KR20170055237A - Antibody for estrus diagnosis in animal and use thereof - Google Patents

Abstract

The present invention relates to antibodies for detecting estrus of animals and uses thereof, and more specifically, to a kit and a method for detecting estrus using the antibodies. In the present invention, an estrus time can be accurately predicted by treating the antibodies and measuring concentration change of luteinizing hormone (LH) protein, and sandwich ELISA method is used to remarkably improve diagnostic efficiency of estrus. Therefore, the present invention accurately diagnoses the estrus time of stocks to improve fertilization rate and production of a surrogate mother, thereby being expected to contribute to improvement of planned breeding, and management efficiency.

Description

Antibodies for the diagnosis of estrogen in animals and uses thereof < RTI ID = 0.0 >

TECHNICAL FIELD The present invention relates to an antibody for estrogenic diagnosis of an animal and use thereof, and more particularly to an estrogenic diagnostic kit and a diagnostic method using the antibody.

In recent years, animal husbandry has been studied in the form of breeding, improvement, and production of high quality meat. In particular, farmers are increasingly interested in production of farmers who are directly connected to income. Numerous studies have reported methods to reduce the production rate of the living and the labor of the farmer by increasing the pregnancy rate through artificial fertilization and adjusting the maternal delivery period by using the collective conception method. In addition, it is possible to improve the fertilization rate and ultimately to increase the yield of the living by accurately diagnosing the fertilization period by diagnosing the estrus timing of the livestock.

As an example, according to reports on the rate of artificially fertilized conception rate by the stage of estrus of cattle, the highest conception rate is shown in mid-estrus period, but the actual ovulation time is 8-14 hours after termination of estrus, It is recommended that artificial insemination be carried out between time points. In other words, 2 hours before the end of the estrus, and 4 hours after the end of the estrus are the most appropriate fertilizer period. Therefore, oocytes and spermatozoa after estrus must be fertilized within the time of fertilization. For this purpose, fertilization should be performed immediately before ovulation time of oocytes by observing the flow of estrus.

Currently, judgment methods based on gross changes in vulva, judgments using mucus secreted from estrus, and judgment methods using autologous pacemakers are widely used to judge estrus and correction modalities. The cow that continues to hung up, while the vagina begins to swell with a bright red color at the beginning of the horn, while the appearance of this appearance gradually disappears at the end of the horn, and the mucus from the vagina to the outside of the vagina is also in the late estrus The amount of secretion becomes smaller. In addition, viscous secretion of mucus secreted by estrus shows a strong tendency to electricity, but when it reaches the end of estrus, it shows a gradually lighter consistency. By observing the periphery of the vulva, it is possible to predict estrus and fertilization period. In addition, the method using the mucus secreted to the estrus period uses a device which notifies the physiological changes of the mucous secreted during the estrus by the electric resistance value sensor, and the judgment method using the self-artificial correction device is the color change To determine whether or not horns are present and flow. However, these methods have a problem that the physiological changes due to the differences of the individuals, age, season, nutritional status, and parity are not sufficiently reflected, and there are limitations in application of the technology.

Under these circumstances, studies have been made actively on a precise diagnosis method of ovulation (Korean Patent Laid-Open No. 10-2015-0100990) in order to solve the above-mentioned problem, but it is still not enough.

Disclosure of the Invention The present invention has been conceived to solve the problems as described above. The present inventors have made intensive studies to anticipate estrogenic timing by treating an antibody specifically binding to LH (Luteinizing hormone) protein, The present invention has been completed on the basis of this finding.

Accordingly, an object of the present invention is to provide a fragment comprising an anti-LH antibody or an antigen-binding region thereof that specifically binds to an LH protein.

Another object of the present invention is to provide a composition for the diagnosis of ovulation of a mammal other than human, comprising the anti-LH antibody or a fragment containing the antigen binding region thereof as an active ingredient.

Yet another object of the present invention is to provide an estrogenic diagnostic kit for a mammal other than human, comprising the diagnostic composition.

It is yet another object of the present invention to provide a method of detecting the level of antigen-antibody reaction between a fragment comprising an LH protein and an anti-LH antibody or antigen-binding region thereof in a biological sample, And the like.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a fragment comprising an anti-LH antibody or an antigen-binding region thereof that specifically binds to an LH (luteinizing hormone) protein.

In one embodiment of the present invention, the anti-LH antibody comprises HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: A heavy chain variable region comprising at least one selected from the group consisting of: And LCDR3 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, And may include variable regions.

In another embodiment of the present invention, the anti-LH antibody may include a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

In another embodiment of the present invention, the anti-LH antibody comprises HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10, and HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: A heavy chain variable region comprising any one or more selected from the group consisting of HCDR3; And LCDR3 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, And may include variable regions.

In another embodiment of the present invention, the anti-LH antibody may comprise a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

In addition, the present invention provides a composition for the diagnosis of ovulation of a mammal other than human, comprising the anti-LH antibody or a fragment comprising the antigen-binding region thereof as an active ingredient.

In addition, the present invention provides an estrogenic diagnostic kit for a mammal other than a human, comprising the diagnostic composition.

In one embodiment of the present invention, the diagnostic kit may be an enzyme-linked immunospecific assay (ELISA) diagnostic kit.

In another embodiment of the present invention, the enzyme-linked immunosorbent assay (ELISA) diagnostic kit may be a sandwich enzyme-linked immunosorbent assay (Sandwich ELISA) diagnostic kit.

The present invention also provides a method for diagnosing an estrogenic condition in a mammal other than a human, comprising the step of, in a biological sample, measuring the level of an antigen-antibody reaction between the LH protein and the fragment comprising the anti-LH antibody or antigen- .

In one embodiment of the present invention, the biological sample may be any one selected from the group consisting of whole blood, serum, plasma, and mucus.

In another embodiment of the present invention, the step of measuring the antigen-antibody reaction level can be performed by an enzyme immunoassay (ELISA), radioimmunoassay (RIA), Western blot, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, and enzyme substrate staining method.

The present invention provides a novel use of said anti-LH antibody or fragment comprising the antigen binding region thereof for producing an estrogenic composition.

The present invention relates to an antibody for estrus diagnosis of animals and its use, wherein the antibody specifically binds to LH (Luteinizing Hormone) protein, and thereby the estrus and flow of the mammal can be specifically predicted.

Conventional diagnostic methods of estrus have a fear that the accuracy of the diagnosis of estrus is lowered due to factors such as the individual, age, season, nutritional status, and parity. However, the present invention targets sex hormone proteins, which are essential for estrus, which can sufficiently reflect the above factors, and the problems of the prior art are improved by significantly increasing the sensitivity using the sandwich ELISA technique. The invention is expected to increase the fertilization rate by accurately predicting the date of livestock estrus, and ultimately to increase the production of live animals.

Fig. 1 shows the result of measuring the fluorescence intensity according to the antigen-antibody reaction when the culture medium of the LH-1 antibody protein diluted to various concentrations was treated.
Fig. 2 shows the result of measuring the fluorescence intensity according to the antigen-antibody reaction when the culture medium of the LH-4 antibody protein diluted to various concentrations was treated.
FIG. 3 shows the results of comparing the fluorescence intensities of the LH-1 antibody proteins when the antigen-antibody reaction was confirmed using an indirect ELISA or a sandwich ELISA.
FIG. 4 shows the results of comparing the fluorescence intensities of the LH-4 antibody proteins when the antigen-antibody reaction was confirmed using an indirect ELISA or a sandwich ELISA.
FIG. 5 shows the results of measurement of changes in the concentration of LH in the serum according to the estrous cycle using the anti-LH antibody protein of the present invention and the sandwich ELISA.

The inventors of the present invention have newly developed a monoclonal antibody protein capable of diagnosing the estrus of a mammal by specifically binding to LH (Luteinizing hormone) protein, and developed an estrogenic diagnostic kit with improved diagnostic efficiency by using the same. It was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a fragment or functional variant comprising an anti-LH antibody or an antigen-binding region thereof that specifically binds to an LH protein.

As used herein, the term "antibody" includes immunoglobulin molecules immunologically reactive with specific antigens and includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering such as antibodies, antibody fragments, chimeric antibodies (e. G., Humanized murine antibodies) and heterologous binding antibodies (e. G., Bispecific antibodies).

Typically, the antibody has a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (also known as a "domain"). The variable regions of the light and heavy chains comprise three variable regions and four "structural regions" (framework regions) called "complementarity-determining regions " The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3 starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.

In the present invention, CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3) of the heavy chain variable region are the amino acids represented by SEQ ID NOS: 1 to 3 or SEQ ID NOS: 9 to 11, CDR1 CDR2 (LCDR2) and CDR3 (LCDR3) may be composed of the amino acids represented by SEQ ID NOS: 4 to 6 or SEQ ID NOS: 12 to 14, respectively, and the amino acid sequences represented by SEQ ID NOS: 1 to 6 and SEQ ID NOS: And more preferably 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the nucleotide sequence.

In the present invention, the heavy chain variable region of the anti-LH antibody may be an amino acid represented by SEQ ID NO: 7 or 15, the light chain variable region may comprise an amino acid represented by SEQ ID NO: 8 or 16, And at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 16 and 16, respectively.

In addition, functional variants of anti-LH antibodies include biological equivalents of the anti-LH antibody sequences described herein, to the extent that the LH protein is specifically recognizable. For example, additional changes can be made to the amino acid or polynucleotide sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include deletion of the amino acid sequence residues of the antibody. Insertion, and / or substitution, and are based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In one embodiment of the present invention, the anti-LH antibody protein (LH-1 antibody protein, LH-4 antibody protein) and LH protein of the present invention can specifically bind (see Examples 1 and 2) , The efficiency of the diagnosis of ovulation can be remarkably improved and the above results are again verified by using an experimental animal. The present invention can be usefully used for accurately predicting the estrus timing of livestock (Examples 3 and 4 Reference).

Accordingly, the present invention provides an estrogenic composition for mammals other than humans, comprising the anti-LH antibody or a fragment comprising the antigen-binding region thereof as an active ingredient, and provides an estrogenic diagnostic kit comprising the diagnostic composition do.

The present invention also provides a method for diagnosing an estrogenic condition in a mammal other than a human, comprising the step of, in a biological sample, measuring the level of an antigen-antibody reaction between the LH protein and the fragment comprising the anti-LH antibody or antigen- .

As used herein, the term "diagnosis" refers to the act of confirming the physiological state, i.e., the state of estrus, by administering the composition according to the present invention.

In addition, the estrogen diagnostic kit and / or diagnostic method of the present invention can diagnose estrogenicity by quantitatively or qualitatively analyzing an antigen of an antibody protein through an antigen-antibody binding reaction. The antigen-antibody binding reaction can be carried out by conventional enzyme immunoassay (ELISA), radioimmunoassay (RIA), Western blot, immunoprecipitation, immunohistochemical staining, A substrate colorimetric method, and the like, preferably by enzyme-linked immunosorbent assay (ELISA), and most preferably by sandwich enzyme-linked immunosorbent assay (Sandwich ELISA).

Meanwhile, the estrogen diagnostic kit of the present invention may further include an additional configuration for measuring the antigen-antibody binding reaction. The marker of the secondary antibody for measuring the antigen-antibody binding reaction is preferably a conventional coloring agent that undergoes a color reaction, and is preferably selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase (alkaline phosphatase), colloidal gold fluorescent substances such as colloidal gold, FITC (poly-L-lysine-fluorine isothiocyanate) and RITC (rhodamine-B-isothiocyanate) , But is not limited thereto. The coloring substrate is preferably used in accordance with a labeling substance that undergoes a color reaction, and preferably TMB (3,3 ', 5,5'-tetramethylbenzidine), ABTS [2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid)], OPD (O-phenylenediamine), and the like. In addition, a solid material, a washing solution, etc. for the antigen-antibody binding reaction may be used or included in a conventional material or solution well known in the art.

In addition, the biological sample may include, but is not limited to, whole blood, serum, plasma, or mucus, such as blood or other liquid samples of biological origin, biopsy specimens, tissue samples of solid tissue such as tissue culture, or cells derived therefrom. And the sample can be obtained from an animal, preferably a mammal. In addition, the sample can be pretreated prior to its use in diagnosis of estrus, and the pretreatment furnace may include filtration, distillation, extraction, concentration, inactivation of disturbance components, and addition of reagents, no.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example 1. Detection of anti-LH antibody protein

The present inventors newly identified a monoclonal antibody protein capable of specifically diagnosing or diagnosing the estrus of a mammal by specifically binding to LH (Luteinizing hormone) protein. In the following Examples, it was confirmed that the anti-LH antibody protein (LH-1 Antibody protein, LH-4 antibody protein).

First, to obtain the anti-LH antibody protein of the present invention, mice were inoculated with 100 ug of LH protein and Complete adjuvant three times. One week after the third inoculation day, the mouse spleen and lymph node The cells were harvested and immunocytes were obtained. Then, the immune cells were fused with SP cells, and cell lines for producing monoclonal antibodies were selected from them. From the selected cell lines, LH-1 antibody protein (heavy chain variable region of SEQ ID NO: 1, light chain of SEQ ID NO: 2 Variable region) and the LH-4 antibody protein (heavy chain variable region of SEQ ID NO: 3, light chain variable region of SEQ ID NO: 4).

Example 2. Identification of specific binding ability of anti-LH antibody protein

In this example, the specific binding ability between the anti-LH antibody protein of the present invention and the LH protein was confirmed by enzyme-linked immunosorbent assay (ELISA).

Specifically, 100 μl of LH antigen (10 μg / ml) was added to a 96-well plate for immunological reaction, coated at 4 ° C. for 24 hours, and washed three times with PBS (-) solution. The LH-1 antibody protein obtained in Example 1 and the culture medium of the LH-4 antibody protein were diluted 5-fold, 10-fold, 50-fold, or 100-fold with PBS (-) solution, Lt; 0 > C for 24 hours. Then, the cells were washed with PBS (-) solution three times to remove unbound antibody protein, and then the secondary antibody, anti-mouse IgG-HRP, was further added and incubated at room temperature for 2 hours. After washing with PBS (-) solution three times, TMB (3,3 ', 5,5'-Tetramethylbenzidine) coloring solution was added. When color development was observed, the fluorescence intensity at 450 mm was measured by Immunoreader. On the other hand, in this example, as a control group, a group to which no LH-1 antibody protein or LH-4 antibody protein was added was used.

As a result, as shown in Figs. 1 and 2, The group to which LH-1 antibody protein or LH-4 antibody protein was added showed a significantly higher fluorescence intensity than the control group, and this tendency was observed to be higher as the dilution ratio was lower. From the above results, it was confirmed that the specific binding ability between the anti-LH antibody protein of the present invention and the LH protein was confirmed.

Example 3: Confirmation of difference in diagnostic efficiency according to diagnostic method

In this example, blood samples collected from dogs were used to identify differences in diagnostic efficacy according to diagnostic methods. Indirect ELISA and sandwich ELISA were used as diagnostic methods.

Specifically, in order to perform an indirect ELISA, 10 μl of serum protein and 90 μl of PBS (-) solution, which were separated from the blood of a dog, were added to a 96 well plate for immunological reaction and coated at 4 ° C. for 24 hours, And washed three times with PBS (-) solution. Then, LH-1 antibody protein and LH-4 antibody protein were added to the plate, reacted with serum protein, and anti-mouse IgG-HRP, a secondary antibody, was added and incubated at room temperature for 2 hours. The fluorescence intensity was measured in the same manner as in Example 2.

In order to perform the sandwich ELISA, 100 μl of LH-1 antibody protein or LH-4 antibody protein was added at 10 ug / ml for 24 hours at 4 ° C, Lt; / RTI > Then, 10 μl of serum protein and 90 μl of PBS (-) solution, which had been separated from the blood of dogs, were added together and coated at 4 ° C. for 24 hours, and then washed three times with PBS (-) solution. Then, biotin-conjugated LH-1 antibody protein or LH-4 antibody protein was added to the plate, followed by incubation at room temperature for 2 hours. After the reaction, the cells were washed three times with PBS (-) solution, and then the Avidin-HRP antibody was added thereto. After incubation at room temperature for 2 hours, the fluorescence intensity was measured in the same manner as above. On the other hand, in this embodiment. The differences in fluorescence intensity between the indirect ELISA and the sandwich ELISA were compared for three blood samples (A527, A531, and A524).

FIG. 3 shows the results obtained when the LH-4 antibody protein was used for antigen capture, the LH-1-biotin antibody was used for detection, the LH-1 antibody protein was used for antigen capture and the LH- As a case, the Sandwich ELISA group showed significantly higher fluorescence intensity than the group using the Indirect ELISA, and this challenge was observed in all three blood samples (A527, A531, and A524). In addition, these differences were more prominent when LH-1-biotin antibody was used for detection. The above results show that the present invention can further improve the detection efficiency of the LH protein and the diagnostic efficiency of the estrus by combining the anti-LH antibody protein of the present invention with the sandwich ELISA technique.

Example 4. Confirmation of estrogenic diagnostic effect using sandwich ELISA diagnostic method

In this example, the effect of the anti-LH antibody protein and the sandwich ELISA of the present invention on the serum concentration of LH according to the estrus cycle was examined.

Specifically, 100 μl of 10 μg / ml LH-1 antibody protein was added to each well, coated at 4 ° C. for 24 hours, and washed three times with PBS (-) solution. Then, 10 μl of serum protein and 90 μl of PBS (-) solution, which had been separated from the blood of dogs, were added together and coated at 4 ° C. for 24 hours, and then washed three times with PBS (-) solution. Then, biotin-conjugated LH-4 antibody protein was added to the plate, followed by incubation at room temperature for 2 hours. After the reaction, the cells were washed with PBS (-) solution three times. Then, Avidin-HRP antibody was added thereto, followed by reaction at room temperature for 2 hours, and then the fluorescence intensity was measured in the same manner as in Example 2. In this embodiment. Three blood samples (A527, A531, and A524) were monitored for changes in serum LH concentration.

As a result, as shown in FIG. 5, the increase and decrease of the LH concentration in the serum was confirmed by using the anti-LH antibody protein and the sandwich ELISA of the present invention, thereby confirming the estrus and the specific estrus day.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> KIM, dong ku <120> Antibody for estrus diagnosis in animal and use thereof <130> MP15-171 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Heavy chain CDR1 region <400> 1 Ala Tyr Arg Phe Ser Arg His Tyr   1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Heavy chain CDR2 region <400> 2 Ile Tyr Pro Gly Ser Gly Asn Thr   1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Heavy chain CDR3 region <400> 3 Asn Trp Ile Ser Pro Ile Cys Cys   1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Light chain CDR1 region <400> 4 Gln Ser Ile Val His Ser Asn Arg Tyr Thr Tyr   1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Light chain CDR2 region <400> 5 Gly Val Ser   One <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Light chain CDR3 region <400> 6 Gly Thr Ser Val Pro Tyr Thr   1 5 <210> 7 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Heavy chain <400> 7 Gln Thr Gln Met Arg Arg Gly Val Pro Gln Lys Thr Lys Arg Gly Asp   1 5 10 15 Ser Phe Thr Asp Ser Ser Lys Asp Leu Ala Tyr Arg Phe Ser Arg His              20 25 30 Tyr Asn His Trp Glu Asn Gln Lys Arg Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Val Ile Tyr Pro Gly Ser Gly Asn Thr Lys Cys Asn Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Lys Arg Gln Thr His Pro Pro Ala Arg Gly Ser  65 70 75 80 Pro Pro His Met Lys Thr Leu Arg Ser Thr Thr Gly Asn Trp Ile Ser                  85 90 95 Pro Ile Cys Cys Arg Glu Glu Leu Leu Gln             100 105 <210> 8 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-1 antibody Light chain <400> 8 Cys Gly Ile Ser Thr Met Lys Leu Pro Val Arg Leu Leu Val Met Met   1 5 10 15 Phe Trp Ile Pro Ala Ser Ser Asp Val Val Met Thr Gln Thr Pro              20 25 30 Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg          35 40 45 Ser Ser Gln Ser Ser Val Ser Ser Asn Arg Tyr Thr Tyr Leu Glu Trp      50 55 60 Tyr Leu Gln Lys Pro Gly Gln Ser Leu Lys Leu Leu Ile Tyr Gly Val  65 70 75 80 Ser Asn Arg Phe Cys Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Gly                  85 90 95 Thr Asp Leu Ser Gly Thr Asp Leu Ser His Lys Asn Asn Pro Ser Glu             100 105 110 Ala Glu Asp Met Gly Val Tyr Tyr Cys Phe Gly Thr Ser Val Pro Tyr         115 120 125 Thr Ser Asp     130 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Heavy chain CDR1 region <400> 9 Gly Tyr Thr Phe Thr Asp Tyr Trp   1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Heavy chain CDR2 region <400> 10 Ile Leu Pro Gly Ser Gly Ser Thr   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Heavy chain CDR3 region <400> 11 Pro Phe Cys Thr Gly Glu Lys Cys Leu   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Light chain CDR1 region <400> 12 Lys Gln Ser Glu His Gly Tyr Ile Val   1 5 <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Light chain CDR2 region <400> 13 Tyr Cys Ile   One <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Light chain CDR3 region <400> 14 Lys His Ile Arg   One <210> 15 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Heavy chain <400> 15 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr              20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile          35 40 45 Gly Ile Leu Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr  65 70 75 80 Met Gln Leu Ser Thr Pro Thr Thr Glu Asp Ser Ala Ile Trp Ile Ser                  85 90 95 Pro Phe Cys Thr Gly Glu Lys Cys Leu             100 105 <210> 16 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> anti-LH-4 antibody Light chain <400> 16 Ile His Val His Ile Arg Gly Pro Pro Cys Gln Thr Gly Pro Ala Lys   1 5 10 15 Gln Ser Glu His Gly Tyr Ile Val Ala Ala Glu Pro Thr Glu Gly Arg              20 25 30 Thr Ala Thr Gln Thr Pro His Leu Tyr Cys Ile Arg Tyr Arg Ile Gly          35 40 45 Gly Ala Cys Gln Val Gln Trp His Gly Val Trp Asp Arg Val Thr Leu      50 55 60 Asn Ile His Pro Val Arg Arg Arg Thr Trp Glu Leu Ile Arg Lys His  65 70 75 80 Ile Arg        

Claims (12)

A fragment comprising an anti-LH antibody or an antigen-binding region thereof, which specifically binds to an LH (Luteinizing hormone) protein.
The method according to claim 1,
The anti-LH antibody may be any one selected from the group consisting of HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: A heavy chain variable region comprising the heavy chain variable region; And LCDR3 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, LH antibody or a fragment thereof comprising an antigen-binding region thereof.
The method according to claim 1,
Wherein the anti-LH antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. A fragment containing a region.
The method according to claim 1,
The anti-LH antibody may be any one selected from the group consisting of HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: A heavy chain variable region comprising the heavy chain variable region; And LCDR3 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, LH antibody or a fragment thereof comprising an antigen-binding region thereof.
The method according to claim 1,
Wherein the anti-LH antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. A fragment containing a region.
A composition for the diagnosis of estrus of a mammal other than a human, which comprises the anti-LH antibody of any one of claims 1 to 5 or a fragment comprising the antigen binding region thereof as an active ingredient.
An estrogenic diagnostic kit for a mammal, excluding human, comprising the diagnostic composition of claim 6.
8. The method of claim 7,
Wherein the diagnostic kit is an enzyme-linked immunospecific assay (ELISA) diagnostic kit.
9. The method of claim 8,
Wherein the enzyme-linked immunosorbent assay (ELISA) diagnostic kit is a sandwich enzyme-linked immunosorbent assay (Sandwich ELISA) diagnostic kit.
In a biological sample, the method comprises measuring the level of an antigen-antibody reaction between a fragment comprising an LH (Luteinizing hormone) protein and an anti-LH antibody of any of claims 1 to 5 or an antigen- Methods for diagnosing ovulation of mammals other than humans.
11. The method of claim 10,
Wherein the biological sample is any one selected from the group consisting of whole blood, serum, plasma, and mucus.
11. The method of claim 10,
The step of measuring the antigen-antibody reaction level may be performed by an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), a Western blot, an immunoprecipitation method, an immunohistochemical staining method, And an enzyme substrate coloring method, wherein the method is characterized in that the measurement is carried out using any one of the methods selected from the group consisting of an enzyme substrate coloring method and an enzyme substrate coloring method.

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