HK1081972A - Antibodies to adipose tissues - Google Patents
Antibodies to adipose tissues Download PDFInfo
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- HK1081972A HK1081972A HK06102180.0A HK06102180A HK1081972A HK 1081972 A HK1081972 A HK 1081972A HK 06102180 A HK06102180 A HK 06102180A HK 1081972 A HK1081972 A HK 1081972A
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Description
Technical Field
The present invention relates to a method for producing antibodies binding to adipose tissues, in particular polyclonal antibodies binding to characteristic components of plasma membranes in adipose tissues in the body of animals (e.g., farm animals) and/or humans. The invention also relates to antibodies obtainable according to the method, the use of the antibodies, a feed additive comprising the antibodies, a medicament comprising the antibodies and a method for modulating the adipose tissue content in a target animal in need of the antibodies.
Background
In animal farms, a main aim is to increase the growth rate of the farm animals so that, under generally the same regulatory conditions, the animals will grow faster and thus increase the productivity of the farm. In the past, prior to the adoption of modern technology in animal farms, it was common for farmers to simply feed the animals with more food, and thus hoped that more food consumption will promote animal growth and weight gain faster. However, this method of helping an animal gain weight has limitations. Moreover, it has the disadvantage that this method increases the overall consumption of animal feed and thus higher operating costs for undesired conversions.
Another method of promoting growth in farm animals is to administer growth hormones to the animals. This approach is undesirable for a number of reasons. First, the auxins from different animals are rarely homogenous and different mammals, for example, react only with a particular auxin type. Since suitable exogenous growth hormones are usually extracted from the pituitary gland, it is rather difficult and uneconomical to prepare suitable exogenous growth hormones sufficient for large-scale application. Although exogenous growth factors can now be produced by recombinant DNA techniques, the production of exogenous growth factors using such methods is still rather expensive. Secondly, the use of exogenous growth hormones for feeding animals is usually performed by direct injection, which is inevitably rather expensive and difficult to apply to large animal farms having thousands of animals. Third, it is quite difficult to control the dosage administered to produce precisely the desired effect, and excessive amounts of exogenous growth hormones may be harmful to the animals. Fourth, the remainder of the exogenous auxin may enter the meat product and, with its consumables, the human body. Even though some scientists are concerned about the negative effects of these exogenous growth hormones on humans, further research is still needed in this regard.
It has also been proposed to add various feed additives to animal feeds so that animals fed these feeds will grow faster. Unfortunately, regardless of which of the above approaches is used, it is often the case that a relatively large proportion of the increased body weight results from an increase in fat content, rather than from an increase in muscle content. Although similar problems occur in other farm animals, this is particularly true in pigs. As humans become increasingly health conscious, there is hardly any need for high fat content meat products. Therefore, there is an increasing demand for meat products with as little fat content as possible (i.e. high muscle content).
Many methods are used to promote the development of farm animals with high muscle content. One very old method is to raise animals in open or semi-open farms so that the animals have more opportunity to exercise and thereby reduce the fat content in the body. However, since open space is at a premium, this method is almost impossible to implement in modern farm practice. In addition, this method is rather difficult to predict. Animals obtained according to this method may still have a high fat content in their body.
Thus, a need still exists for a substance that regulates and reduces the fat content in farm animals. Preferably, the substance should be easy to use and natural and should not have side effects similar to those caused by artificial or exogenous auxins. In other words, the substance should be safe to use. The substances to be applied to the farm animals should also be preferably applied to humans after modification.
It is therefore an object of the present invention to address the above issues, or at least to provide the public with a useful choice.
Disclosure of Invention
According to a first aspect of the present invention there is provided a non-therapeutic method of producing antibodies which bind adipose tissues in a target animal in need of antibodies to modulate the adipose tissue content of the target animal, the method comprising the steps of: (1) preparing an antigen from adipose tissue of a source animal; (2) administering an antigen to the egg-laying animal to cause production of antibodies; and (3) obtaining antibodies from eggs of the egg-laying animal, wherein the source animal and the egg-laying animal belong to different species.
The antibody is a characteristic component for binding the plasma membrane of the adipose tissues of the target animal. In particular, the antibodies may bind to the granular mucin and/or the fibrous mucin of the adipose tissue of the target animal. The antibody is adapted to specifically bind the characteristic component. By specifically binding to a characteristic component is meant that the antibody has low, or no, significant cross-reactivity.
The method includes the step of causing the production of antibodies from within the body of the egg-laying animal. Advantageously, the method comprises a step of causing the antibody to be deposited in the eggs of the egg-laying animal. In particular, the step of obtaining antibodies from the egg may comprise the step of isolating antibodies from the yolk of the egg. The antigen suitably comprises plasma membrane of adipose tissue of the source animal, plasma membrane surface proteins of adipose tissue thereof, or fragments thereof.
Preferably, the antibody is a polyclonal antibody.
Preferably, the source animal and the egg-laying animal belong to distinctly different species. The egg-laying animal is preferably an avian animal and the source animal may be a non-avian animal. In particular, the egg-laying animal may be a hen and the source animal may be a mammal, such as a pig.
Preferably, the target animal and the egg-laying animal belong to different species. More preferably, the target animal and the egg-laying animal belong to distinctly different species. By distinctly different species is meant that they are phylogenetically distinct. For example, pigs are mammals, hens are birds, and they are distinctly different species.
Preferably, the target animal and the source animal belong to the same species. Alternatively, the target animal and the source animal belong to close species. Close species means that they are phylogenetically related. For example, pigs and mice are close species, they are mammals, and antibodies produced in response to antigens prepared from pig adipose tissues can very well act to modulate the adipose tissue content in mice.
In one embodiment, the target animal is a farm animal. In particular, the source animal and/or the target animal may be a pig. The fat content of pigs is often higher than that of other farm animals and the adipose tissues in pigs administered antibodies are significantly reduced. However, the target animal may be other animals whose adipose tissue needs to be adjusted. For example, the target animal may be a cow to produce leaner beef.
After the method is performed, one or all of the animals may die.
In another embodiment, the target animal is a patient.
According to a second aspect of the present invention there is provided an antibody obtainable according to the method defined above. According to a third aspect of the invention, antibodies are provided for use in therapeutic and diagnostic methods.
According to a fourth aspect of the present invention there is provided the use of an antibody as defined above in the manufacture of a medicament for the treatment of a condition which causes excessive adipose tissue.
According to a fifth aspect of the present invention there is provided a feed additive comprising an effective amount of antibodies as defined above. In particular, the feed additive may be adapted to reduce the content of adipose tissue in the target animal.
In one embodiment, the feed additive may comprise egg yolk of the egg containing the antibody.
According to a sixth aspect of the invention there is provided a medicament containing an effective amount of an antibody as defined above. Preferably, the medicament is suitable for use by consumption. Alternatively, the medicament is also suitable for use by injection.
According to a seventh aspect of the present invention, there is provided a method for modulating the adipose tissue content in a target animal in need of an antibody, comprising the steps of: (1) preparing an antigen from adipose tissue of a source animal, (2) administering the antigen to an egg-laying animal, (3) producing antibodies from the egg-laying animal to which the antigen corresponds, (4) obtaining the antibodies from eggs of the egg-laying animal, and (5) administering a pharmaceutically effective amount of the antibodies to a target animal, wherein the source animal and the egg-laying animal belong to different species. In particular, the step of obtaining comprises isolating the antibodies from the yolk of the egg.
Preferably, the method comprises administering the antibody by consumption. Alternatively, the method comprises the use of the antibody by injection.
The method of modulation comprises the step of binding the antibody to a characteristic component of the plasma membrane of adipose tissues of the target animal. The method may comprise the step of binding the antibodies to the granular mucin of the adipose tissue of the target animal. The method may further comprise the step of binding the antibody to the fibrous mucin of the adipose tissue of the target animal. In particular, the antibodies are adapted to specifically bind the characteristic components. By specifically binding to a characteristic component is meant that the antibody has low or no significant interactive capacity.
Preferably, the antigen comprises plasma membrane of adipose tissue of the source animal, its adipocyte plasma membrane surface proteins, or fragments thereof.
Preferably, the antibody is a polyclonal antibody.
Preferably, the source animal and the egg-laying animal belong to distinctly different species. The egg-laying animal is preferably an avian animal and the source animal may be a non-avian animal. In particular, the egg-laying animal may be a hen and the source animal may be a mammal, such as a pig.
Preferably, the target animal and the egg-laying animal belong to different species. More preferably, the target animal and the egg-laying animal belong to distinctly different species. By distinctly different species is meant that they are phylogenetically distinct. For example, pigs are mammals, hens are birds, and they are distinctly different species.
Preferably, the target animal and the source animal belong to the same species. Alternatively, the target animal and the source animal belong to close species. Close species means that they are phylogenetically related. For example, pigs and mice are close species, they are mammals, and antibodies produced in response to antigens prepared from pig adipose tissues can very well act to modulate the adipose tissue content in mice.
The method of modulation may be a non-therapeutic method.
In one embodiment, the target animal is a farm animal. In particular, the source animal and/or the target animal may be a pig.
Preferably, the egg-laying animal is an avian animal and the source animal may be a non-avian animal. In particular, the egg-laying animal may be a hen and the source animal may be a pig.
After the method is performed, one or more of the animals may die.
In another embodiment, the target animal is a patient.
According to an eighth aspect of the present invention, there is provided a method for producing an antibody which binds to adipose tissues in a target animal in need of the antibody to regulate the content of the adipose tissues in the target animal, comprising the steps of: (1) preparing an antigen from adipose tissue of a source animal, (2) administering the antigen to an egg-laying animal to cause production of antibodies, and (3) obtaining the antibodies from eggs of the egg-laying animal, wherein the source animal and the egg-laying animal belong to different species.
According to a further aspect of the present invention, there is provided an antibody specifically binding to adipose tissue in a target subject, the target subject being a farm animal or a patient in need of the antibody to modulate the adipose tissue content in the target subject. The antibodies bound characteristic components of plasma membranes of adipose tissues. In particular, the antibodies may bind to the granular mucin of adipose tissue. Antibodies may also bind to the fibrous mucin of adipose tissue.
The antibodies are obtained from and/or contained in eggs of an egg-laying animal. The antibodies are deposited on the eggs of the egg-laying animal. Producing antibodies from within the body of the egg-laying animal. Antibodies are produced in response to an antigen administered to an egg-laying animal.
The antigen is prepared from adipose tissue of the source animal. The antigen includes plasma membrane and/or its adipocyte plasma membrane surface protein of the adipose tissue of the source animal.
Preferably, the target animal and the source animal belong to the same species. Alternatively, the target animal and the source animal belong to close species.
Preferably, the source animal and the egg-laying animal belong to distinctly different species.
Preferably, the antibody is a polyclonal antibody.
According to a further aspect of the invention there is provided a feed additive comprising an effective amount of an antibody as defined above. The feed additive is suitable for reducing the content of adipose tissue in a target animal.
According to a further aspect of the invention there is provided a medicament comprising a pharmaceutically effective amount of an antibody as defined above. The medicament is suitable for use by eating. The medicament is suitable for use by injection.
According to a further aspect of the present invention there is provided a method of modulating the adipose tissue content in the body of a target subject in need of an antibody, the target subject being a farm animal or a patient, the method comprising the step of administering a pharmaceutically effective amount of an antibody as defined above, which specifically binds adipose tissue in the target subject.
The method comprises the step of binding antibodies to characteristic components of plasma membranes of adipose tissues. In particular, the method may comprise the step of binding the antibodies to the granular mucin of the adipose tissue. The method may further comprise the step of binding the antibody to a fibronectin within the adipose tissue.
The method comprises the step of administering said composition by ingestion.
The antibody is a polyclonal antibody.
According to a further aspect of the invention there is provided a method of making a composition comprising a pharmaceutically effective amount of an antibody as defined above, the method comprising the step of obtaining the antibody from the eggs of an egg-laying animal. Preferably, the method comprises the step of depositing the antibody in the eggs of the egg-laying animal.
Preferably, the method comprises the step of causing the production of antibodies from within the body of the egg-laying animal. In particular, the method includes the step of producing within the egg-laying animal an antibody corresponding to an antigen prepared from adipose tissue of the source animal. The method includes the step of administering an antigen to the egg-laying animal.
Preferably, the antigen comprises plasma membrane and/or its adipocyte plasma membrane surface proteins of the adipose tissues of the source animal.
Preferably, the target animal and the source animal belong to the same species. Alternatively, the target animal and the source animal belong to close species.
Preferably, the source animal and the egg-laying animal belong to distinctly different species.
Preferably, the antibody is a polyclonal antibody.
The composition may comprise egg yolk containing the antibody.
Detailed description of the invention
Biological substances (e.g., auxins) can be produced and extracted from the pituitary gland of "production" animals. By production animal is meant an animal that is a machine used to produce the desired biological substance. Depending on the kind or nature of the biological substances, they can in fact be obtained or isolated using different methods. For example, if the biological substance is an auxin present as milk colostrum in the production animal, a suitable separation method of its auxin will be performed. Alternatively, if the auxin is present as serum in the production animal, another suitable isolation of its auxin will be performed. However, whatever isolation method is used, it has been found that isolation of sufficient quantities of biological material from animal sources for commercial use is very difficult. Firstly, difficulties arise from the very small quantities of biological material that are usually produced, and secondly, the isolation of biological material from animals is very expensive. The same difficulties similarly exist in the extraction or isolation of specific antibodies from production animals.
In the present invention, it is demonstrated that antibodies prepared according to the present invention, when used in a target animal, which may be a feed animal, reduce or at least modulate the overall fat content in its body to a more desirable level, thereby producing lean meat. When the present invention is used for human use, the target animal means a patient in need of the antibody.
Typically, adipose tissue is first removed from the source animal. Then, plasma membranes of the adipose tissues were separated from the adipose tissues. The isolated plasma membrane includes all of the adipocyte plasma membrane proteins and recognition sites such as granular and fibrous mucin proteins. The separated plasma membrane is used for preparing a substance used as an antigen. Preferably, the substance is formed into a form suitable for injection and is designed to have an immunologically effective concentration of the antigen suitable for eliciting the desired immune response in the production animal.
In the present invention, the substance is used for production animals of egg-laying animals such as hens. The use of hens as production animals is particularly preferred, since hens typically produce more eggs than other egg-laying poultry. For example, an average hen in an economic farm can typically lay 200 to 300 eggs per year. The amount of egg yolk produced is thus very considerable. However, other egg-laying animals, such as ducks, may also be used.
Once the antigen-containing material is used in the production animal, for example by injection, the production animal reacts with the antigen and elicits an immune response to produce antibodies. As described above, the antigen in the application substance actually includes the plasma membrane from the adipose tissue of the source animal, or a fragment thereof. Thus, the antibodies produced by egg-laying animals are polyclonal and suitable for binding various characteristic components, namely the plasma membrane proteins of the adipocytes of the plasma membrane. In the research and development of the present invention, it was determined that a significant amount of the antibodies produced in the production animals were deposited in eggs subsequently laid by the production animals. It was further determined that the concentration of antibodies in the yolk of the egg was much higher than in the albumen to show that the antibodies preferentially sediment in the yolk. In other words, the problem of producing and isolating biologically effective substances from animal sources is addressed in the present disclosure. In particular, eggs can be considered as a warehouse from which antibodies can be recovered quite easily. Also for this reason, hens are preferably used as production animals because they can produce a considerable amount of eggs.
The antibodies produced can then be isolated from the egg yolk. In other words, the original egg yolk containing the antibody may be used directly or after treatment, e.g., dried to form egg yolk powder. An effective amount of the isolated antibody, i.e., the original or treated egg yolk containing the antibody, is then applied to the target animal. One of the main applications of the invention is intended for animal farming and wherein the target animal may be a farm animal. However, as described above, the present invention may be used for human use, and then the target animal may be a patient in need of the antibody.
When used in a target animal, the antibodies will bind to a similar characteristic structure or domain (e.g., surface proteins of cells within the target animal) as the adipocyte plasma membrane proteins of the adipose tissues of the source animal. For example, if the source animal is a pig and the target animal is of the same species as the pig, the antibodies used will bind to the adipocyte plasma membrane of the adipose tissue in the body of the target animal and interfere with the physiological development of its adipose tissue. It has been determined that in the research and development of the present invention, such binding and/or interference significantly reduces the amount of adipose tissue in the target animal, both in terms of weight percent and absolute weight thereof.
As mentioned above, the source animal and the target animal may belong to the same species of animal. The more closely related the source animal and the target animal, the more effective the produced antibodies are to target the adipose tissue of the target animal and ultimately reduce or at least modulate the fat content in the body of the target animal. However, the source animal and the target animal need not belong to the same species. For example, the source animal may be a cow, but the target animal may be a pig. Since both cattle and swine are mammals, their adipose tissues, and in particular their plasma membranes, have more resemblance than plasma membranes between birds and mammals. In summary, the more closely related the source animal and the target animal, the more effective the antibody is in binding, interfering, modulating and/or reducing the fat content in the body of the target animal.
It is to be noted, however, that the source animal and the production animal should preferably be completely different. Otherwise, the antigen-containing material used in the production animal cannot elicit an effective immune response to produce sufficient amounts of antibodies. For example, if the source animal is a duck, the antigen prepared from its adipose tissue will elicit a relatively low immune response in a hen, because both the duck and the hen are avian animals and their adipose tissues are relatively similar. The source animal and the production animal should be different because the animal is unable to produce antibodies that are considered "self antigens.
The invention is further described by the following tests.
Test of
Test of1 : method for producing antibodies for porcine adipose tissue in hens
A. Isolation of plasma membranes from adipose tissue of source animals
Adipose tissue was removed from the back of the source animal. The source animal used in the test was twoA anthopodium brevifolium. Adipose tissue was treated and homogenized in extraction medium at about 37 ℃ for 5 minutes at 2000rmp with a waring blender and then sonicated for 10 minutes. The extraction medium is composed of 0.25M sucrose, 0.01M Na2HPO40.002M EDTA and 0.2mM PMSF, and adjusted to pH 7.4 at 40 ℃. The homogeneous mixture was then centrifuged at 5000rmp for 30 minutes at 37 ℃ to separate the triglycerides from the other components.
Then, the supernatant containing triglyceride was removed from the centrifuge, and the remainder, i.e., the supernatant, was centrifuged at 10000rpm at 4 ℃ for 30 minutes. The supernatant was then centrifuged at 10000rpm at 4 ℃ for 30 minutes and the supernatant was retained. The supernatant was then separated at 38000rpm for 1 hour at 4 ℃. Plasma membranes containing the adipose cell membrane proteins were obtained from adipose tissues. Thereafter, the membrane proteins were stored at-20 ℃ until use.
B. Production of antibodies to porcine adipocyte plasma membranes and their proteins
The plasma membrane obtained from the above method is used for preparing an antigen that elicits an immune response from a production animal. In this experiment, egg-laying hens were used as production animals. In the experiment, initial injections containing the antigen were prepared containing approximately 80 μ g of plasma membrane and its proteins initially suspended in 0.5ml of complete Freund's adjuvant. A second injection comprising the same antigen suspended in incomplete Freund's adjuvant to facilitate the immune response can then also be administered by direct injection. Every four weeks at intervals, each round was performed at 20 different positions between the mucocutaneous regions of the hens. Egg yolk was obtained from the eggs subsequently laid by the hens after the third and fourth challenge injections. The antibody response of the egg yolk was then estimated.
C. Enzyme immunoassay for yolk antibody of porcine adipose tissue plasma membrane
Egg yolk antibodies for antibody titer to appropriate adipose tissue plasma membranes were prepared and screened, and used to determine the degree of cross-reactivity of liver, kidney, red blood cells and skeletal muscle by immunoassay. Will be provided withMu.l of plasma membrane containing 0.25. mu.g of adipose tissue plasma membrane protein in carbonate buffer solution was smeared onto each well of a 96-well polystyrene plate. The plate was left overnight in a humidifier at 4 ℃. The wells were then decanted and plugged three times with PBS containing 0.05% tween 20. 100 μ l egg yolk diluted with PBST was added to each well. Plates were left at 37 ℃ for 1 hour and then rinsed three times with PBST. Mu.l of rabbit anti-chicken 1gGHRP conjugated antibody diluted 1: 5000 in PBST was added to each well. Plates were incubated for 1 hour at 37 ℃. Plates were washed three times with PBST. Then 100. mu.l of o-phenylenediamine zymolyte (OPD) (1.5mg/ml) was added to each well. Plates were then incubated for 5-10 minutes at 37 ℃ and 50. mu.l of 2MH was added2SO4The reaction in each well was stopped. The absorbance measured using an enzyme immunoassay analyzer was 490 nm. Each experiment was performed in two rounds and repeated three times. It was found that the titer of antibodies in egg yolk was higher than 1: 12800, which in the case of the present invention was considered to be a rather high concentration value.
Test 2: effect of adipose tissue antibodies in body weight of target animal
A. Background of the invention
The target animal used in this experiment was a laboratory mouse. 96 developing mother mice with an average body weight of 140g were used in the experiment. Mice were randomly divided into 4 groups. Mice were housed in cages in three subgroups. The rats were fed with normal rat feed. The test started at 30/9 in 2001 and ended at 14/12 in 2001.
B. Method of producing a composite material
The four groups included two test groups and two separate control groups. The first test group was included in two test groups, in which raw egg yolk containing adipose tissue antibodies was injected at different positions on the back of each mouse. Antibodies were generated according to the procedure of test 1 above. In particular, antibodies are produced in response to antigens prepared from porcine adipose tissue. Egg yolk adipose tissue antibodies were obtained according to a similar procedure as described in test 1 above. The dose of each injection was 1ml per mouse per day. Each round of administration included once daily injections for four consecutive days. Egg yolk was used once a month in the experiment. The concentration of the antibody in the raw egg yolk is higher than 1: 12800.
The same dose, concentration and frequency of egg yolk adipose tissue antibody was used for the second test group, but not by injection, but by oral administration.
There was a corresponding control group for both mouse test groups. Control groups of mice were given ordinary raw egg yolk.
C. Results
The following table shows the test results.
Table 1: influence of yolk adipose tissue antibody in body weight and feed conversion ratio (X + -SE)
| At the beginning | At the end of | Of body weight | Food product | Feed stuff |
| Body weight (g) | Body weight (g) | Increase (g) | Amount of food (g) | Conversion rate | |
| First test group (by injection) | 163.42±2.55 | 297.64±5.23 | 133.91±4.23 | 22.25±0.23 | 6.25±0.20 |
| First control group (by injection) | 162.88±2.28 | 289.00±5.33 | 126.62±4.40 | 21.87±0.26 | 6.27±0.30 |
| Second test group (by oral administration) | 159.10±2.70 | 281.56±7.43 | 122.25±5.02 | 21.75±0.23 | 5.87±0.22 |
| Second control group (by oral administration) | 164.21±2.00 | 292.82±6.54 | 127.18±6.20 | 21.72±0.52 | 6.52±0.21 |
Table 2: effect of yolk adipose tissue antibodies on fat content in different parts of the mouse
(X±SE)
| Fat content of omentum and mesenterium (%) | Periuterine connective tissue fat content (%) | Perirenal fat content (%) | Gastrocnemius fat content (%) | |
| First test group (by injection) | 18.06±0.72Aa | 26.43±1.72Aa | 17.95±1.48Aa | 6.03±0.11Aa |
| First control group (by injection) | 18.75±0.87Aa | 27.58±1.78Aa | 19.18±1.32Aa | 5.73±0.06b |
| Second test group (by oral administration) | 14.22±1.02Bb | 18.63±1.98Bb | 12.01±1.17Bb | 5.89±0.11 |
| Second control group (by oral administration) | 17.16±1.05a | 24.58±2.24a | 15.32±1.25a | 5.83±0.09 |
Note: values with different superscripts have different meanings; A. b means P < 0.01; a. b means P < 0.05
Table 3: yolk adipose tissue antibody pair in blood of mice
Effect on the triglyceride, Cholesterol and fatty acid layer (X + -SE)
| Triglyceride (mg/dl) | Total cholesterol (mg/dl) | Total fatty acids (mg/dl) | |
| First test group | 33.83±1.70Aa | 61.05±3.56 | 140.69±9.73 |
| (by injection) | |||
| First control group (by injection) | 45.42±2.67B | 58.91±2.44 | 135.29±7.31 |
| Second test group (by oral administration) | 32.00±1.60Aa | 61.35±2.61 | 161.21±8.05A |
| Second control group (by oral administration) | 41.20±2.48b | 54.64±4.21 | 121.72±7.47B |
Note: values with different superscripts have different meanings; A. b means P < 0.01; a. b means P < 0.05
D. Results and discussion
Table 1 shows that the administration of the antibody by injection increased the body weight and food consumption of the first test group by 5.8% and 1.7%, respectively, compared to the corresponding control group of the rats. However, the feed conversion rate decreased by 0.32%. It was also shown that the oral administration of the antibody resulted in a 3.9% reduction in body weight and a 0.14% increase in food consumption in the first test group, compared to the corresponding control group of rats. The feed conversion rate was reduced by about 10%. The experimental data on the first test group and the control group show that the body weight increased slightly by the injection of the antibody, although the feed conversion rate decreased very little. A low feed conversion rate indicates that a smaller amount of feed is required to produce one weight unit of body weight. The experimental data on the second test group and the control group show that the body weight was slightly increased and the feed conversion rate was significantly reduced by 10% by the administration of the antibody. This is important and demonstrates that administration of antibodies by ingestion is somewhat more effective in reducing overall body weight, and is more pronounced in reducing feed conversion rates.
With respect to table 2, it is shown that the fat content of omentum and mesentery, parametrial and perirenal tissues was reduced by 3.7%, 4.2% and 6.4% respectively due to the administration of the antibody by injection, relative to the corresponding control group. However, the gastrocnemius fat content increased by 5.2%. It was also shown that for the respective control groups, their omentum and mesentery, parametrial and perirenal fat content was reduced by 17.1%, 24.2% and 2.16%, respectively, due to the administration of antibodies by ingestion.
It is clear from this experiment that either way of using, injecting or administering the antibody is generally effective in reducing fat content at different sites in the animal's body. In particular, it has been shown that administration by way of ingestion is significantly more effective in reducing the common fat content of animals.
With respect to table 3, it is shown that the use of the antibody by injection caused a significant 25.5% reduction in the triglyceride layer. Resulting in a small increase in cholesterol and free fatty acid layers of 3.6% and 4.0%, respectively. With respect to the administration of the antibody by ingestion, the triglyceride layer was significantly reduced by 22.3%. Resulting in a 12.3% or 32.4% increase in cholesterol and free fatty acids, respectively.
When all three data sets were considered together, it was clearly shown that the use of antibodies in animals was able to reduce the total fat content in their bodies, and this was supported by the overall fat content of the test group of mice shown in table 2 and the reduction of triglycerides shown in table 3. In particular, it has been shown that administration of the antibody in a manner that is more effective than direct injection.
The above results are significant in two respects. First, it is surprising that the antibodies produced according to the invention are more effective when administered orally. This is important because antibodies can theoretically be mixed with standard feed in farms and thus their use becomes very simple and effective, even reaching the planned function of reducing fat content. Second, there are no significant side effects to the animals. For example, the whole body weight is not affected any significant, however the fat content is reduced. The feed conversion ratio was also slightly improved. In other words, the target animal has a lower fat content and a higher lean meat content.
Table 3 shows a significant increase in free fatty acid layer. It can be explained as follows: triglycerides are composed of fatty acids and glycerol. When the triglyceride layer (i.e., fat content) is caused to decrease, the equilibrium shown below shifts to the right.
Triglyceride fatty acid + Glycerol
→
For this reason, an increase in the free fatty acid layer is caused.
Test of3 : effect of adipose tissue antibodies on mouse body weight
A. Background and methods
The target animal used in this experiment was a laboratory mouse. 150 developing mother rats with an average body weight of 110g were used in the experiment. Mice were randomly divided into 5 groups (i.e., groups I to V) and housed in cages, 3 mice per cage. Group I is the control group and groups II to V are the test groups. The rats were fed with normal rat feed. The composition of the general feed is as follows.
Table 4: common feed composition
| Composition (I) | By weight% |
| Protein | 24.02 |
| Fat | 3.94 |
| 7.9 | |
| Calcium carbonate | 1.4 |
| Phosphorus (P) | 0.8 |
| Salt (salt) | 1.31 |
In the experiment, mice of group I were fed with a normal diet. Groups II to V were fed with normal food supplemented with various amounts of adipose tissue antibodies prepared according to the method described in test 1 above. The antibody was produced according to the production method of the above test 1. In particular, antibodies were produced based on antigen injections prepared from porcine adipose tissues.
The test started at 28 days 11/2002 and ended at 18 days 2/2003.
B. Results
Table 5: effect of antibodies on adipose tissue (X. + -. SE)
| Antibodies in body weight foods | Comparison of | 75ppm | 500ppm | 1000ppm | 6000ppm |
| Initial body weight (g) | 115.19±6.78 | 115.24±7.81 | 114.72±7.54 | 114.81±6.65 | 115.65±9.19 |
| Ending body weight (g) | 304.13±14.65 | 302.23±21.38 | 296.77±22.68 | 296.66±23.17 | 304.00±23.16 |
| Weight gain (g) | 188.94±16.37 | 187.00±21.75 | 182.04±21.52 | 181.68±25.09 | 188.05±18.92 |
| P value | 0.70 | 0.17 | 0.19 | 0.84 | |
| Food consumption (g/day/mouse) | 24.68±2.18 | 24.42±2.63 | 23.93±1.90 | 25.53±2.17 | 25.90±1.33 |
| P value | 0.81 | 0.42 | 0.39 | 0.15 | |
| Periuterine connective tissue fat (g) | 6.04±1.66 | 5.02±1.72 | 5.33±1.93 | 5.97±2.10 | 5.14±1.79 |
| P value | 0.025 | 0.137 | 0.887 | 0.053 | |
| Periuterine connective tissue fat index | 19.80±5.20 | 16.44±4.96 | 18.11±6.27 | 20.00±6.69 | 16.90±5.24 |
| P value | 0.014 | 0.266 | 0.898 | 0.041 | |
| Mesenteric fat (g) | 4.64±0.86 | 4.28±0.99 | 4.42±0.96 | 4.44±1.16 | 4.15±1.00 |
| P value | 0.149 | 0.362 | 0.458 | 0.052 | |
| Mesenteric fat index | 15.21±2.56 | 14.20±2.80 | 14.98±2.89 | 14.82±2.98 | 13.69±2.74 |
| P value | 0.155 | 0.739 | 0.588 | 0.033 | |
| Perirenal fat (g) | 4.80±1.39 | 4.23±1.47 | 4.06±1.37 | 4.01±1.35 | 3.83±1.37 |
| P value | 0.144 | 0.054 | 0.041 | 0.013 | |
| Perirenal fat index | 15.79±4.40 | 13.90±4.36 | 13.85±4.54 | 13.55±4.46 | 12.66±4.35 |
| P value | 0.113 | 0.115 | 0.071 | 0.011 | |
| Abdominal fat (g) | 15.48±3.36 | 13.53±3.79 | 13.74±3.80 | 14.21±4.67 | 13.46±4.07 |
| P value | 0.041 | 0.069 | 0.240 | 0.044 | |
| Abdominal fat index | 50.76±10.25 | 44.53±10.67 | 46.77±12.20 | 47.72±14.21 | 44.33±11.94 |
| P value | 0.026 | 0.182 | 0.352 | 0.032 | |
| Gastrocnemius (g) | 1.85±0.11 | 1.85±0.13 | 1.81±0.12 | 1.83± | 1.87±0.17 |
| P value | 0.946 | 0.319 | 0.521 | 0.561 | |
| Gastrocnemius muscle index | 6.09±0.37 | 6.09±0.37 | 6.10±0.45 | 6.17±0.32 | 6.15±0.41 |
| P value | 0.995 | 0.973 | 0.428 | 0.582 | |
| Serum glycerol hydrochloride (mg/dl) | 56.76±24.33 | 58.59±19.15 | n/a | n/a | 44.95±15.76 |
| P value | 0.756 | 0.040 | |||
| Serum free fatty acid (umol/l) | 151.08±79.12 | 163.36±82.92 | n/a | n/a | 209.45±125.76 |
| P value | 0.573 | 0.041 | |||
| Serum leptin (ng/ml) | 1.89±0.66 | 1.73±0.63 | n/a | n/a | 1.68±0.52 |
| P value | 0.29 | 0.18 | |||
| Serum insulin (uU/ml) | 25.78±7.63 | 20.11±4.87 | n/a | n/a | 22.49±5.83 |
| P value | 0.0017 | 0.0882 |
C. Discussion and conclusions
The above results illustrate that: mice fed diets supplemented with 75ppm antibody had a 16.89% reduction in periuterine connective tissue fat, a 7.76% reduction in mesenteric fat, an 11.88% reduction in perirenal fat and a 12.60g reduction in the abdomen relative to control mice. The rats fed the diet to which 75ppm of the antibody had been added had no significant difference in gastrocnemius muscle weight relative to the control rats.
The above results illustrate that: mice fed diet with 6000ppm antibody had 14.90% less periuterine connective tissue fat, 10.56% less mesenteric fat, 20.21% less perirenal fat and 13.05g less abdominal fat relative to control mice. The rats fed the diet to which 6000ppm of antibody was added had an increase in gastrocnemius muscle weight of 1.08% relative to the control rats. However, this increase is significant.
The test data shows that: food consumption was statistically approximately the same for the different groups of rats.
The test data shows that: the antibodies made and used according to the present invention are effective for modulating, especially reducing, adipose tissues in a target animal.
According to the results of the above experiments, when the antibodies are used in animal farms (e.g., through animal feed), they can produce animals with more lean meat. In most farm animals supplied for human consumption as meat production, pigs tend to have a considerable fat content. Therefore, the invention is particularly suitable for application in pig farming.
When applied in human use, the antibodies can be used in the preparation of a medicament or composition for the treatment or prevention of obesity and/or related conditions. In other words, the antibodies can be added to a food supplement suitable for human consumption. Medicaments comprising this antibody can also be produced.
The contents of each of the above-mentioned references and UK patent No.0212876.7 are incorporated herein by reference in their entirety. It is noted that numerous variations, modifications and further embodiments are possible and contemplated, and all such variations, modifications and embodiments are to be regarded as being within the scope of the invention and as would be understood by those skilled in the art.
Claims (48)
1. A non-therapeutic method for producing antibodies that bind adipose tissues in a target animal in need of said antibodies to modulate the content of adipose tissues in said target animal, comprising the steps of:
(1) preparing an antigen from adipose tissue of a source animal;
(2) administering an antigen to the egg-laying animal to cause production of said antibody; and
(3) obtaining the antibodies from the eggs of the egg-laying animal, wherein the source animal and the egg-laying animal belong to different species.
2. A method according to claim 1, comprising the step of causing production of said antibodies from within the body of said egg-laying animal.
3. A method according to claim 1 or 2, comprising the step of causing the antibody to be deposited in the eggs of the egg-laying animal.
4. A method according to any preceding claim, wherein said step of obtaining comprises the step of isolating said antibodies from the yolk of said eggs.
5. A method according to any preceding claim, wherein the antigen comprises: a plasma membrane of said adipose tissue of said source animal, an adipocyte plasma membrane surface protein thereof, or a fragment thereof.
6. A method according to any preceding claim, wherein the antibody is a polyclonal antibody.
7. The method according to any of the preceding claims, wherein said source animal and said egg-laying animal belong to distinctly different species.
8. The method according to any of the preceding claims, wherein said target animal and said egg-laying animal belong to different species.
9. The method according to claim 8, wherein the target animal and the egg-laying animal belong to distinctly different species.
10. A method according to any preceding claim, wherein the target animal and the source animal belong to the same species.
11. The method according to any of the preceding claims 1 to 9, wherein the target animal and the source animal belong to close species.
12. A method according to any preceding claim, wherein the target animal is a farm animal.
13. The method according to any of the preceding claims, wherein the source and/or target animal is a pig.
14. The method according to any of the preceding claims 1 to 11, wherein the target animal is a patient.
15. The method according to any of the preceding claims, wherein said egg-laying animal is an avian animal and said source animal is a non-avian animal.
16. A method according to any preceding claim, wherein the source animal is a mammal.
17. A method according to any preceding claim, wherein one or more of said animals die after said method is performed.
18. An antibody obtained according to the method defined in any one of claims 1 to 17.
19. The antibody of claim 18 for use in a method of treatment or diagnosis.
20. Use of an antibody as defined in claim 18 for the preparation of a medicament for the treatment of a condition caused by excessive adipocytes.
21. A feed additive comprising an effective amount of an antibody as defined in claim 18.
22. A feed additive according to claim 21 adapted to reduce the level of said adipose tissue in said target animal.
23. A feed additive according to claim 21 or 22 wherein the composition comprises the egg yolk of the egg containing the antibody.
24. A medicament containing a pharmaceutically effective amount of an antibody as defined in claim 18.
25. A medicament according to claim 24, which is suitable for use by eating.
26. A medicament according to claim 24, which is suitable for administration by injection.
27. A method of modulating the adipose tissue content in a target animal in need of an antibody comprising the steps of:
(1) preparing an antigen from adipose tissue of a source animal;
(2) administering the antigen to an egg-laying animal;
(3) allowing the egg-laying animal to produce the antibody in response to the antigen;
(4) obtaining the antibody from the eggs of the egg-laying animal;
(5) administering said pharmaceutically effective amount of said antibody to said target animal, wherein said source animal and said egg-laying animal belong to different species.
28. The method according to claim 27, wherein said step of obtaining comprises the step of isolating said antibodies from the yolk of said egg.
29. A method according to claim 27 or 28, comprising the step of administering said antibody by consumption.
30. A method according to claim 27 or 28, comprising the step of administering said antibody by injection.
31. A method according to any one of claims 27 to 30 including the step of binding said antibody to a characteristic component of said adipose tissue plasma membrane of said target animal.
32. The method according to claim 31, comprising the step of binding said antibody to a granular mucin of said adipose tissue of said target animal.
33. The method according to claim 31, comprising the step of binding said antibody to a fibrous mucin of said adipose tissue of said target animal.
34. A method according to any one of claims 27 to 33, wherein the antigen comprises: the plasma membrane of adipose tissue of said source animal, the plasma membrane surface protein of the adipocyte thereof, or a fragment thereof.
35. A method according to any one of claims 27 to 34, wherein the antibody is a polyclonal antibody.
36. A method according to any one of claims 27 to 35, wherein said source animal and said egg-laying animal belong to distinctly different species.
37. A method according to any one of claims 27 to 36, wherein said target animal and said egg-laying animal belong to different species.
38. The method according to claim 37, wherein said target animal and said egg-laying animal belong to distinctly different species.
39. A method according to any one of claims 27 to 38, wherein the target animal and the source animal belong to the same species.
40. A method according to any one of claims 27 to 38, wherein the target animal and the source animal belong to close species.
41. A method according to any one of claims 27 to 40, wherein the method is a non-therapeutic method.
42. A method according to any one of claims 27 to 41 wherein the target animal is a farm animal.
43. A method according to any one of claims 27 to 42, wherein the source animal and/or the target animal is a pig.
44. A method according to any one of claims 27 to 41, wherein the target animal is a patient.
45. The method according to any one of claims 27 to 44, wherein said egg-laying animal is an avian animal and said source animal is a non-avian animal.
46. A method according to any one of claims 27 to 45, wherein the source animal is a mammal.
47. A method according to any one of claims 27 to 46 wherein one or more of the animals dies following the practice of the method.
48. A method for producing an antibody that binds to adipose tissues in a target animal in need of the antibody to modulate the content of adipose tissues in the target animal, comprising the steps of:
(4) preparing an antigen from adipose tissue of a source animal;
(5) administering said antigen to an egg-laying animal to cause production of said antibody; and
(6) obtaining the antibodies from the eggs of the egg-laying animal, wherein the source animal and the egg-laying animal belong to different species.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0212876.7 | 2002-06-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HK1081972A true HK1081972A (en) | 2006-05-26 |
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