KIT-OF-PARTS TO IDENTIFY MOTHER'S MILK MISSING FUCOSYLTRANSFERASE-2 DEPENDENT GLYCANS AND FEEDING DOSES WITH SAID GLYCANS

Abstract

Described is a matrix to identify mother's milk missing fucosyltransferase-2 (FUT2) dependent glycans which comprise a line of a glycan binding protein such as a lectin or antibody suitable to bind 2fucosyl-glycans and a line of a positive control to bind a mother's milk protein. For instance, this matrix is porous and allows for transport by capillary force of compounds of mother's milk such as glycoproteins. Especially, the glycan binding protein is the plant lectin from Ulex europaeus. A diagnostic kit comprising one or more of such matrices and a device suitable to receive such a matrix provided with a milk application window and a read-out window has been described as well. Finally a kit-of-parts comprising such a diagnostic kit and one or more feeding doses of 2fucosyl-glycans has been disclosed.

Claims

1. Matrix to identify mother's milk missing fucosyltransferase-2 (FUT2) dependent glycans comprising a line of a glycan binding protein and a line of a positive control to bind a mother's milk protein.

2. Matrix according to claim 1 which is porous and allows for transport by capillary force of compounds of mother's milk.

3. Matrix according to claim 1 wherein the glycan binding protein is the plant lectin from Ulex europaeus.

4. Matrix according to claim 1 wherein the positive control comprises an antibody or a fragment thereof specific for a specific milk protein like lactalbumin.

5. Matrix according to claim 1 wherein both the glycan binding protein and the positive control have been labelled with different dyes such as fluorescent dyes.

6. Diagnostic kit comprising one or more matrices to identify mother's milk missing fucosyltransferase-2 (FUT2) dependent glycans comprising a line of a glycan binding protein and a line of a positive control to bind a mother's milk protein and a device suitable to receive such a matrix provided with a milk application window and a read-out window.

7. Diagnostic kit according to claim 6 wherein the intensity of the band or of the colour seen on the read-out window indicates the relative amount of 2fucosyl-glycans.

8. Kit-of-parts comprising a diagnostic kit comprising one or more matrices to identify mother's milk missing fucosyltransferase-2 (FUT2) dependent glycans comprising a line of a glycan binding protein and a line of a positive control to bind a mother's milk protein and a device suitable to receive such a matrix provided with a milk application window and a read-out window and one or more feeding doses of 2fucosyl-glycans such as of 100-5000 mg.

9. Kit-of-parts according to claim 8 wherein the 2fucosyl-glycans comprise 2 -fucosyllactose.

10. Kit-of-parts as defined in claim 8 for use both to identify a deficiency of 2fucosyl-glycans in mother's milk and to supplement this deficiency.

11. Kit-of-parts according to claim 10 to supplement the 2fucosyl-glycans to a daily dose such as of 100-5000 mg.

Description

FIGURES

[0017] FIG. 1 represents a diagnostic kit comprising

[0018] a matrix (1) with a line of plant lectin from Ulex europaeus and an antibody specific for lactalbumin and

[0019] a plastic device (3) in the form of a stick comprising a milk application window (6) and a read-out window (5). The matrix (1) is integrated in the device indicated with the dotted line (4).

DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein, the following terms have the following meanings.

[0021] The terms 2fucosyl-glycans or 2-fucosylated oligosaccharides or alpha-1,2-linked fucosylated oligosaccharides have interchangeable been used and have all the same meaning: HMO's comprising a 2 fucosyl-epitope.

[0022] The term HMO or HMOs refers to human milk oligosaccharide(s). These carbohydrates are highly resistant to enzymatic hydrolysis, indicating that they may display essential functions not directly related to their caloric value. It has especially been illustrated that they play a vital role in the early development of infants and young children, such as the maturation of the immune system. Many different kinds of HMOs are found in the human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milkover 130 such structures have been identified so far. Almost all of them have a lactose moiety at their reducing end while sialic acid and/or fucose (when present) occupy terminal positions at the non-reducing ends. The HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide).

[0023] A fucosylated oligosaccharide is an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are 2-FL (2-fucosyllactose), 3-FL (3-fucosyllactose), difucosyllactose, lacto-N-fucopentaose (e.g. lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II and any combination thereof. Without wishing to be bound by theory it is believed that the fucosyl-epitope of the fucosylated oligosaccharides may act as decoy at the mucosal surface. By a competition effect, it may prevent and/or limit the action of the pathogens responsible of infections (of viral or bacterial origin) or of their secreted components (e.g. toxins), especially by avoiding their binding to natural ligands, and this will therefore reduce the risk of infections, and particularly of URT infections or allergies.

[0024] The expressions fucosylated oligosaccharides comprising a 2-fucosyl-epitope and 2-fucosylated oligosaccharides encompass fucosylated oligosaccharides with a certain homology of form since they contain a 2-fucosyl-epitope, therefore a certain homology of function can be expected. Without wishing to be bound by theory the 2-fucosyl-epitope of these fucosylated oligosaccharides is believed to be particularly specific to pathogens (or their secreted components) involved in URT infections or allergies. Some examples are 2-FL (2 fucosyllactose), difucosyllactose, lacto-N-fucopentaose, lacto-N-fucohexaose, lacto-N-difucohexaose, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose difuco-lacto-N-neohexaose, difucosyllacto-N-neohexaose, fucosyl-para-Lacto-N-hexaose and any combination thereof.

[0025] An example of lacto-N-fucopentaose is lacto-N-fucopentaose I.

[0026] The term infant means a child under the age of 12 months.

[0027] The expression young child means a child aged between one and three years, also called toddler.

[0028] The expressions to prevent URT infections, preventing URT infections or prevention of URT infections mean avoiding that URT infections occur and/or decreasing the incidence of the URT infections (reduction of the frequency, i.e. the number of URT infections). The prevention of URT infections is during the treatment (i.e. during the administration of the composition of the present invention). It can also encompass the prevention of URT infections later in life. The term later in life encompasses the effect after the termination of the intervention or treatment. The effect later in life can be from 1 week to several months, for example from 2 to 4 weeks, from 2 to 6 weeks, from 2 to 8 weeks, from 1 to 6 months or from 2 to 12 months.

[0029] The expressions to treat URT infections, treating URT infections or treatment of URT infections should be understood as comprising the decrease of the duration of the URT infections (number of days/weeks/years the infants or young children will suffer from URT infections), of the severity of URT infections (the consequences and/or the seriousness of URT infections). These expressions also encompass the relieve of the symptoms such as cough, sore throat, runny nose, nasal congestion, headache, low grade fever, facial pressure, sneezing, and combinations thereof, and/or the decrease of complications caused by URT infections on the infant or young child health, such as otitis media and sometimes bronchitis, and/or the decrease of pain, and/or the decrease of tiredness, and/or the ease of the sleep and/or the stabilization of the activity of the infants or young children suffering from URT infections.

[0030] The mother's milk should be understood as the breast milk or the colostrum of the mother.

[0031] By a deficiency of 2fucosyl-glycans' in mother's milk or mother's milk missing fucosyltransferase-2 (FUT2) dependent glycans it is meant a mother's milk that lacks, is depleted of or is poor in 2-fucosylglycans.The milk may contain no (or almost no) fucosylated oligosaccharides, or a low amount of fucosylated oligosaccharides such as an amount which represents less than 50%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 3%, or less than 2% of the mean (or average) amount that is generally found in the mother's milk or mother's milk fed to the infant or young child is deficient in at least one 2-fucosylated oligosaccharide(s) in the sense that it has a concentration of said 2-fucosylated oligosaccharide(s) of less than 3000 mg/L, less than 2000 g/L, less than 1000 mg/l, less than 500 mg/L, less than 300 mg/L, less than 100 mg/L, less than 50 mg/L, less than 10 mg/L, less than 1 mg/L.

[0032] The 2fucosylated-oligosaccharide to be used in the feeding dose may be isolated by chromatography or filtration technology from a natural source such as animal milks. Alternatively, it may be produced by biotechnological means using specific 2fucosyltransferases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and/or mixed cultures may be used. Fucosylated oligosaccharide formation can be initiated by acceptor substrates starting from any degree of polymerization (DP), from DP=1 onwards. Alternatively, 2fucosylated oligosaccharides may be produced by chemical synthesis from lactose and free fucose. Fucosylated oligosaccharides are also available for example from Kyowa, Hakko, Kogyo of Japan. Alternatively, fucosyl-glycans might be produced by employing the reverse reaction of fucosylhydrolases and suitable fucose donor substrates such as fucose linked to a carrier.

[0033] All percentages are by weight unless otherwise stated.

[0034] The invention will now be described in further details. It is noted that the various aspects, features, examples and embodiments described in the present application may be compatible and/or combined together.

[0035] In addition, in the context of the invention, the terms comprising or comprises do not exclude other possible elements. The composition of the present invention, including the many embodiments described herein, can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise depending on the needs.

[0036] A first object of the invention is to provide a diagnostic kit to analyse the presence or absence and/or the amount of 2fucosyl-glycans in mother's milk in an easy and convenient way which can be applied at home as well.

[0037] In a first embodiment this diagnostic kit comprises [0038] one or more matrices which allow the transport of compounds of mother's milk by capillary force, for instance made of silica, a line of a plant lectin from Ulex europaeus suitable to bind 2fucosyl-glycans and a line of a positive control to bind a mother's milk protein, both labelled with different dyes and [0039] a device suitable to receiving a matrix with a milk application window and a read out window to analyse, where the device may be used several times by receiving various matrices.

[0040] With one type of matrix, the application of a milk sample to the application window will lead to migration of small milk components such as fucosyl-glycans. When reaching the lectin and antibody lines these will migrate with the milk. Lectins and antibodies that bound their respective substrate (fucosyl-glycans or specific milk protein) will migrate less and reach the height of the read-out window. Appearance of two bands indicates that the test worked (positive control) and that 2fucosyl-glycans are present. If no 2fucosyl-glycans are present the second band will migrate outside the read-out window and not be seen.

[0041] If two bands appear the intensity of the band will indicate the relative amount of 2fucosyl-glycans. A scale such as from 1 to 4 corresponding to band intensity seen on the read-out window will be provided on the device. The scale will allow to select the optimal supplementation dose of the provided 2fucosyl-glcans.

[0042] In an alternative embodiment of the invention the matrix will have the glycan binding protein as lectin and antibody positioned in such a way that application of milk will lead to appearance or quenching of colour without migration of the lectin or antibody. In this embodiment preferably labelling of the glycan binding protein and antibody with fluorescent dyes as well as a light source should be provided to read the colour appearance and quenching on the device. The intensity of the colour will indicate the relative amount of 2fucosyl-glycans.

[0043] The principle of the application of the diagnostic kit may be compared with other generally known immune tests.

[0044] A second object of the invention is to provide a kit-of-parts to analyse the content of 2fucosyl-glycans in mother's milk in a convenient, easy and cheap way, and to provide feeding doses of said glycans to supplement an eventual deficiency.

[0045] This kit-of-parts according to the present invention comprises: [0046] a diagnostic kit as disclosed above [0047] one or more feeding doses with 2fucosyl-glycans to supplement the feeding with the mother's milk if necessary.

[0048] If the line coloured with the dye labelled to the 2fucosyl-glycans did not move while the control one did as exemplified in FIG. 1, the mother's milk lacks 2fucosyl-glycans.

[0049] In that case one or more feeding doses of 2fucosyl-glycans should be given to the infant or young child fed with the mother's milk in such an amount that an amount of 0.1 g-5 g per day, especially 2-5 g/day be achieved.

[0050] Furthermore the line coloured with the dye labelled to the 2fucosyl-glycans does not indicate only that 2fucosyl-glycan is present but the intensity of the colour of the quenching is related to the amount of 2fucosyl-glycans as well.

[0051] This feeding with extra 2fucosyl-glycans may take place by offering the feeding dose or feeding doses in several ways as for instance:

[0052] dissolved in water or a small amount of breast milk to allow for application of a spoon or a syringe preferably during the normal breast feeding session, in an amount to have at least the minimum amount as indicated above given to the infant or young child.

[0053] However, the feeding dose of 2fucosyl-glycans may be given added to other nutrition compositions as fruit or vegetables as well.

[0054] As indicated, the device may repeatedly be used to receiving a new matrix each time to read out the amount of 2fucosyl-glycans present in the mother's milk.

EXAMPLE

[0055] FIG. 1 shows a diagnostic kit according to the invention comprising a rectangular matrix (1) with a line of plant lectin from Ulex europaeus and a line of an antibody specific for lactalbumin, both bound to a dye. This matrix may be put into the milk with the side where the lines are. Then the matrix should be put into the transparent device (3) at position (4) with the side where the lines are at the height of the milk application window (6). With the read-out window (5) the position of the lines and therewith the presence of the 2fucosyl-glycans should be determined provided that the position of the control line denoting lactalbumin is found on the expected place.