Antibody pairs binding to mature prolactin

Abstract

The present invention provides an antibody pair that binds human mature prolactin, comprising: a monoclonal antibody 1 or an antigen-binding fragment thereof that binds to an N-terminal region of human mature prolactin, and a monoclonal antibody 2 or an antigen-binding fragment thereof that binds to a C-terminal region of human mature prolactin. The present invention also provides use of the antibody pair of the present invention in manufacturing a kit for measuring prolactin in a biological sample. The present invention also provides a monoclonal antibody or an antigen-binding fragment thereof that binds to an N-terminal region of human mature prolactin and a monoclonal antibody or an antigen-binding fragment thereof that binds to a C-terminal region of human mature prolactin, respectively. The present invention also provides a method for measuring prolactin in a biological sample using the antibody pair of the present invention. The present invention also provides a method for producing a monoclonal antibody in the antibody pair of the present invention.

Description

[Technical Field]

[0001] This invention relates to an antibody pair binding to human mature prolactin, comprising: monoclonal antibody 1 or an antigen-binding fragment thereof binding to the N-terminal region of human mature prolactin, and monoclonal antibody 2 or an antigen-binding fragment thereof binding to the C-terminal region of human mature prolactin. The invention also relates to the use of the antibody pair of the invention in the manufacture of a kit for determining prolactin in a biological sample. The invention further relates to a monoclonal antibody or an antigen-binding fragment thereof binding to the N-terminal region of human mature prolactin and a monoclonal antibody or an antigen-binding fragment thereof binding to the C-terminal region of human mature prolactin, respectively. The invention also relates to a method for determining prolactin in a biological sample using the antibody pair of the invention. The invention further relates to a method for generating the monoclonal antibody in the antibody pair of the invention. [Background Technology]

[0002] Prolactin (PRL), also known as "prolactin" or "pituitary prolactin," is a polypeptide hormone secreted by acidophilic cells in the anterior pituitary gland. Mature (signal peptide-free) prolactin consists of 199 amino acids (SEQ ID NO: 1) with a molecular weight of approximately 23 kDa. Since PRL is the only biochemical indicator of hyperprolactinemia, it is used clinically for its diagnosis. Increased PRL levels can also lead to oligomenorrhea or even amenorrhea, infertility, recurrent miscarriage, osteopenia, and galactorrhea in women, and impotence and infertility in men. When tumors are present, it can cause headaches, visual field defects, nausea, vomiting, abnormal appetite, drowsiness, and coma. PRL exists in the human body mainly in the following three forms:

[0003] • Monomeric PRL: accounts for approximately 80%-90% of total PRL, and has the highest biological and immunomodulatory activity;

[0004] • Dimer PRL: Molecular weight approximately 50KD, accounting for about 8%-20% of total PRL, with low biological and immunomodulatory activity;

[0005] • Macro PRL: It is a complex of autoantibody IgG and PRL, with a molecular weight greater than 100KD, accounting for about 1-5% of the total PRL, and has low or no biological and immunological activity.

[0006] Due to the polymorphism, varying proportions, and differences in activity of prolactin (PRL) in the bloodstream, clinical manifestations often contradict serum PRL levels. In particular, because giant PRL has a large molecular weight, it cannot freely pass through capillary walls and cannot bind to target receptors, resulting in low or no biological activity in vivo. However, due to its long half-life, it easily accumulates in the bloodstream, creating a false impression of increased PRL. The problem is that currently available commercially available PRL assays cannot specifically measure monomeric PRL, easily misdetecting the nearly inactive giant PRL accumulated in the bloodstream as biologically active monomeric PRL. This could lead to misdiagnosis of hyperprolactinemia in healthy individuals, and even mistreatment.

[0007] In this field, methods have also been developed to eliminate the aforementioned interference caused by giant PRL, including gel filtration chromatography and polyethylene glycol (PEG) precipitation.

[0008] The principle of gel filtration chromatography is to separate monomeric PRL from total PRL through gel filtration chromatography, thereby determining the true monomeric PRL level in human blood circulation. This method is reliable and is considered the "gold standard" for completely eliminating the interference caused by giant PRL. However, it also has obvious disadvantages such as being cumbersome and time-consuming to operate, requiring specific equipment resources, demanding high technical skills, and being difficult to carry out in the laboratory.

[0009] The principle of the PEG precipitation method is to precipitate larger molecular weight PRLs from the total PRL using PEG precipitation, and then remove the precipitated large PRLs by centrifugation and collecting the supernatant. This method is faster than gel filtration chromatography, but it is also cumbersome and slightly less reliable than gel filtration chromatography.

[0010] The present invention aims to develop a method for the more rapid and specific determination of monomer PRL than gel filtration chromatography and PEG precipitation. [Summary of the Invention]

[0011] This invention includes the following:

[0012] 1. Antibody pairs that bind to human prolactin, among which...

[0013] The antibody pair includes:

[0014] Monoclonal antibody 1 or its antigen-binding fragment, which binds to the N-terminal region of human mature prolactin, and

[0015] Monoclonal antibody 2 or its antigen-binding fragment, which binds to the C-terminal region of human mature prolactin, and

[0016] The human maturation prolactin consists of the amino acid sequence shown in SEQ ID NO: 1.

[0017] 2. The antibody pair according to the above embodiments, wherein...

[0018] The monoclonal antibody 1 or its antigen-binding fragment binds to the region of amino acids 1 to 13 of SEQ ID NO: 1, and / or

[0019] The monoclonal antibody 2 or its antigen-binding fragment binds to the region of amino acids 183 to 199 of SEQ ID NO: 1.

[0020] 3. The antibody pair according to the above embodiments, wherein...

[0021] In the region of amino acids at positions 1 to 13 of SEQ ID NO: 1, the portion outside positions 1 to 3 and positions 10 to 13 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions, and / or

[0022] In the region of amino acids at positions 183 to 199 of SEQ ID NO: 1, the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions.

[0023] 4. The antibody pair according to the above embodiments, wherein...

[0024] The monoclonal antibody 1 or its antigen-binding fragment comprises:

[0025] Heavy chain variable region, which contains:

[0026] CDR1 contains the amino acid sequence of GFIFX1WYV.

[0027] CDR2, which contains the amino acid sequence ISGGX2SHT, and

[0028] CDR3, which contains the amino acid sequence TRHSPYDHGDFGY, and

[0029] The light chain variable region, which includes:

[0030] CDR1 contains the amino acid sequence QTLVHSLGNTY.

[0031] CDR2, which contains the amino acid sequence of KVSIA, and

[0032] CDR3, which contains the amino acid sequence SQTTHVPTF; and / or

[0033] The monoclonal antibody 2 or its antigen-binding fragment comprises:

[0034] Heavy chain variable region, which contains:

[0035] CDR1 contains the amino acid sequence of GASITSDYA.

[0036] CDR2, which contains the amino acid sequence ISFGGGHT, and

[0037] CDR3, which contains the amino acid sequence of ARHSPNDHGDTNF, and

[0038] The light chain variable region, which includes:

[0039] CDR1 contains the amino acid sequence QDISX3NG.

[0040] CDR2, which contains the amino acid sequence of KTSX4V, and

[0041] CDR3 contains the amino acid sequence of QQGNTVPLT.

[0042] in,

[0043] X1 is selected from: S, T, A, or G;

[0044] X2 is selected from: D, E, Q, or N;

[0045] X3 is selected from: D or E; and / or

[0046] X4 is selected from: G, A, S or T.

[0047] 5. The antibody pair according to the above embodiments, wherein...

[0048] X1 is either S or A;

[0049] X2 is either D or Q;

[0050] X3 is D or E; and / or

[0051] X4 is G, A, or S.

[0052] 6. The antibody pair according to the above embodiments, wherein...

[0053] The monoclonal antibody 1 or its antigen-binding fragment comprises:

[0054] The heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 5 and 32–46, and

[0055] The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 10; and / or

[0056] The monoclonal antibody 2 or its antigen-binding fragment comprises:

[0057] The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and

[0058] The light chain variable region contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20 and 53-59.

[0059] 7. Use of the antibody pair according to the above embodiments in the manufacture of a kit for determining prolactin in biological samples.

[0060] 8. In the use described in the above embodiments, the prolactin is determined by a double-antibody sandwich chemiluminescent immunoassay.

[0061] 9. The application according to the above embodiments, wherein...

[0062] The monoclonal antibody 1 or its antigen-binding fragment is used as a capture antibody, and

[0063] The monoclonal antibody 2 or its antigen-binding fragment is used as the detection antibody.

[0064] 10. The use according to the above embodiments, wherein the determination does not depend on the isolation of monomeric prolactin from biological samples.

[0065] 11. A monoclonal antibody or its antigen-binding fragment that binds to the N-terminal region of human prolactin, comprising:

[0066] Heavy chain variable region, which contains:

[0067] CDR1 contains the amino acid sequence of GFIFX1WYV.

[0068] CDR2, which contains the amino acid sequence ISGGX2SHT, and

[0069] CDR3, which contains the amino acid sequence TRHSPYDHGDFGY, and

[0070] The light chain variable region, which includes:

[0071] CDR1 contains the amino acid sequence QTLVHSLGNTY.

[0072] CDR2, which contains the amino acid sequence of KVSIA, and

[0073] CDR3 contains the amino acid sequence SQTTHVPTF.

[0074] in,

[0075] X1 is selected from: S, T, A, or G;

[0076] X2 is selected from: D, E, Q or N.

[0077] 12. The monoclonal antibody or its antigen-binding fragment according to the above embodiments, wherein...

[0078] X1 is either S or A; and

[0079] X2 is either D or Q.

[0080] 13. The monoclonal antibody or its antigen-binding fragment according to the above embodiments, comprising:

[0081] The heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 5 and 32–46, and

[0082] The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 10.

[0083] 14. Use of the monoclonal antibody or antigen-binding fragment thereof as described in the above embodiments in the manufacture of a kit for determining prolactin in biological samples.

[0084] 15. A monoclonal antibody or its antigen-binding fragment that binds to the C-terminal region of human prolactin, comprising:

[0085] Heavy chain variable region, which contains:

[0086] CDR1 contains the amino acid sequence of GASITSDYA.

[0087] CDR2, which contains the amino acid sequence ISFGGGHT, and

[0088] CDR3, which contains the amino acid sequence of ARHSPNDHGDTNF, and

[0089] The light chain variable region, which includes:

[0090] CDR1 contains the amino acid sequence QDISX3NG.

[0091] CDR2, which contains the amino acid sequence of KTSX4V, and

[0092] CDR3 contains the amino acid sequence of QQGNTVPLT.

[0093] in,

[0094] X3 is selected from: D or E; and

[0095] X4 is selected from: G, A, S or T.

[0096] 16. The monoclonal antibody or its antigen-binding fragment according to the above embodiments, wherein...

[0097] X3 is either D or E; and

[0098] X4 is G, A, or S.

[0099] 17. The monoclonal antibody or its antigen-binding fragment according to the above embodiments, comprising:

[0100] The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and

[0101] The light chain variable region contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20 and 53-59.

[0102] 18. Use of the monoclonal antibody or antigen-binding fragment thereof as described in the above embodiments in the manufacture of a kit for determining prolactin in biological samples.

[0103] 19. A method for determining prolactin in biological samples, comprising:

[0104] (a) Contacting a biological sample suspected of containing prolactin with an antibody pair according to any one of claims 1 to 6, and

[0105] (b) Quantify the prolactin in the biological sample based on the signal generated in step (a).

[0106] 20. The method according to the above embodiments, wherein prolactin in the biological sample is quantified based on the chemiluminescent signal generated in step (a).

[0107] 21. The method according to the above embodiments, wherein...

[0108] (1) Prior to step (a), the biological sample is pre-divided into:

[0109] (1A) Part 1, which is unprocessed, and

[0110] (1B) The second part is the supernatant obtained by mixing the biological sample with an equal volume of a solution containing polyethylene glycol, shaking, and then centrifuging.

[0111] (2) The concentrations of prolactin in the first and second portions are determined by steps (a) and (b) and respectively used as the first and second concentrations; and

[0112] (3) The recovery rate of prolactin after treatment in (1B) is calculated using the following formula:

[0113]

[0114] The recovery rate is greater than 60%, preferably greater than 80%, and more preferably greater than 90%.

[0115] 22. A method for producing monoclonal antibodies, comprising immunizing animals with the following peptides:

[0116] The N-terminal region of human prolactin, and / or

[0117] The C-terminal region of human prolactin,

[0118] The human maturation prolactin consists of the amino acid sequence shown in SEQ ID NO: 1.

[0119] 23. The method according to the above embodiments, wherein...

[0120] The N-terminal region of the human prolactin is the region of amino acids 1 to 13 of SEQ ID NO: 1, and / or

[0121] The C-terminal region of the human maturation prolactin is the region of amino acids 183 to 199 of SEQ ID NO: 1.

[0122] 24. The method according to the above embodiments, wherein...

[0123] In the region of amino acids at positions 1 to 13 of SEQ ID NO: 1, the portion outside positions 1 to 3 and positions 10 to 13 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions, and / or

[0124] In the region of amino acids at positions 183 to 199 of SEQ ID NO: 1, the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions.

[0125] [Technical Effects]

[0126] Antibody kits made from antibody pairs consisting of antibodies binding to the N-terminal region of mature human PRL (especially the region aa1-13 of SEQ ID NO: 1, where residues aa1-3 and aa10-13 are key residues) and the C-terminal region (especially the region aa183-199 of SEQ ID NO: 1, where residues aa184-186 and aa199 are key residues) are less susceptible to interference from giant PRLs present in the sample when measuring the concentration of monomeric PRLs in the sample. That is, giant PRLs are less likely to be misidentified as monomeric PRLs, thus having a significant advantage in the specific determination of monomeric PRLs in the sample. [Brief Description of the Attached Image]

[0127]

【Figure 1a A schematic diagram of the three-dimensional structure of prolactin.

[0128]

【 Figure 1b Design of multiple consecutive peptide fragments of approximately 30 aa (30-35 aa) covering the full sequence of mature human PRL.

[0129]

【 Figure 2A Comparison of this product with commercially available products based on the recovery rate of PRL after PEG precipitation.

[0130]

【 Figure 2B Comparison of this product with commercially available products based on a dotted line graph of the concentration of monomer PRL in samples obtained by gel filtration chromatography.

[0131]

【 Figure 2C Comparison between this product and the reference product.

[0132]

【 Figure 3 PRL assay results for variants 1-4.

Detailed Implementation Methods

[0133] Unless otherwise specified, the terms used in this specification are used in the sense commonly used in the art.

[0134] The monoclonal antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody or antigen-binding fragment thereof having the CDR or heavy chain variable region / light chain variable region described in this specification. The antibody or antibody fragment may also be derived from any class (e.g., IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecules, for example, from any species including mice, rats, rabbits, pigs, hamsters, camels, alpacas, goats, or humans. In the present invention, the antigen-binding fragment may be selected from: Fab, Fab', F(ab')2, scFv, and dsFv, etc., whose definitions follow those commonly used in the art.

[0135] The monoclonal antibodies in the antibody pairs of the present invention can be manufactured using any monoclonal antibody manufacturing process well known in the art. In specific embodiments, the monoclonal antibodies in the antibody pairs of the present invention can be recovered from cloned hybridoma cells using methods such as protein A / G affinity chromatography and ion exchange.

[0136] [The epitopes bound to the monoclonal antibodies in PRL and the antibody pairs of the present invention]

[0137] The GenBank accession number ACT52574.1 (https: / / www.ncbi.nlm.nih.gov / protein / ACT52574.1) contains the amino acid sequence of human (Homo sapiens) prolactin precursor, which is 227 amino acids long (227 aa). Among them, aa1-28 are the amino acid sequence of the signal peptide, and aa29-227 are the amino acid sequence of mature human PRL (SEQ ID NO: 1).

[0138] [Amino acid sequence of mature human PRL (SEQ ID NO: 1)]

[0139]

[0140] This invention is the first to discover that by using a sandwich immunoassay consisting of antibody 1 and antibody 2, which respectively bind to the N-terminal region or a conserved variant thereof and the C-terminal region or a conserved variant thereof of mature human PRL (SEQ ID NO: 1), monomeric PRL can be bound from total PRL in the blood circulation with significantly higher specificity.

[0141] In one embodiment, antibody 1 can bind to the region of amino acids 1 to 13 of SEQ ID NO: 1 (SEQ ID NO: 2), and antibody 2 can bind to the region of amino acids 183 to 199 of SEQ ID NO: 1 (SEQ ID NO: 3).

[0142] [human mature PRL aa1~13 (SEQ ID NO: 2)]

[0143] LPI CPGGAA RCQV

[0144] In one embodiment, in the region of amino acids at positions 1 to 13 of SEQ ID NO: 1 (SEQ ID NO: 2), the portion outside positions 1 to 3 and positions 10 to 13 of SEQ ID NO: 1 (the underlined amino acid residues mentioned above) may have one or more conserved amino acid insertions, deletions, or substitutions.

[0145] [human mature PRL aa183~199 (SEQ ID NO: 3)]

[0146] D NYL KLLKCR IIHNNN C

[0147] In one embodiment, in the region of amino acids at positions 183 to 199 of SEQ ID NO: 1 (SEQ ID NO: 3), the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 (the underlined amino acid residues mentioned above) may have one or more conserved amino acid insertions, deletions, or substitutions.

[0148] In one embodiment, the amino acid sequences of the CDRs of the heavy chain variable region and the light chain variable region of antibody 1 and antibody 2 are as follows:

[0149]

[0150] in,

[0151] X1 is selected from: S, T, A or G, preferably S or A;

[0152] <![CDATA[X1]]> Heavy chain CDR1 in antibody 1 SEQ ID NO: 24 S GFIFSWYV SEQ ID NO: 6 T GFIFTWYV SEQ ID NO: 25 A GFIFAWYV SEQ ID NO: 26 G GFIFGWYV SEQ ID NO: 27

[0153] X2 is selected from: D, E, Q or N, preferably D or Q;

[0154] <![CDATA[X2]]> Heavy chain CDR2 in antibody 1 SEQ ID NO: 28 D ISGGDSHT SEQ ID NO: 7 E ISGGESHT SEQ ID NO: 29 Q ISGGQSHT SEQ ID NO: 30 N ISGGNSHT SEQ ID NO: 31

[0155] X3 is selected from: D or E; and

[0156] <![CDATA[X3]]> CDR1 in antibody 2 (light chain) SEQ ID NO: 47 D QDISDNG SEQ ID NO: 21 E QDISENG SEQ ID NO: 48

[0157] X4 is selected from: G, A, S or T.

[0158]

[0159]

[0160] thus,

[0161] The specific sequence of the variable region of antibody 1 can be as follows:

[0162] Antibody 1 <![CDATA[X1]]> <![CDATA[X2]]> Heavy chain variable region Light chain variable region Antibody 1a S D SEQ ID NO: 5 SEQ ID NO: 10 Antibody 1b S E SEQ ID NO: 32 SEQ ID NO: 10 Antibody 1c S Q SEQ ID NO: 33 SEQ ID NO: 10 Antibody 1d S N SEQ ID NO: 34 SEQ ID NO: 10 Antibody 1e T D SEQ ID NO: 35 SEQ ID NO: 10 Antibody 1f T E SEQ ID NO: 36 SEQ ID NO: 10 1g antibody T Q SEQ ID NO: 37 SEQ ID NO: 10 Antibody 1h T N SEQ ID NO: 38 SEQ ID NO: 10 Antibody 1i A D SEQ ID NO: 39 SEQ ID NO: 10 Antibody 1j A E SEQ ID NO: 40 SEQ ID NO: 10 Antibody 1k A Q SEQ ID NO: 41 SEQ ID NO: 10 Antibody 1l A N SEQ ID NO: 42 SEQ ID NO: 10 Antibody 1m G D SEQ ID NO: 43 SEQ ID NO: 10 Antibody 1n G E SEQ ID NO: 44 SEQ ID NO: 10 Antibody 1o G Q SEQ ID NO: 45 SEQ ID NO: 10 Antibody 1p G N SEQ ID NO: 46 SEQ ID NO: 10

[0163] *As long as the sequences of the same HCDR1-3 and LCDR1-3 are contained, the sequences of the heavy chain variable region and the light chain variable region are not limited to the above sequences.

[0164] The specific sequence of the variable region of antibody 2 can be as follows:

[0165] Antibody 2 <![CDATA[X3]]> <![CDATA[X4]]> Heavy chain variable region Light chain variable region Antibody 2a D G SEQ ID NO: 15 SEQ ID NO: 20 Antibody 2b D A SEQ ID NO: 15 SEQ ID NO: 53 Antibody 2c D S SEQ ID NO: 15 SEQ ID NO: 54 Antibody 2d D T SEQ ID NO: 15 SEQ ID NO: 55 Antibody 2e E G SEQ ID NO: 15 SEQ ID NO: 56 Antibody 2f E A SEQ ID NO: 15 SEQ ID NO: 57 antibody 2g E S SEQ ID NO: 15 SEQ ID NO: 58 Antibody 2h E T SEQ ID NO: 15 SEQ ID NO: 59

[0166] *As long as the sequences of the same HCDR1-3 and LCDR1-3 are contained, the sequences of the heavy chain variable region and the light chain variable region are not limited to the above sequences.

[0167] In one embodiment, antibody 1 is a capture antibody and antibody 2 is a detection antibody; in another embodiment, antibody 2 is a capture antibody and antibody 1 is a detection antibody. In a preferred embodiment, antibody 1 is a capture antibody and antibody 2 is a detection antibody.

[0168] In one embodiment, antibody 1 of the present invention can be obtained by immunizing animals with the following polypeptide:

[0169] • A peptide containing the amino acid sequence shown in SEQ ID NO: 2, or

[0170] • A peptide comprising, compared to the amino acid sequence shown in SEQ ID NO: 2, a portion other than aa1-3 and aa10-13 containing one or more conserved amino acid insertions, deletions or substitutions.

[0171] In one embodiment, antibody 1 of the present invention is obtained by immunizing an animal with the following polypeptide:

[0172] • A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or

[0173] • A peptide consisting of an amino acid sequence other than aa1-3 and aa10-13 that includes one or more conserved amino acid insertions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 2.

[0174] In one embodiment, antibody 2 of the present invention is obtained by immunizing animals with the following polypeptide:

[0175] • A peptide containing the amino acid sequence shown in SEQ ID NO: 3, or

[0176] • A peptide comprising, compared to the amino acid sequence shown in SEQ ID NO: 3, a portion other than aa2–4 and aa17 (as per SEQ ID NO: 3) containing one or more conserved amino acid insertions, deletions, or substitutions.

[0177] In one embodiment, antibody 2 of the present invention is obtained by immunizing animals with the following polypeptide:

[0178] • A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, or

[0179] • A peptide consisting of an amino acid sequence other than aa2-4 and aa17 (as per SEQ ID NO:3) that includes one or more conserved amino acid insertions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO:3.

[0180] In one embodiment, the present invention relates to a method for generating monoclonal antibodies, comprising immunizing an animal with the N-terminal region and / or the C-terminal region of human mature prolactin, said human mature prolactin comprising the amino acid sequence shown in SEQ ID NO: 1. In a further embodiment, the N-terminal region of said human mature prolactin is the region of amino acids 1 to 13 of SEQ ID NO: 1, and / or the C-terminal region of said human mature prolactin is the region of amino acids 183 to 199 of SEQ ID NO: 1. In a further embodiment, in the region of amino acids 1 to 13 of SEQ ID NO: 1, the portion other than positions 1 to 3 and 10 to 13 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions, and / or in the region of amino acids 183 to 199 of SEQ ID NO: 1, the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions.

[0181] In the above embodiments, the insertion, deletion or substitution of one or more conserved amino acids includes, but is not limited to, insertion, deletion or substitution of one or more, two or more, three or more, four or more or five or more conserved amino acids.

[0182] In one embodiment, the invention further relates to a polynucleotide encoding the heavy chain, heavy chain variable region, light chain, and light chain variable region of each antibody in the antibody pair described above. In one embodiment, the invention further relates to an expression vector comprising the aforementioned polynucleotides.

[0183] In this invention, the heavy / light chain variable region or its CDR may "comprise" the amino acid sequence shown in SEQ ID NO: ..., or consist of the amino acid sequence shown in SEQ ID NO: ... Similarly, the nucleotide sequence encoding the heavy / light chain variable region or its CDR may "comprise" the nucleotide sequence shown in SEQ ID NO: ..., or consist of the nucleotide sequence shown in SEQ ID NO: ...

[0184] [PRL Measurement]

[0185] In one embodiment, the antibody pair of the present invention is used to determine prolactin in a biological sample. In a further embodiment, the determination of prolactin in a biological sample can be performed by various methods known in the art. In a preferred embodiment, the determination of prolactin in a biological sample can be performed by a double-antibody sandwich chemiluminescence immunoassay (CLIA).

[0186] The basic principle of the double antibody sandwich method is as follows: one antibody immobilized on a solid-phase support is used as a capture antibody to capture the antigen to be detected, and another antibody labeled with an enzyme is used as a detection antibody to bind to the antigen to be detected captured by the capture antibody. Then, the colorimetric / chemiluminescent substrate of the enzyme is added to develop color / chemiluminescence. The intensity of the color development / chemiluminescence is positively correlated with the concentration of the antigen to be detected.

[0187] In this invention, the solid-phase support used to immobilize the captured antibody is not particularly limited, and may be, for example, but not limited to, magnetic beads, polyvinyl chloride, polystyrene, polyacrylamide, and cellulose.

[0188] In this invention, the enzyme used to label the detection antibody is not particularly limited, and may be, for example, but not limited to, alkaline phosphatase, horseradish peroxidase, etc.

[0189] In this invention, the choice of the chromogenic / chemiluminescent substrate depends on the enzyme used to label the detection antibody. In one embodiment, when labeling the detection antibody with alkaline phosphatase, any substrate that can be acted upon by alkaline phosphatase to produce chromogenic / chemiluminescent results can be used, such as, but not limited to, 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane (AMPPD). In one embodiment, AMPPD loses a phosphate group under the action of alkaline phosphatase to generate an unstable intermediate product, which generates a methyl m-oxobenzoate anion through intramolecular electron transfer. The methyl m-oxobenzoate anion in the excited state returns to the ground state, producing chemiluminescence. In another embodiment, when labeling the detection antibody with horseradish peroxidase, any substrate that can be acted upon by horseradish peroxidase to produce chromogenic results can be used, such as, but not limited to, 3,3',5,5'-tetramethylbenzidine (TMB).

[0190] In one embodiment, the present invention relates to a method for determining prolactin in a biological sample, comprising:

[0191] (a) Contacting a biological sample suspected of containing prolactin with the antibody pair of the present invention, and

[0192] (b) Quantify the prolactin in the biological sample based on the signal generated in step (a).

[0193] In a preferred embodiment, prolactin in the biological sample is quantified based on chemiluminescence signals.

[0194] In a further implementation,

[0195] (1) Prior to step (a), the biological sample is pre-divided into:

[0196] (1A) Part 1, which is unprocessed, and

[0197] (1B) The second part is the supernatant obtained by mixing the biological sample with an equal volume of a solution containing polyethylene glycol, shaking, and then centrifuging.

[0198] (2) The concentrations of prolactin in the first and second portions are determined by steps (a) and (b) and respectively used as the first and second concentrations; and

[0199] (3) The recovery rate of prolactin after treatment in (1B) is calculated using the following formula:

[0200]

[0201] The recovery rate is greater than 60%, preferably greater than 80%, and more preferably greater than 90%.

[0202] In one implementation, the biological sample for measuring PRL may be selected from whole blood, serum, or plasma, but is not limited thereto.

[0203] In one embodiment, the present invention relates to the use of the above-described antibody pairs and each antibody in the antibody pairs in the manufacture of a kit for determining prolactin in biological samples.

[0204]

Example

[0205] [Example 1: Obtaining a monoclonal antibody and determining the epitope it binds to on mature human PRL]

[0206] (1.1) Obtaining monoclonal antibodies

[0207] Human mature PRL (SEQ ID NO: 1) was injected into Balb / c mice as an immunogen to stimulate their immune system to produce antibodies. Antibody-producing B cells were isolated from the spleen of the immunized mice. The isolated B cells were fused with SP2 / 0 myeloma cells to form hybridoma cells using standard methods. Monoclonal antibodies that bind well to human mature PRL (as antigen) were screened using limiting dilution and cloned. Monoclonal antibodies were recovered from the cloned hybridoma cells using affinity chromatography with protein A.

[0208] The binding specificity of the obtained monoclonal antibodies to human mature PRL was tested, and antibodies with better binding specificity to human mature PRL were screened. Two of these antibodies were named "Antibody 1" and "Antibody 2" (more specifically "Antibody 1a" and "Antibody 2a"), and their amino acid sequences of heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 are shown in the table below.

[0209]

[0210]

[0211]

[0212] (1.2) Determination of the epitope on the mature human PRL bound by the monoclonal antibody

[0213] For the antibodies with good binding specificity to human mature PRL obtained in (1.1) (especially antibody 1 and antibody 2 mentioned above), the epitopes on human mature PRL bound by these antibodies are analyzed as follows:

[0214] (a) such as Figure 1b As shown, based on the amino acid sequence of mature human PRL (SEQ ID NO: 1), multiple consecutive peptide fragments of approximately 30 aa (30-35 aa) each, covering its full sequence, were synthesized, with approximately 10 amino acids overlapping between adjacent approximately 30 aa peptide fragments. These approximately 30 aa peptide fragments were then used to competitively bind to the aforementioned antibody 1 and antibody 2 against the full-length mature human PRL. The results showed that:

[0215] Antibody 1 preferentially binds to the N-terminal aa1-30 peptide fragment corresponding to the mature human PRL (SEQ ID NO: 1);

[0216] Antibody 2 preferentially binds to the C-terminal aa176-199 peptide fragment corresponding to the mature human PRL (SEQ ID NO: 1).

[0217] (b) Furthermore, based on the amino acid sequences of the N-terminal aa1–30 peptide fragment and the C-terminal aa1–199 peptide fragment of the mature human PRL, several shorter peptide fragments (e.g., 15aa, 13aa, 10aa, etc.) capable of covering them were synthesized, with several amino acid overlaps between adjacent peptide fragments. These shorter peptide fragments were then used to competitively bind to antibodies 1 and 2 against the N-terminal aa1–30 peptide fragment and the C-terminal aa1–199 peptide fragment of the mature human PRL. It was found that:

[0218] • Antibody 1 preferentially binds to the N-terminal aa1-13 peptide fragments corresponding to the mature human PRL (SEQ ID NO: 1), that is, antibody 1 binds to the aa1-13 (SEQ ID NO: 2) region of the mature human PRL (SEQ ID NO: 1);

[0219] Antibody 2 preferentially binds to the C-terminal aa183-199 peptide fragment corresponding to the mature human PRL (SEQ ID NO: 1), that is, antibody 2 binds to the region of aa183-199 (SEQ ID NO: 3) of the mature human PRL (SEQ ID NO: 1).

[0220] (c) Manufacturing mutants of human mature PRL (SEQ ID NO: 1) by deletion / substitution of one or more amino acids, specifically the N-terminal aa1-13 peptide fragment and the C-terminal aa183-199 peptide fragment. The binding affinity of these deletion / substitution mutants to antibodies 1 and 2 was determined, revealing that:

[0221] • The aa1-3 and aa10-13 residues of mature human PRL (SEQ ID NO: 1) (underlined amino acid residues in the following sequences) are key residues in the epitope that antibody 1 binds to (aa1-13 of SEQ ID NO: 1 (SEQ ID NO: 2)):

[0222] [human mature PRL aa1~13 (SEQ ID NO: 2)]

[0223] LPI CPGGAA RCQV

[0224] • The residues aa184–186 and aa199 of mature human PRL (SEQ ID NO: 1) (underlined amino acid residues in the following sequences) are key residues in the epitope that antibody 2 binds to (aa183–199 of SEQ ID NO: 1 (SEQ ID NO: 3)):

[0225] [human mature PRL aa183~199 (SEQ ID NO: 3)]

[0226] D NYL KLLKCRIIHNNN C

[0227] Antibody 1 and antibody 2 were used for further experiments.

[0228] [Example 2: Determination of monomeric PRL in a sample using an antibody kit]

[0229] (2.1) Antibody kit

[0230] Antibody kits, referred to as "Product 1" and "Product 2", were prepared using antibodies 1 and 2 obtained in Example 1, as shown in the table below.

[0231]

[0232] As a control antibody kit, an antibody pair consisting of a capture antibody and a detection antibody, which binds to the non-N-terminal and non-C-terminal regions, is used and is referred to as a "control".

[0233]

[0234] As comparative antibody kits, commercially available products 1 (Abbott), 2 (Roche), and 3 (Beckman) with the following compositions were used.

[0235] In the above antibody kit,

[0236] • The above-mentioned capture antibody was coated onto superparamagnetic microparticle beads to serve as a capture reagent.

[0237] • The above-mentioned detection antibodies are labeled with alkaline phosphatase and used as detection reagents.

[0238] (2.2) Sample

[0239] Initial samples: Human serum samples from clinical settings were used as the initial samples. These samples included: positive samples with PRL concentrations outside the normal range, normal samples with PRL concentrations within the normal range, and suspected samples with PRL concentrations possibly affected by giant PRL interference.

[0240] Samples obtained by gel filtration chromatography: The initial sample was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and passed through an Agilent advanceBio SEC 300A column on a liquid chromatograph (Cat.1260, Agilent) to collect only the monomer PRL fraction as the sample obtained by gel filtration chromatography.

[0241] Samples precipitated by PEG: The initial sample was mixed with 25% PEG 6000 solution at a volume ratio of 1:1 and shaken. The mixture was then centrifuged at 8000 rpm for 5 min and the supernatant was collected as the sample precipitated by PEG. (2.3) Determination of monomer PRL in the sample (2.3.1) The PRL in the sample was determined using this product 1 and this product 2, as well as reference product 1 and reference product 2.

[0242] The above samples were measured in a chemiluminescence analyzer (Mindray) using product 1 and product 2, as well as reference product 1 and reference product 2, according to the standard procedure.

[0243] (2.3.2) The PRL in the samples was determined using commercially available products 1, 2 and 3.

[0244] Commercially available products 1, 2, and 3 were loaded onto their respective instruments, and the samples were measured according to their instructions.

[0245] (2.4) Evaluation criteria (2.4.1) Determination of samples precipitated with PEG

[0246] The recovery rate of PRL for each sample after PEG precipitation was calculated using the following formula:

[0247]

[0248] Based on the following recovery rates, all PEG-precipitated samples (100%) were divided into:

[0249] • Samples with a recovery rate >60% are defined as: samples whose PRL determination results are not affected by giant PRL (blue bar, percentage x%).

[0250] • Samples with a recovery rate of <40% are defined as: samples whose PRL determination results are affected by giant PRL (orange column, percentage y%).

[0251] • Samples with a recovery rate between 40% and 60% are defined as those that require further investigation using other methods (such as gel filtration chromatography or clinical manifestations) to determine whether the PRL measurement results are affected by giant PRL (gray column, percentage z%).

[0252] Where x + y + z = 100.

[0253] (2.4.2) Determination of samples obtained by gel filtration chromatography

[0254] A dotted-line graph was created by connecting the PRL concentration values ​​measured from each sample (sample number = 1, 2, ..., n, where n = sample size) with line segments. The similarity between the dotted-line graph obtained from measuring the initial sample using each antibody kit and the dotted-line graph obtained from measuring the monomeric PRL sample obtained by gel filtration chromatography was compared. The closer the dotted-line graph obtained from measuring the initial sample using each antibody kit is to the dotted-line graph obtained from measuring the monomeric PRL sample obtained by gel filtration chromatography, the higher the accuracy of the antibody kit in measuring monomeric PRL in the sample, i.e., the less affected by macroprolactin interference. Those skilled in the art will understand that samples precipitated with PEG, having already removed macroprolactin interference, will yield similar monomeric PRL concentrations when measured using different kits than the initial sample.

[0255] (2.5) Results

[0256] (2.5.1) Results of this product vs. commercially available products (2.5.1.1) PEG-precipitated samples

[0257] like Figure 2A As shown,

[0258] • Among the PEG-precipitated samples tested with this product 1, the percentage of samples whose PRL determination results were not affected by giant PRL (up to 92.0%) was the highest, the percentage of samples whose PRL determination results were affected by giant PRL (only 1.0%) and the percentage of samples whose PRL determination results required further determination by other means (only 7.0%) were the lowest.

[0259] • Among the PEG-precipitated samples tested with this product 2, the percentage of samples whose PRL determination results were not affected by giant PRL (up to 88.0%) was the second highest, the percentage of samples whose PRL determination results were affected by giant PRL (only 5.0%) and the percentage of samples whose PRL determination results needed to be further determined by other means (only 7.0%) were the second lowest.

[0260] In contrast, among the PEG-precipitated samples measured using commercially available products 1, 2, and 3, the percentages of samples whose PRL measurements were not affected by giant PRL (only 71.0%, 78.0%, and 75.0%, respectively) were significantly lower, while the percentages of samples whose PRL measurements were affected by giant PRL (as high as 18.0%, 12.0%, and 13.0%, respectively) and the percentages of samples requiring further investigation to determine whether PRL measurements were affected by giant PRL (as high as 11.0%, 10.0%, and 12.0%, respectively) were significantly higher.

[0261] (2.5.1.2) Determination results of samples obtained by gel filtration chromatography

[0262] like Figure 2B As shown,

[0263] • The concentration of monomer PRL in the gel filtration chromatography sample determined using this product 1 is almost identical to the concentration of PRL in the initial sample;

[0264] In contrast, the concentrations of monomeric PRL in the gel filtration chromatography samples determined using commercially available products 1, 2, and 3 all showed significant deviations from the concentrations of PRL in the initial samples to varying degrees.

[0265] (2.5.2) This product vs. reference product

[0266] Samples from those PEG-precipitated samples tested with commercially available product 1 where "PRL determination results were interfered with by giant PRL" were selected to compare the assay effects of products 1 and 2 with those of reference products 1 and 2. The results are as follows: Figure 2C As shown:

[0267] • Among the PEG-precipitated samples tested with this product 1, the percentage of samples whose PRL determination results were not affected by giant PRL (up to 84%) was the highest, the percentage of samples whose PRL determination results were affected by giant PRL (only 5%) and the percentage of samples whose PRL determination results needed to be further determined by other means (only 11%) were the lowest.

[0268] • Among the PEG-precipitated samples tested with this product 2, the percentage of samples whose PRL determination results were not affected by giant PRL (up to 78%) was the second highest, the percentage of samples whose PRL determination results were affected by giant PRL (only 8%) and the percentage of samples whose PRL determination results needed to be further determined by other means (only 14%) were the second lowest.

[0269] In contrast, the PEG-precipitated samples measured with reference standards 1 and 2 were all samples whose PRL measurement results were interfered with by giant PRL (accounting for as high as 97% and 100%, respectively) and samples whose PRL measurement results were interfered with by giant PRL required further determination by other means (accounting for 3% and 0%, respectively). The samples with no PRL measurement results were not interfered with by giant PRL (accounting for 0% of all samples).

[0270] (2.6) Conclusion

[0271] above Figure 2A and 2B The results showed that when using the currently mainstream commercially available products 1, 2, and 3 to determine the concentration of monomeric PRL in samples, interference from giant PRLs present in the samples was still easily encountered, meaning that giant PRLs were easily misidentified as monomeric PRLs. In contrast, when using the antibody kits composed of the antibody pairs of the present invention (products 1 and 2) to determine the concentration of monomeric PRL in samples, interference from giant PRLs present in the samples was less likely, meaning that giant PRLs were less likely to be misidentified as monomeric PRLs, thus demonstrating a significant advantage in the specific determination of monomeric PRL in samples.

[0272] above Figure 2C The results showed that, for samples precipitated with PEG and measured using commercially available product 1, "the PRL measurement results were affected by giant PRL".

[0273] • Controls 1 and 2, which are used in the kits containing anti-human mature PRL antibodies that bind to the non-N-terminal and non-C-terminal regions, have no anti-interference resolution capability.

[0274] In contrast, the antibody kits (products 1 and 2) made from antibody pairs of the present invention, which consist of antibodies binding to the N-terminal region of mature human PRL (especially the region of aa1-13 (SEQ ID NO: 2) of SEQ ID NO: 1, more specifically residues aa1-3 and aa10-13) and the C-terminal region (especially the region of aa183-199 (SEQ ID NO: 3) of SEQ ID NO: 1, more specifically residues aa184-186 and aa199), have good anti-interference resolution.

[0275] This further confirms the significant advantage of the antibody of the present invention in specifically measuring monomeric PRL in the sample. [Example 3: Validation of the efficacy of the variant monoclonal antibody]

[0276] (3.1) Preparation of variant monoclonal antibodies

[0277] In addition to "antibody 1a" and "antibody 2a" used in Examples 1 and 2, other variants of antibody 1 and antibody 2 (i.e., "antibody 1b-p" and "antibody 2b-h") as shown in the table below were further prepared:

[0278]

[0279] in,

[0280] X1 is selected from: S, T, A, or G, thus the specific sequence of HCDR1 of antibody 1 is as follows:

[0281] <![CDATA[X1]]> Heavy chain CDR1 in antibody 1 SEQ ID NO: 24 S GFIFSWYV SEQ ID NO: 6 T GFIFTWYV SEQ ID NO: 25 A GFIFAWYV SEQ ID NO: 26 G GFIFGWYV SEQ ID NO: 27

[0282] X2 is selected from D, E, Q, or N, thus the specific sequence of HCDR2 of antibody 1 is as follows:

[0283] <![CDATA[X2]]> Heavy chain CDR2 in antibody 1 SEQ ID NO: 28 D ISGGDSHT SEQ ID NO: 7 E ISGGESHT SEQ ID NO: 29 Q ISGGQSHT SEQ ID NO: 30 N ISGGNSHT SEQ ID NO: 31

[0284] X3 is selected from D or E, thus the specific sequence of LCDR1 of antibody 2 is as follows:

[0285]

[0286]

[0287] X4 is selected from: G, A, S, or T, thereby enabling the LCD of antibody 2. The specific sequence of R2 is as follows:

[0288] <![CDATA[X4]]> CDR2 light chain in antibody 2 SEQ ID NO: 49 G KTSGV SEQ ID NO: 22 A KTSAV SEQ ID NO: 50 S KTSSV SEQ ID NO: 51 T KTSTV SEQ ID NO: 52

[0289] thus,

[0290] The specific sequence of the variable region of antibody 1 is as follows:

[0291] Antibody 1 <![CDATA[X1]]> <![CDATA[X2]]> Heavy chain variable region Light chain variable region Antibody 1a S D SEQ ID NO: 5 SEQ ID NO: 10 Antibody 1b S E SEQ ID NO: 32 SEQ ID NO: 10 Antibody 1c S Q SEQ ID NO: 33 SEQ ID NO: 10 Antibody 1d S N SEQ ID NO: 34 SEQ ID NO: 10 Antibody 1e T D SEQ ID NO: 35 SEQ ID NO: 10 Antibody 1f T E SEQ ID NO: 36 SEQ ID NO: 10 1g antibody T Q SEQ ID NO: 37 SEQ ID NO: 10 Antibody 1h T N SEQ ID NO: 38 SEQ ID NO: 10 Antibody 1i A D SEQ ID NO: 39 SEQ ID NO: 10 Antibody 1j A E SEQ ID NO: 40 SEQ ID NO: 10 Antibody 1k A Q SEQ ID NO: 41 SEQ ID NO: 10 Antibody 1l A N SEQ ID NO: 42 SEQ ID NO: 10 Antibody 1m G D SEQ ID NO: 43 SEQ ID NO: 10 Antibody 1n G E SEQ ID NO: 44 SEQ ID NO: 10 Antibody 1o G Q SEQ ID NO: 45 SEQ ID NO: 10 Antibody 1p G N SEQ ID NO: 46 SEQ ID NO: 10

[0292] The specific sequence of the variable region of antibody 2 is as follows:

[0293] Antibody 2 <![CDATA[X3]]> <![CDATA[X4]]> Heavy chain variable region Light chain variable region Antibody 2a D G SEQ ID NO: 15 SEQ ID NO: 20 Antibody 2b D A SEQ ID NO: 15 SEQ ID NO: 53 Antibody 2c D S SEQ ID NO: 15 SEQ ID NO: 54 Antibody 2d D T SEQ ID NO: 15 SEQ ID NO: 55 Antibody 2e E G SEQ ID NO: 15 SEQ ID NO: 56 Antibody 2f E A SEQ ID NO: 15 SEQ ID NO: 57 antibody 2g E S SEQ ID NO: 15 SEQ ID NO: 58 Antibody 2h E T SEQ ID NO: 15 SEQ ID NO: 59

[0294] (3.2) Preparation of variant antibody kit

[0295] The antibody kits shown in the table below were prepared using the variant monoclonal antibodies prepared in (3.1).

[0296]

[0297]

[0298] In the above antibody kit,

[0299] • The above-mentioned capture antibody was coated onto superparamagnetic microparticle beads to serve as a capture reagent.

[0300] • The above-mentioned detection antibodies are labeled with alkaline phosphatase and used as detection reagents.

[0301] (3.3) Determination of monomer PRL in the sample

[0302] As a sample, the sample from the PEG-precipitated sample measured in Example 2 with commercially available product 1, in which "PRL measurement results were interfered with by giant PRL", was used.

[0303] The above samples were measured using variants 1 to 4 as described in Example 2, and the results were compared with those obtained by measuring the above samples using this product 1.

[0304] (3.4) Results

[0305] like Figure 3 As shown, the results obtained using this product 1 are similar to those obtained using variants 1-4 in the above-mentioned PEG-precipitated samples.

[0306] The percentage of samples whose PRL measurements were not affected by giant PRL (as high as 81.1%, 78.4%, 81.1%, and 83.8%, respectively) was also relatively high;

[0307] The percentage of samples whose PRL measurements were affected by giant PRL (only 5.4%, 5.4%, 5.4%, and 5.4%, respectively) was also low;

[0308] The percentage of samples requiring further investigation using other methods to determine whether the PRL measurement results were affected by giant PRL (only 13.5%, 16.2%, 13.5%, and 10.8%, respectively) was also low.

[0309] (3.5) Conclusion

[0310] Antibody kits (variants 1-4) made from antibody pairs consisting of conserved variants of antibodies binding to the N-terminal region of mature human PRL (especially the region of aa1-13 (SEQ ID NO: 2) of SEQ ID NO: 1, more specifically residues aa1-3 and aa10-13) and conserved variants of antibodies binding to the C-terminal region (especially the region of aa183-199 (SEQ ID NO: 3) of SEQ ID NO: 1, more specifically residues aa184-186 and aa199) can also achieve the same / similar effects as those achieved with this product 1.

Claims

1. Antibody pairs that bind to human prolactin, among which... The antibody pair includes: Monoclonal antibody 1 or its antigen-binding fragment, which binds to the N-terminal region of human mature prolactin, and Monoclonal antibody 2 or its antigen-binding fragment, which binds to the C-terminal region of human mature prolactin, and The human maturation prolactin consists of the amino acid sequence shown in SEQ ID NO:

1.

2. The antibody pair according to claim 1, wherein The monoclonal antibody 1 or its antigen-binding fragment binds to the region of amino acids 1 to 13 of SEQ ID NO: 1, and / or The monoclonal antibody 2 or its antigen-binding fragment binds to the region of amino acids 183 to 199 of SEQ ID NO:

1.

3. The antibody pair according to claim 2, wherein... In the region of amino acids at positions 1 to 13 of SEQ ID NO: 1, the portion outside positions 1 to 3 and positions 10 to 13 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions, and / or In the region of amino acids at positions 183 to 199 of SEQ ID NO: 1, the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions.

4. The antibody pair according to claim 1, wherein The monoclonal antibody 1 or its antigen-binding fragment comprises: Heavy chain variable region, which contains: CDR1 contains the amino acid sequence of GFIFX1WYV. CDR2, which contains the amino acid sequence ISGGX2SHT, and CDR3, which contains the amino acid sequence TRHSPYDHGDFGY, and The light chain variable region, which includes: CDR1 contains the amino acid sequence QTLVHSLGNTY. CDR2, which contains the amino acid sequence of KVSIA, and CDR3, which contains the amino acid sequence SQTTHVPTF; and / or The monoclonal antibody 2 or its antigen-binding fragment comprises: Heavy chain variable region, which contains: CDR1 contains the amino acid sequence of GASITSDYA. CDR2, which contains the amino acid sequence ISFGGGHT, and CDR3, which contains the amino acid sequence of ARHSPNDHGDTNF, and The light chain variable region, which includes: CDR1 contains the amino acid sequence QDISX3NG. CDR2, which contains the amino acid sequence of KTSX4V, and CDR3 contains the amino acid sequence of QQGNTVPLT. in, X1 is selected from: S, T, A, or G; X2 is selected from: D, E, Q, or N; X3 is selected from: D or E; and / or X4 is selected from: G, A, S or T.

5. The antibody pair according to claim 4, wherein X1 is either S or A; X2 is either D or Q; X3 is D or E; and / or X4 is G, A, or S.

6. The antibody pair according to claim 4, wherein The monoclonal antibody 1 or its antigen-binding fragment comprises: The heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 5 and 32–46, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 10; and / or The monoclonal antibody 2 or its antigen-binding fragment comprises: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and The light chain variable region contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20 and 53-59.

7. Use of the antibody pair according to any one of claims 1 to 6 in the manufacture of a kit for determining prolactin in biological samples.

8. The use according to claim 7, wherein the prolactin is determined by a double-antibody sandwich chemiluminescent immunoassay.

9. The use according to claim 9, wherein The monoclonal antibody 1 or its antigen-binding fragment is used as a capture antibody, and The monoclonal antibody 2 or its antigen-binding fragment is used as the detection antibody.

10. The use according to any one of claims 7 to 9, wherein the determination is independent of isolating monomeric prolactin from a biological sample.

11. A monoclonal antibody or its antigen-binding fragment that binds to the N-terminal region of human prolactin, comprising: Heavy chain variable region, which contains: CDR1 contains the amino acid sequence of GFIFX1WYV. CDR2, which contains the amino acid sequence ISGGX2SHT, and CDR3, which contains the amino acid sequence TRHSPYDHGDFGY, and The light chain variable region, which includes: CDR1 contains the amino acid sequence QTLVHSLGNTY. CDR2, which contains the amino acid sequence of KVSIA, and CDR3 contains the amino acid sequence SQTTHVPTF. in, X1 is selected from: S, T, A, or G; X2 is selected from: D, E, Q or N.

12. The monoclonal antibody or its antigen-binding fragment according to claim 11, wherein... X1 is either S or A; and X2 is either D or Q.

13. The monoclonal antibody or its antigen-binding fragment according to claim 11 or 12, comprising: The heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 5 and 32–46, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO:

10.

14. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 11 to 13 in the manufacture of a kit for determining prolactin in biological samples.

15. A monoclonal antibody or its antigen-binding fragment that binds to the C-terminal region of human prolactin, comprising: Heavy chain variable region, which contains: CDR1 contains the amino acid sequence of GASITSDYA. CDR2, which contains the amino acid sequence ISFGGGHT, and CDR3, which contains the amino acid sequence of ARHSPNDHGDTNF, and The light chain variable region, which includes: CDR1 contains the amino acid sequence QDISX3NG. CDR2, which contains the amino acid sequence of KTSX4V, and CDR3 contains the amino acid sequence of QQGNTVPLT. in, X3 is selected from: D or E; and X4 is selected from: G, A, S or T.

16. The monoclonal antibody or its antigen-binding fragment according to claim 15, wherein... X3 is either D or E; and X4 is G, A, or S.

17. The monoclonal antibody or antigen-binding fragment thereof according to claim 15 or 16, comprising: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and The light chain variable region contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20 and 53-59.

18. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 15 to 17 in the manufacture of a kit for determining prolactin in biological samples.

19. A method for determining prolactin in biological samples, comprising: (a) Contacting a biological sample suspected of containing prolactin with an antibody pair according to any one of claims 1 to 6, and (b) Quantify the prolactin in the biological sample based on the signal generated in step (a).

20. The method of claim 19, wherein prolactin in the biological sample is quantified based on the chemiluminescent signal generated in step (a).

21. The method according to claim 19 or 20, wherein (1) Prior to step (a), the biological sample is pre-divided into: (1A) Part 1, which is unprocessed, and (1B) The second part is the supernatant obtained by mixing the biological sample with an equal volume of a solution containing polyethylene glycol, shaking, and then centrifuging. (2) The concentrations of prolactin in the first and second portions are determined by steps (a) and (b) and respectively used as the first and second concentrations; and (3) The recovery rate of prolactin after treatment in (1B) is calculated using the following formula: The recovery rate is greater than 60%, preferably greater than 80%, and more preferably greater than 90%.

22. A method for producing monoclonal antibodies, comprising immunizing animals with the following peptides: The N-terminal region of human prolactin, and / or The C-terminal region of human prolactin, The human maturation prolactin consists of the amino acid sequence shown in SEQ ID NO:

1.

23. The method of claim 22, wherein The N-terminal region of the human prolactin is the region of amino acids 1 to 13 of SEQ ID NO: 1, and / or The C-terminal region of the human maturation prolactin is the region of amino acids 183 to 199 of SEQ ID NO:

1.

24. The method of claim 23, wherein In the region of amino acids at positions 1 to 13 of SEQ ID NO: 1, the portion outside positions 1 to 3 and positions 10 to 13 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions, and / or In the region of amino acids at positions 183 to 199 of SEQ ID NO: 1, the portion other than positions 184 to 186 and 199 of SEQ ID NO: 1 has one or more conserved amino acid insertions, deletions, or substitutions.

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