504,587; U.S. Pat. No. 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al., European Journal of Clinical Investigation, 9:451-459 (1979). EP Patent Application No. 0298210 and No. 0339443 both describe immunological determination of procollagen peptide type III and fragments thereof. Further, a method based on the measurement of procollagen is disclosed in EP Patent Application No. 0465104.
The use of synthetic peptides with sequences derived from type IX collagen for the development of immunological reagents is disclosed in PCT Patent Application No. WO 90/08195. Likewise the application describes the use of the antibodies thus produced for the determination of type IX collagen fragments in body fluids.
U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof in a synovial fluid sample.
Dodge, J. Clin Invest 83:647-661 (1981) discloses methods for analysing type II collagen degradation utilising a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. The degradation products of collagen were not detected in a body fluid, but histochemically by staining of cell cultures, i.e. by xe2x80x9cin situxe2x80x9d detection.
WO 94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments in a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-terminal or N-terminal domain of collagen is incubated with an antibody to the linear synthetic peptide and the sample, and wherein the binding of the antibody to the binding partner is determined.
WO 95/08115 relates to assay methods in which collagen fragments in a body fluid are determined by reaction with an antibody which is reactive with a synthetic peptide. The assay may be a competition assay in which the sample and such a peptide compete for an antibody, possibly a polyclonal antibody raised against fragments of collagen obtained by collagenase degradation of collagen. Alternatively, it may be an assay in which an antibody, possibly a monoclonal anti-body, is used which has been raised against such a synthetic peptide.
As disclosed in WO 91/08478, one particular type of peptide fragment found in body fluid, particularly urine, is of the formula:
In the above formula, K-K-K is disclosed as representing a hydroxypyridinium cross-link but in fact it may be any naturally occurring cross-link and specifically any of those discussed in the above referenced paper of Last et al.
As further discussed below, larger peptide fragments including the above smaller fragment are also disclosed in this document.
A proportion of the xe2x80x9cpeptidexe2x80x9d fragments in body fluid are related to peptides of equivalent amino acid sequence, e.g. peptides of formula 1, by the isomerisation of aspartic acid in the formula to isoaspartic acid. We put xe2x80x9cpeptidesxe2x80x9d in quotes here as of course the isomerisation means that these species are no longer properly regarded as being peptides.
The isomerisation of proteins containing aspartic acid has been reported previously to be a spontaneous reaction occurring under physiological conditions.
See for instance Brennan et al Protein Science 1993, 2, 331-338, Galletti et al, Biochem. J. 1995, 306, 313-325, Lowenson et al, Blood Cells 1988, 14, 103-117 and Oliya et al, Pharmaceutical Research, Vol. 11, No. 5, 1994, p.751.
The isomerisation has the effect of transferring that part of the peptide chain which runs downstream of the aspartic acid residue in the carboxy terminus direction from the alpha carboxylic acid of the aspartic acid to which it is bonded via a peptide bond in the normal protein to the side chain carboxylic acid in a non-peptide amide bond, as shown below:
The non-peptide bonded aspartic acid residue is termed xe2x80x9cisoaspartic acidxe2x80x9d or xcex2-aspartic acid (xcex2D).
Similar isomerisation can occur in proteins containing asparagine residues (i.e. with xe2x80x94NH2 instead of xe2x80x94OH in the starting protein in the above reaction scheme).
The above discovery indicates that this isomerisation also occurs in bone tissue and the extent of isomerisation is expected therefore to be marker for the age of the bone tissue concerned.
Furthermore, the presence amongst such bone peptide fragments of the isomerised peptides provides confirmation that the fragments indeed derive from bone degradation and not some other source such as the degradation of newly formed collagen never incorporated into bone.
J. Macek and M. Adam xe2x80x9cDetermination of collagen degradation products in human urinexe2x80x9d, Z. Rheumatol. 46:237-240 (1987) reports the presence of pyridinoline containing collagen cross-linked peptides in urine having a molecular weight above 10,000 but provides no sequence information relating to the peptide chains present or the collagen type to which the fragments belong.
As mentioned above WO 91/08478 discloses that a number of fragments of type 1 collagen can be found in urine. These include a pyridinium crosslink, which may be hydroxylysyl pyridinoline or lysyl pyridinoline. Attached to the crosslink are peptide chains of defined sequence derived from the collagen molecule. The crosslink has three points at which it may bear peptide chains. The fragment of Formula 1 above (Formula VI in WO 91/08478) bears two chains, each having the sequence EKAHDGGR (SEQ ID No.1). Two other fragments are described in WO 91/08748 which each have a third chain, that shown in Formula IV of that specification being 7 amino acids longer than that in Formula V, but otherwise of the same sequence. The amino-acid sequence of the chains of type 1 collagen has been published elsewhere as described above, as has the location of the trivalent crosslinks between the collagen molecules. The third chain depicted in the said Formulae IC and V of WO 91/08748 does not correspond to that of any collagen chain at the location of the crosslink and is believed to be an error, possibly caused by an artefact of the isolation procedure used.
The only fragment for which a credible formula has been given is there for that of Formula VI (equivalent to Formula 1 herein) having two identical peptide chains.
DE-A-4225038 discloses sandwich assays for collagen breakdown products in body fluids. Antibodies are to be produced by immunisation with haptens containing a linear sequence of amino acids. One proposed sequence is FDFSFLP (SEQ ID No.2) and another is PPQEKAHDGGR (SEQ ID No.3), although these were not suggested for use in combination to make two antibodies for use in the same sandwich assay. Indeed although the sequence PPQEKAHDGGR (SEQ ID No.3) is given, no antibodies made against it are specifically described and therefore no disclosure is provided of their actual utility and properties. The only specific sandwich assay described combines an antibody against the C-terminal sequence FDFSFLP (SEQ ID No.2) with one against the sequence GMKGHRGF (SEQ ID No.4) (from the helical region crosslink site).
However, DE-A-4225038 asserts that there is a close correlation between results obtained using an assay based on the sequence FDFSFLP (SEQ ID No.2) and a commercial assay known as the ICTP assay. It has been shown however that the ICTP assay in serum does not appear to reflect bone resorption in that the results it produces do not successfully track the effect of therapeutic treatment (Hassager et al. Calcif. Tissue. Int. (1994) 54:40-33). This of course would imply that the population of molecules containing the sequence FDFSFLP (SEQ ID No.2) in serum would not reflect bone resorption in a useful way.
We have now established that body fluids do in fact contain larger collagen fragments containing not only the sequence EKAHDGGR (SEQ ID No. 1) but also further amino acid residues. These may be present in a third chain attached to the crosslink with two chains incorporating the sequence EKAHDGGR (SEQ ID No. 1) and/or as extensions of N-amino terminal direction of the sequence EKAHDGGR (SEQ ID No. 1) of one or both of the two chains containing that sequence.
We have further established that surprisingly it is possible to obtain binding of two distinct antibodies to a single collagen degradation fragment where both antibodies are specific for an epitope in the sequence EKAHDGGR (SEQ ID No.1) or a variant of it.
Accordingly, the present invention provides a method of measurement of the rate of type I collagen resorption comprising measuring in a sample (e.g. a body fluid) the amount of a population of collagen fragments by a sandwich assay using a first antibody reactive with a first epitope located in the collagen amino acid sequence EKAHDGGR (SEQ ID No.1) or in isomerised and/or racemised variants thereof and a second antibody reactive with a second collagen epitope located in said fragments.
Preferably, the body fluid on which the assay is conducted is other than urine.
Preferably said epitope is treated in the sequence AHDGGR or a said variant of it (SEQ ID No.7).
Optionally, said second epitope is located in the N-amino terminal direction with respect to said first epitope in the same or a different collagen chain. If so, it may include at least a part of the amino acid sequence FDFSF (SEQ ID No. 8).
The assay may therefore be based on the sequence FDFSFLP (SEQ ID No.2) and on the sequence EKAHDGGR (SEQ ID No.1).
Preferably the molecular weight of fragments detected in the assay exceeds 1500 Da, more preferably 5000 Da. However, the molecular weight of the fragments may exceed 10000 Da or even 25000 Da.
Said second collagen epitope is preferably also located in the amino acid sequence EKAHDGGR (SEQ ID No. 1) or in isomerised and/or racemised variants thereof. Each said epitope is therefore preferably present in a respective amino acid chain attached to a crosslink. Preferably each said epitope is located in the amino acid sequence EKAH-xcex2D-GGR (SEQ ID No.5). The antibodies employed are preferably in each case a monoclonal antibody raised against a peptide analogue containing the amino acid sequence EKAH-xcex2D-GGR (SEQ ID No.5).
The invention includes a sandwich assay for collagen degradation products in which antibodies of essentially identical specificity are used on both sides of the sandwich, e.g. antibodies each specific for the same amino acid sequence within the C- or N- telopeptide region of collagen, especially collagen type I or type II or type III.
One or both antibodies may be mAb1H11 described in WO96/36645 or may have similar specificity thereto, i.e. specificity for an epitope contained in the metabolite with which said in mAb1H11 is immunologically reactive.
In a further aspect, the invention includes a method of measurement of the concentration of collagen degradation products in a sample comprising conducting a sandwich assay using first and second immunological binding partners (which may be the same or different) each immunologically reactive with an epitope in an N-terminal telopeptide fragment produced upon collagen degradation in vivo. Such N-terminal fragments may be as described in U.S. Pat. No. 5,455,179 and one or both of the antibodies may be mAb1H11 or have equivalent specificity. The second antibody may be reactive with an eptiope containing amino acid sequence crosslinked in collagen type I degradation products with the mAb1H11 epitope sequence. Accordingly, the fragments may be Type I collagen fragments. Preferably, at least one of the antibodies in specific for an epitope containing an isomerised aspartic acid or asparagine residue.
The preparation of antibodies reactive with the sequence EKAHDGGR has been described in WO95/08115, including methods for the preparation of monoclonal antibodies. As described in WO96/30765 this sequence may be isomerised at the aspartate residue such that this is linked to the following glycine by its xcex2 carboxylic acid group. The preparation of monoclonal antibodies to that isomerised sequence is taught there. As described in PCT/EP97/04372 either the normal sequence or the isomerised sequence may become racemised at the aspartate residue so that one or both chains contains a D-aspartic acid residue either bonded normally or via the xcex2 carboxylic acid group. The production of monoclonal antibodies to the normal or iso-D forms of the sequence may be carried out by processes analogous to those described in WO95/08115 or in WO96/30765. Any of these monoclonal antibodies may be used in the present invention as either or both of the two antibodies, which may have identical specificity. Generally, one antibody will be coupled to a substrate, e.g. a solid or particulate support, and the other will be coupled to a direct or indirect label.
The second antibody for use in the invention may be a monoclonal antibody produced by immunisation with bacterial collagenase treated collagen (CTC) followed by selection for antibodies reactive with a selected peptide sequence such as that set out below taken from the sequence of the collagen molecule upstream (i.e. in the N-terminal direction) from the sequence EKAHDGGR (SEQ ID No.1). Preferably in this case, the sequence is one which embraces the immunogenic sequence FDFSFL (SEQ ID No.2). A preferred peptide for this purpose is one that contains the sequence FDFSFL (SEQ ID No.2) plus additional amino acids at one or more preferably both ends such as CSAGFDFSFLPQPPQE (SEQ ID No.6).
Alternatively, immunisation may be carried out with the peptide itself conjugated to a suitable carrier according to known techniques.
Where both epitopes are contained in the same amino acid sequence, e.g. EKAHDGGR (SEQ ID No. 1) or its variants, both antibodies may be the same or may be raised in the same way.
The methods of preparation of monoclonal antibodies are well known in the art. For example, see Campbell, A. M., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 12 (1986). It is possible to produce antibodies to synthetic peptides or their isomerised or racemised variants by immunisation. However, because of the relatively small molecular weight of these compounds it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin, thyroglobulin, ovalbumin, tetanus toxoid, and keyhole limpet haemocyanin. The preferred carrier is bovine serum albumin. To present the hapten in its most immunogenic form to the antibody producing cells of the immunised animal a number of alternative coupling protocols can be used. Suitable procedures include, but are not limited to, glutaraldehyde, carbodiimide, and periodate. Preferred binding agents are glutaraldehyde and carbodiimide.
The preparation of antibodies may also be carried out by conventional techniques including immunisation with collagen fragments which may contain natural isomerisation or with an adjuvant before injection. Examples of adjuvants include, but are not limited to, aluminium hydroxide, Freund""s adjuvant, and immune-stimulating complexes (ISCOMs). ISCOMs can be made according to the method described by Morein, B. et al., Nature 308:457-460 (1984).
Either monoclonal or polyclonal antibodies to the haptencarrier molecule can be produced. For the production of monoclonal antibodies it is preferred that mice are immunised. Spleen cells from the immunised mouse are harvested, homogenised, and thereafter fused with cancer cells in the presence of polyethylene glycol to produce a cell hybrid which produces monoclonal antibodies specific for isomerised peptide fragments derived from collagen. Suitable cancer cells include, but are not limited to, myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of the production of monoclonal antibodies are provided in Goding, J. W., in Monoclonal Antibodies: Principles and Practice, (1986). A preferred preliminary screening protocol comprises the use of synthetic peptides or isomerised or racemised peptide analogues conjugated to a carrier and coated on to the solid surface of a microtitre plate.
For the preparation of polyclonal antibodies, different animal species can be immunised. Suitable species include, but are not limited to, chicken, rabbit and goat. Chicken and rabbit are preferred.
Antibodies so produced may be screened for suitability for use according to the invention by testing for reactivity with a synthetic peptide or peptide analogue of appropriate sequence.
Antibody fragments are prepared by methods known in the art (see E. Ishikawa. Journal of Immunoassay 3:209-327 (1983)).
It is possible to omit (or add) one or more amino acid residues from (or to) the crosslink site sequences without substantial loss of the ability to raise antibodies recognising the corresponding native collagen fragment. It is possible to use longer collagen fragments and/or chimeric peptide analogues to raise the antibodies. It is possible to make substitution of one or more amino acids which are not critical for antibody recognition.
The preparation of synthetic peptides and peptide analogues may be performed according to procedures well known in the art, e.g. by solid-phase peptide synthesis techniques commonly described as xe2x80x9cMerrifield synthesisxe2x80x9d. Also classical solution phase techniques may be used.
The sandwich assay according to the present invention may be conducted according to any of the known sandwich assay formats. These include formats in which one antibody is provided on a solid support and the other is labelled in one of many known ways including the use of radio-isotope labels. The antibodies and revealing reagents for the conduct of an immunoassay using standard detection protocols, for example radioisotope labelling, fluorescent labelling or ELISA, may conveniently be supplied as kits which include the necessary components and instructions for the assay. In one embodiment of the invention such a kit includes a microtitre plate coated with a relevant antibody, standard solutions for preparation of standard curve, a body fluid (e.g. serum) control for quality testing of the analytical run, a second antibody reactive with a second epitope in fragments to be detected conjugated to an enzyme such as horse radish peroxidase or otherwise labelled, a substrate solution, a stopping solution, a washing buffer and an instruction manual.
Both antibodies may be bound to a micro-particle, e.g. in a latex emulsion, so that binding of the antibodies to the target produces an agglutination which can be observed by known techniques such as light transmission or scattering measurements.
However, the two antibodies can be mixed with the sample before one of the antibodies is bound to a capture substrate such as a microtitre plate or other form of solid support such as micro-beads. For such use one antibody may be coupled to a capture moiety which has affinity for and can be captured on a capture substrate. It is preferred that the other antibody is coupled to a direct or indirect label.
The capture moiety may for instance be biotin. Biotinylated antibodies may be captured on a capture substrate bearing avidin or streptavidin.
The use of such a format is particularly suitable where each antibody has the same or an overlapping specificity, there being two identical or mutually interfering epitopes in each of the collagen degradation fragments to be detected. Addition of the target fragments to either one of the antibodies separately may result in both epitopes being bound by the first antibody, so that the second antibody when added does not bind. This may occur even when the first antibody is immobilised on a solid surface. If both antibodies are mixed with the target fragments at the same time, sandwiches containing the first antibody, the target fragment and the second antibody can be formed. These may then be captured to a capture substrate by the capture moiety present on one antibody and the captured sandwich can then be detected via the label of the other antibody.
Accordingly the invention provides in a separate aspect a method of conducting a sandwich assay comprising:
mixing a target antigen containing at least two antigenically similar epitopes with a first antibody reactive with both said epitopes, which first antibody is coupled to a capture moiety and with a second antibody reactive with both said epitopes, which second antibody is coupled to a label, so as to form a first antibodyxe2x80x94target antigenxe2x80x94second antibody sandwich, capturing said sandwich to a capture substrate having an affinity for said capture moiety of said first antigen,
and detecting the capture of said sandwich by detection of the label of the second antibody.
Since immunoassays can be constructed using antibodies specific for synthetic isomerised and/or racemised peptide analogues, the ratios of the corresponding collagen fragment sequences in an appropriate biological fluid can be determined as well as their individual levels and their total. Thus, the assay can be designed to include antibodies which will result in determination of fragments containing several isoaspartic acid containing and/or racemised peptide analogues and optionally the native peptide sequences or determination of a single isoaspartic acid containing and/or racemised peptide analogue sequence, or any desired combination thereof.
In addition to the use of the herein specified peptides as indicators of bone resorption, bone metabolic balance is advantageously determined by the substantially simultaneous determination of a marker of the formation of bone in the same or other appropriate biological fluid form the same individual. xe2x80x9cSubstantially simultaneousxe2x80x9d means the same day, preferably within 4 hours. For example such markers include osteocalcin (also known as bone GLA protein of BGP), propeptides of procollagen type I, bone alkaline phosphatase and total alkaline phosphatase. Suitable methods for the determination of these markers can be found, for example, in Delmas, P. D., et al., J. Bone Min. Res. (1986) 1:333-337.
The assay of the present invention which provides an index to determination of the metabolic status of tissues, which generate collagen-derived peptides and isomerised and/or racemised peptide analogues when degradation occurs, is useful in a variety of contexts. The assays may be used to assess an abnormal condition of a subject by indicating excessive bone resorption. This may show the presence of an osteoporotic condition or the metastatic progress of an malignancy. Other conditions characterised by excessive bone resorption include Paget""s disease and hyperparathyroidism.

Sandwich assays for collagen type I fragments