Application of TRPM8 protein, related peptide fragment and their antibodies

Abstract

A TRPM8 related peptide fragment, comprising amino acid sequence as shown in SEQ ID No: 1-18 is provided. Furthermore, application of TRPM8 protein, TRPM8 related peptide fragment and their antibodies in preparing diagnostic reagent for chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS) is provided. By detecting level of TRPM8 protein molecule, TRPM8 related peptide fragment and their antibodies, the chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS) is effectively diagnosed, and the present invention is capable of effectively making a distinction between CP/CPPS and other diseases of prostate. In addition, by intravenously or subcutaneously injecting 1˜30000 IU TRPM8 protein or TRPM8 related polypeptide fragments with or without combining nanoparticles for desensitization therapy, or monoclonal or polyclonal antibodies of the TRPM8 protein or polypeptide fragments, the present invention is capable of curing or significantly relieve clinical symptoms of the chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS) and having an therapeutic effect.

Claims

1. A TRPM8 related peptide fragment, comprising amino acid sequence as shown in SEQ ID No: 1-18.

2. A method for preparing diagnostic reagent for chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS) comprising introducing TRPM8 protein, TRPM8 related peptide fragment and their antibodies, wherein an amino acid sequence of the TRPM8 related peptide fragment is as shown in SEQ ID No: 1-18.

3. The method as recited in claim 2, wherein the diagnostic reagent is immunodiagnostics or biochemical diagnostic reagents.

4. The method as recited in claim 3, wherein an immunology method utilizes TRPM8 protein or TRPM8 related peptide fragment to detect expressing level of TRPM8 protein antibody or TRPM8 related peptide fragment antibody in humor of an organism; or adopting polyclonal antibody or monoclonal antibody of TRPM8 protein or TRPM8 related peptide fragment synthesized by engineering method or prepared by any kinds of animals.

5. The method, as recited in claim 4, wherein the immunology method comprises enzyme immunoassay, fluorescent immunoassay, electrochemiluminescence immunoassay method or chemiluminescence immunoassay for measuring the radiation immunoassay, immune turbidity analysis, time-resolved fluorescence immunoassay technology and method.

6. An ELISA diagnostic kit for chronic prostatitis/chronic pelvic pain syndromes, CP/CPPS, which is characterized in comprising: (1) solid phase carrier coated by TRPM8 protein or TRPM8 related polypeptide fragments and enzyme marked anti-IgG antibody; or solid phase carrier coated by anti IgG, enzyme marked TRPM8 protein or antibody of TRPM8 related polypeptide fragments, wherein amino acid sequence of the TRPM8 related polypeptide fragments has amino acid sequence is as shown in SEQ ID No: 1-18; (2) substrate for the enzyme; (3) negative control, positive control and/or reference standards and control serum; (4) composition and specimen dilution; (5) washing solution; and (6) enzyme reaction-terminated liquid.

7. An diagnosis strip for chronic prostatitis/chronic pelvic pain syndromes, CP/CPPS, which is characterized in that the strip is fixed to the test line and the control line C T strip fiber chromatographic material as stationary phase, test line T punishable strip like coated with TRPM8 protein or TRPM8 polypeptide fragments associated control lines C punishable by a strip coated with IgG antibodies specific immune response in TRPM8 antibody test sample occurs in the test line T at the sample of other free was immune reaction at the C line; alternatively, the strip is fixed to the test line and the control line C T strip fiber chromatographic material as stationary phase, test line T punishable strip is coated with an antibody or TRPM8 protein TRPM8 associated polypeptide fragments corresponding antibody, control line C punishable strip coated with IgG antibodies in the test sample of TRPM8 protein or polypeptide fragments associated TRPM8 specific immune response occurs in the test line T at the sample was free of other immune reactions in the line of C; wherein the TRPM8 amino acid sequence of related polypeptides is as shown in SEQ ID No: 1-18.

8. A method for preparing diagnostic reagent for chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS) comprising introducing 8TRPM8 protein, TRPM8 related polypeptide fragments and corresponding antibodies of TRPM8 and TRPM8 related polypeptide fragments, wherein an amino acid sequence of the TRPM8 related polypeptide fragment is as shown in SEQ ID No: 1-18.

9. The method, as recited in claim 8, wherein an immunology method adopts polyclonal antibody or monoclonal antibody of TRPM8 protein or TRPM8 related peptide fragment are synthesized by engineering method or prepared by any kinds of animals; or TRPM8 protein or TRPM8 related peptide fragments are synthesized by chemical or genetic engineering methods. Nanoparticles combined with TRPM8 protein, TRPM8 related polypeptide fragments and corresponding antibodies of TRPM8 and TRPM8 related polypeptide fragments is made or purchased by or from other companies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 is an HE staining result of each group of prostate pathological section.

[0063] FIG. 2 is a pathological evaluation result of each group of prostate tissue. (**p<0.01 VS control group).

[0064] FIG. 3 is a CD3 immunofluorescence staining result of each group of prostate tissue.

[0065] FIG. 4 is a HE staining result of each group of prostate pathological section after adopting TRPM8 related peptide fragment.

[0066] FIG. 5 is a pathological evaluation result of each group of prostate tissue after adopting TRPM8 related peptide fragment. (**p<0.01 VS control group).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiment 1

[0067] Preparation of TRPM8 Related Peptide Fragment

[0068] From transmembrane analysis of TRPM8, following sections are positioned outside the membrane: 1-692, 758-796, 849-862, 916-953 and 1025-1104. From a comprehensive analysis of various kinds of software, 18 extracellular fragments of transmembrane protein are finally chosen, and the chosen amino acid sequence substantially covered extracellular fragments fully, which is:

[0069] T-1: Biotin-KINTKANDTSEEMRHRFRQLDTKLND (26AA); wherein “Biotin-” represents that sequence of KINTKANDTSEEMRHRFRQLDTKLND is capable of combining with Biotin, i.e., Vitamin H, so as to form a stable structure, hereafter inclusive. T-2: Biotin-FKNEDNETLAWEGVMKENYL (20AA);

TABLE-US-00006 T-3: Biotin-DGTTYDFAHCTFTGNESKPL (20AA); T-4: Biotin-VSRNLGPKIIMLQ (13AA); T-5: Biotin-DEVRQWYVNGVNYFTD (16AA); T-6: Biotin-LTVIKMEEAGDEIVSNA (17AA). T-7: CKEKN MESSY CCFKN EDNET T-8: CSEEM RHRFR QLDTK LNDLKG T-9: CFTGNE SKPLCV ELDEH NLPRFPE T-10: NRRND TLDST RTLYSS T-11: VEVED ALTSS AVKEK LVRFC T-12: CEMDI ELHDV SPITRH T-13: KEKNMESSVCCFKNEDNET T-14: SEEMRHRFRQLDTKLNDLKG T-15: FTGNESKPLCVELDEHNLPRFPE T-16: NRRNDTLDSTRTLYSSASRSTDLSYS T-17: VEVEDALTSSAVKEKLVRF T-18: EMDIELHDVSPITRH

[0070] The peptide fragment mentioned above is synthesized by bioyeargene biological technology co., LTD. The TRPM8 was purchased from ProteinTech Group.

Embodiment 2

[0071] Antigenicity Detection of TRPM8 Protein and TRPM8 Related Peptide Fragment

[0072] In order to prove that TRPM8 protein is the key to CP/CPPS induction and antigen pathogenicity of TRPM8 related peptide fragment. Experiment is designed and performed as follows. Firstly, pure TRPM8 protein is combined with Complete Freund's adjuvant (CFA) to establish an animal model of CP/CPPS. The animal model is compared with a prostatein homogenate modeling method. Meanwhile, TRPM8 in prostatein homogenate is removed by immunomagnetic beads; wherein the prostatein homogenate is molded for serving as a negative control. HE staining method is utilized to evaluate the severity of inflammation in prostate tissue of each modeling group of animal, and the severity of inflammation in prostate tissue is performed with pathological score. CD3 immunofluorescence method is adopted to observe infiltrating of T lymphocyte in prostate of the modeling animal.

[0073] Take 40 8-week-old SPF level male SD rats, with weights at a range of 250±20 g. The rats are purchased from Shanghai Sijie laboratory Co., Ltd with a license number of SCXK (Shanghai) 2012-0006. Under an aseptic condition, prostate of an SD rats is taken to prepare prostatein homogenate and TRPM8 removed homogenate protein. Grouping of immune animals in experiments is as follows. 40 8-week-old SD rats are randomly classified into 4 groups, i.e., a homogenate protein group, a TRPM8 removed homogenate protein group, a pure TRPM8 protein group and a control group; wherein each group comprises 10 rats. After 8 weeks of immune, each group of the rats are injected with 10% chloral hydrate on abdomen for anesthesia, and plasma and prostate of each group of rats are respectively collected. Preparation of prostate pathological section: the paraffin embedded prostate sections of the rats in each experimental group are sent to a drying oven, heated under 60° C. for 30 min. Then according to the conventional method, the prostate pathological sections are sent to each staining jar, dewaxed by dimethylbenzene, hydrated by gradient ethanol, stained by hematoxylin-and-eosin-stain, dehydrated by gradient ethanol and hyalinized by xylene. The prostate pathological sections are covered by neutral resin and then observed and photographed under a microscope. The covered prostate pathological sections are preserved under 4° C.

[0074] In addition, the prostate tissue sections of each group of animals to be tested are taken, baked in an oven for 30 minutes at 60° C., then subjected to CD3 immunofluorescence, and finally added with anti-fluorescence quenching agent for sealing, observed under a microscope, wherein photos are taken.

[0075] According to the method mentioned above, TRPM8 protein related peptide fragments are adopted to immune rats, so as to produce corresponding prostate tissue sections.

[0076] Group T-1 is corresponding to a TRPM8 related peptide fragment: Biotin-KINTKANDTSEEMRHRFRQLDTKLND (SEQ ID: No 1): wherein “Biotin-” represents that sequence of KINTKANDTSEEMRHRFRQLDTKLND is capable of combining with Biotin, i.e., Vitamin H, so as to form a stable structure, hereafter inclusive.

[0077] Group T-2 is corresponding to a TRPM8 related peptide fragment: Biotin-FKNEDNETLAWEGVMKENYL, (SEQ ID: No 2);

[0078] Group T-3 is corresponding to a TRPM8 related peptide fragment: Biotin-DGTTYDFAHCTFTGNESKPL (SEQ ID: No 3);

[0079] Group T-4 is corresponding to a TRPM8 related peptide fragment: Biotin-VSRNLGPKIIMLQ (SEQ ID: No 4);

[0080] Group T-5 is corresponding to a TRPM8 related peptide fragment: Biotin-DEVRQWYVNGVNYFTD (SEQ ID: No 5);

[0081] Group T-6 is corresponding to a TRPM8 related peptide fragment: Biotin-LTVIKMEEAGDEIVSNA (SEQ ID: No 6).

[0082] Group T-7 is corresponding to a TRPM8 related peptide fragment CKEKN MESSV CCFKN EDNET (SEQ ID: No 7).

[0083] Group T-8 is corresponding to a TRPM8 related peptide fragment CSEEM RHRFR QLDTK LNDLKG (SEQ ID: No 8).

[0084] Group T-9 is corresponding to a TRPM8 related peptide fragment CFTGNE SKPLCV ELDEH NLPRFPE (SEQ ID: No 9).

[0085] Group T-10 is corresponding to a TRPM8 related peptide fragment NRRND TLDST RTLYSS (SEQ ID: No 10).

[0086] Group T-11 is corresponding to a TRPM8 related peptide fragment VEVED ALTSS AVKEK LVRFC (SEQ ID: No 11).

[0087] Group T-12 is corresponding to a TRPM8 related peptide fragment CEMDI ELHDV SPITRH (SEQ ID: No 12).

[0088] Group T-13 is corresponding to a TRPM8 related peptide fragment KEKNMESSVCCFKNEDNET (SEQ ID: No 13).

[0089] Group T-14 is corresponding to a TRPM8 related peptide fragment SEEMRHRFRQLDTKLNDLKG (SEQ ID: No 14).

[0090] Group T-15 is corresponding to a TRPM8 related peptide fragment FTGNESKPLCVELDEHNLPRFPE (SEQ ID: No 15).

[0091] Group T-16 is corresponding to a TRPM8 related peptide fragment NRRNDTLDSTRTLYSSASRSTDLSYS (SEQ ID: No 16).

[0092] Group T-17 is corresponding to a TRPM8 related peptide fragment VEVEDALTSSAVKEKLVRF (SEQ ID: No 17).

[0093] Group T-18 is corresponding to a TRPM8 related peptide fragment EMDIELHDVSPITRH (SEQ ID: No 18).

[0094] Control group: blank control group.

[0095] According to HE staining result, prostatitis degree of each group of rats are pathologically scored under standards as follows.

[0096] Score 0: Without any inflammatory cell infiltration and without any signs of inflammation;

[0097] Score 1: A small amount of acinar epithelial shrinks;

[0098] Score 2: A plurality of acinar epithelial shrinks and a small amount of inflammatory cell infiltration;

[0099] Score 3: Severely shrinks of acinar epithelial, severe congestion in the prostate body and a plurality of inflammatory cell infiltration.

[0100] Each prostate section is scored by three associate professors in pathology according to the standards mentioned above under a randomized and double-blind principle, and an average value calculated is a histological score indicating degree of the prostatitis of the animal.

[0101] Statistical analysis was performed using excel 2007, average values of the pathological evaluation indicating the degree of the prostatitis of the animal is performed with homogeneity test of variance and one-way analysis of variance (One-Way ANOVA). p<0.05: there is a significant difference; p<0.01 there is a highly significant difference.

[0102] HE Staining and Pathological Valuation Result

[0103] Referring to FIG. 1, (1: under a 10× objective lens; 2: under a 20× objective lens; 3: under a 40× objective lens; B: protein homogenate group; C: pure TRPM8 protein group; D protein homogenate group; TRPM8 protein-free homogenate group) and FIG. 2, a plurality of inflammatory cell infiltration is seen in prostate stromal of all animals in the protein homogenate group, severe congestion appears in gland. An average value of the histological score is 2.7778±0.4410 (M±SD), which is also much lower than the protein homogenate modeling group.

[0104] In order to research that whether the effect of inducing CP/CPPS morbidity is caused by the TRPM8 in prostatein homogenate, the TRPM8 protein is removed from protein homogenate by immunomagnetic beads method. The result shows that in the TRPM8 protein-free homogenate combining with CFA modeling group, pathophysiological changes of the prostate and the inflammatory cell infiltration are greatly decreased. The histological score (0.8000±0.9190) indicates that level of prostatitis in animals of the TRPM8 protein-free group does not have a significant difference compared with the control blank group.

[0105] In addition, pure TRPM8 protein is combined with CFA for immuring SD rat, so as to further verify the effects of TRPM8 protein in CP/CPPS morbidity. FIG. 1 shows that TRPM8 protein successfully induces generation of CP/CPPS. There is serious congestion in the prostate, and there is a plurality of inflammatory cell infiltration in mesenchyme and particularly around blood vessels. The pathological evaluation (2.7000±0.4830) is very close to the protein homogenate.

[0106] 2. Immunofluorescence Staining Results of CD3

[0107] Seen from HE staining results, prostate tissue of rats in the protein homogenate group and the pure TRPM8 protein group has a plurality of inflammatory cell infiltration. In order to improve the inflammatory cell containing a plurality of T-lymphocytes, the CD3 molecules are performed with immunofluorescence staining. The results are shown in FIG. 3: A: control blank group; B: protein homogenate group; C: pure TRPM8 protein group; D: TRPM8 protein free homogenate group. A large area of CD3 molecule marked by red luminescence can be seen in prostate mesenchyme, which indicates that most of the infiltrative inflammatory cells are T-lymphocytes, not innate immune cells such as neutrophils or macrophages. However, in the blank control group and the TRPM8 protein-free homogenate group, there is almost no inflammatory cell infiltration in the prostate stromal, and the immunofluorescence staining is negative, indicating that no T lymphocyte is in the prostate stromal.

[0108] Testing results of antigen pathogenicity of TRPM8 and related peptide fragment are as shown in FIG. 4 and FIG. 5. Experimental results of studies of antigen pathogenicity of TRPM8 related peptide fragment indicate that all TRPM8 related peptide fragments show strong pathogenicity causing CP/CPPS.

[0109] In summary, both the TRPM8 protein and the TRPM8 related peptide fragment have strong pathogenicity causing CP/CPPS.

Embodiment 3

[0110] (I) Preparation of TRPM8 Protein and TRPM8 Related Peptide Fragments

[0111] Animal Selection and Immunization

[0112] 1. Animal selection: pure BALB/C mice.

[0113] 2. Immunization programs

[0114] Selecting an appropriate immunization program is essential for success of cell fusion and hybridization and obtaining high quality McAb. Usually, primary immunization is started according to the immunization program two month before the fusion. The immunization program is determined based on different characteristics of the antigen.

[0115] Complete sequence of human-derived TRPM8 protein and peptide fragment as shown in SEQ ID No: 1-6 are selected for serving as antigen. Freund's adjuvant is adopted for respectively immunizing the mice.

[0116] 1-50 μg of primary immune antigen adding with freund's complete adjuvant is applied to the mice by multiple sites subcutaneous injection or spleen injection (usually 0.8˜1 ml, 0.2 ml/site. After 3 weeks, a secondary immunization is performed by an identical dosage as mentioned above, incomplete Freund's adjuvant subcutaneously or ip (intraperitoneal injection) (ip dose should not exceed 0.5 ml). Three weeks after the third immunization is performed with an identical dose, without adjuvant, (5-7 days after, blood is collected for testing its potency). 2 to 3 weeks after, enhancing the immune by a dosage of 500 μg is appropriate, intravenously, three days later, performing spleen integration.

[0117] (II) Cell Fusion

[0118] 1. Preparation before cell fusion

[0119] (1) Selection of myeloma cell line

[0120] The myeloma cells are derived from mice. Inoculating hybridoma on abdominal cavity of the same strains of mice, and a plurality of McAb is generated.

[0121] (2) Feeder cells

[0122] In tissue culture, adding feeder cells comprises mice peritoneal macrophage. An amount of the feeder cell is 2×10.sup.4 or 10.sup.5 per pore.

[0123] 2. The cell fusion comprises steps as follows:

[0124] (1) preparing feeder layer cell comprising: selecting mice peritoneal macrophage, adopting 6-10 week old BALB/C mice which is in an identical stain with the immune mice, necking, killing, immersing in 75% ethyl alcohol for 3-5 min, cutting skins of the mice by sterile scissors to expose peritoneum, injecting 5-6 ml pre-cooling nutrient solution by a sterile syringe (strictly forbidding piercing intestinal canal, washing repeatedly, extracting flushing fluid, sending the flushing fluid into 10 ml centrifuge tube, centrifuging for 5-6 min under 1200 rpm, suspending in nutrient solution of 20% calf serum (NCS) or fetal calf serum (FCS), regulating cell number to 1×10.sup.5/ml, adding 100 μl/pore to a 96 pore plate, and sending to a CO.sub.2 incubator culturing under 37° C.;

[0125] (2) preparing immunized splenic cells

[0126] after booster immunization for 3 days, necking the mice for killing, taking spleen under sterile conditions, washing by nutrient solution, grinding the spleen, passing through a cell strainer, centrifuging, washing the cell twice by nutrient solution, counting and taking 10.sup.8 splenic lymphocyte suspension for reserving;

[0127] (3) preparing myeloma cell

[0128] taking logarithmic growth myeloma cell, centrifuging, washing twice by serum free nutrient solution, counting to obtain ×10.sup.7 cells for reserving;

[0129] (4) fusing

[0130] {circle around (1)} mixing myeloma cells and splenic cells according to a proportion of 1:10 or 1:5, washing once in a serum-free culture solution in a 50 ml centrifuge tube, centrifuging under 1200 rpm for 8 min; abandoning supernatant, sucking all residual liquid with a pipette, so as to prevent affecting concentration of polyethylene glycol (PEG), gently flicking a bottom of the 50 ml centrifuge tube, so as to make cell deposition become more loose;

[0131] {circle around (2)} adding 1 ml 45% PEG solution which is preheated at 37° C. and with a molecular weight of 4000 in 90 s into the centrifuge tube, slightly shaking the centrifuge tube while adding the PEG solution, and keeping in a 37° C. water bath for 90 s;

[0132] {circle around (3)} adding a 37° C. preheated incomplete culture solution to terminate PEG effects, wherein the incomplete culture solution is respectively added with dosages of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml at each two-minute interval;

[0133] {circle around (4)} centrifuging under 800 rpm for 6 min;

[0134] {circle around (5)} taking the supernatant to be re-suspended utilizing HAT selection culture solution containing 20% calf serum;

[0135] {circle around (6)} adding the cells mentioned above to a 96-pore plate with an existing feeder cell layer, adding 100 μl for each pore; wherein usually one immune spleen can be inoculated on 4 pieces of 96-pore plate; and

[0136] {circle around (7)} sending the 96-pore plate to an incubator at 37° C. with 5% CO.sub.2.

[0137] (III) Selection of Hybridoma Cells and Test of Antibodies

[0138] 1. HAT selection of hybridoma cell comprises steps of:

[0139] PEG treating spleen cells and myeloma cells to form a mixture of a variety of cells, wherein only the hybridoma cells formed by the spleen cells and myeloma cells are selected;

[0140] wherein during the process of culturing in HAGT selection solution, since the myeloma cells lack thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, and the myeloma cells are not capable of growing and reproducing; the hybridoma cells have the thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase and thus are capable of growing and reproducing in HAT selection culture solution;

[0141] wherein in 1-2 days of HAT selection culture, a large amount of cells are died, after 3-4 days tumor cells disappear and the hybrid cells form small colonies, maintaining the HAT selection culture solution for 7-10 days and then the HAT selection culture solution is changed to HT culture solution, maintain for 2 weeks and conventional culture solution is changed; during the selection culture period, when the hybridoma cells cover 1/10 area of a pore bottom, specific antibodies are detected to select required hybridoma cell lines; wherein during the process of selection culture, half of the culture solution is changed in every 2-3 days.

[0142] 2. Detection of TRPM8 antibody: utilizing a radioimmunoassay (RIA) method.

[0143] (IV) Hybridoma Clone

[0144] The present invention utilizes a limited dilution clone method comprising steps of:

[0145] (1) preparing a feeder cell layer one day before clone, which is identical to cell fusion;

[0146] (2) blow-drying hybridoma to be cloned in the culture hole and counting;

[0147] (3) regulating the cells to a density of 3-10/ml;

[0148] (4) taking a cell culture plate in which the feeder cell layer is cultured, adding 100 ml dilution cells in each pore to be incubated at an incubator at 37° C. and with 5% CO.sub.2;

[0149] (5) changing liquid in the seventh day, wherein the liquid is changed in every 2-3 days;

[0150] (6) in 8-9 days, when clone cells are formed, timely detecting activity of the antibody;

[0151] (7) moving cells in positive holes to a 24-pore plate for expanded culture; and

[0152] (8) frozen preserving each clone as soon as possible.

[0153] (V) Frozen Preservation and Recovery of Hybridoma Cell

[0154] (1) Cryopreservation of hybridoma cells

[0155] cryopreservation solution: 50% fetal bovine serum; 40% incomplete culture solution and 10% DMSO (dimethyl sulfoxide).

[0156] The cryopreservation solution is preferably cooled, wherein cooling operation is gentle and fast, wherein during the cryopreservation process, temperature is dropped from a room temperature to 0° C. and immediately sent to an ultra-low temperature freezer, and then converted to liquid nitrogen the next day.

[0157] (2) Cell recovery method comprising steps of:

[0158] taking glass ampoule from liquid nitrogen carefully, putting in a water bath at 37° C., unfreezing frozen cells, washing the cells twice with completed culture solution, and sending to a cell flask for feeder layer prepared one-day before, sending to an incubator at 37° C. and with 5% CO.sub.2 for culturing, when the cells form colonies, detecting activity of the antibody.

[0159] (VI) Mass Production of Monoclonal Antibodies

[0160] The present invention adopts a solid tumor method comprising:

[0161] incubating logarithmic phase hybridoma cells by 1-3×10.sup.7/ml subcutaneously in the back of a mice, injecting 0.2 ml at each site for a total of 2-4 sites, when the tumor reaches a certain size, generally after 10 to 20 days, blood sampling, wherein serum obtained from the monoclonal antibody content can reach 1-10 mg/ml.

[0162] Antibody Identification

TABLE-US-00007 TABLE 3 Dosage Specificity Affinity Valence TRPM8  1 μg High 10.sup.12 L/mol 1:128 T-1 35 ng High 10.sup.12 L/mol 1:128 T-2 35 ng High 10.sup.12 L/mol 1:128 T-3 35 ng High 10.sup.12 L/mol 1:128 T-4 35 ng High 10.sup.12 L/mol 1:128 T-5 35 ng High 10.sup.12 L/mol 1:128 T-6 35 ng High 10.sup.12 L/mol 1:128 T-7 35 ng High 10.sup.12 L/mol 1:128 T-8 35 ng High 10.sup.12 L/mol 1:128 T-9 35 ng High 10.sup.12 L/mol 1:128 T-10 35 ng High 10.sup.12 L/mol 1:128 T-11 35 ng High 10.sup.12 L/mol 1:128 T-12 35 ng High 10.sup.12 L/mol 1:128 T-13 35 ng High 10.sup.12 L/mol 1:128 T-14 35 ng High 10.sup.12 L/mol 1:128 T-15 35 ng High 10.sup.12 L/mol 1:128 T-16 35 ng High 10.sup.12 L/mol 1:128 T-17 35 ng High 10.sup.12 L/mol 1:128 T-18 35 ng High 10.sup.12 L/mol 1:128

Embodiment 4

[0163] ELISA diagnostic kit for chronic prostatitis or chronic pelvic pain syndrome, comprising:

[0164] (1) TRPM8 protein which is coated (1 μg) or solid carrier of TRPM8-related polypeptide fragment (35 ng), solid carrier coated by anti-human IgG antibody (1 μg) and enzyme marked TRPM8 protein antibody or antibody TRPM8 protein related peptide fragments (1 μg), wherein amino acid sequences of the TRPM8 protein related peptide fragments are as shown in SEQ ID No: 1-18 below;

[0165] (2) substrate for the enzyme;

[0166] (3) negative control, positive control and/or reference standards and control serum, 0.5 ml in each;

[0167] (4) 1.0 m conjugate and 1.0 ml dilution of the respective samples 1;

[0168] (5) 5 ml washing solution;

[0169] (6) 2 ml enzyme reaction terminated liquid.

[0170] 60 clinical persons are tested. Utilizing the kit of the TRPM8 and TRPM8 related peptide fragments to detect plasma of the persons who are tested, comprising 15 healthy persons, 15 clinically diagnosed CP/CPPS patients, 15 bacterial prostatitis patients, 15 prostatic cancer patients, so as to verify the accuracy rate of the kit of the present invention. The results are as shown in Table. 5, wherein testing values of 12 of the 15 bacterial prostatitis patients are smaller than 10 ng/ml and the elimination rate is 80%; testing values of the 15 prostatic cancer patients are all greater than 500 ng/ml, and the elimination rate is 100%. Thus, it can be seen that the kit of the present invention is capable of effectively diagnosing the CP/CPPS patients and effectively identifying CP/CPPS and other protastic disorders such as bacterial prostatitis and prostatic cancer.

TABLE-US-00008 TABLE 4 Diagnostic Criteria Detection value of Antibody detection TRPM8 protein or value of TRPM8 related peptide protein or related Detection group fragment (ng/ml) peptide fragment (ng/ml) Chronic prostatitis 10 ≦ detection 250 ≦ detection or chronic pelvic value < 200, judged value < 500, judged pain syndrome positive CP/CPPS positive CP/CPPS Normal individuals Detection Detection value < 10, judged value < 5, judged negative CP/CPPS negative CP/CPPS Bacterial prostatitis <10 <5 Prostate cancer ≧500 ≧2000

TABLE-US-00009 TABLE 5 Plasma samples TRPM8 protein TRPM8 antibody detection accuracy detection accuracy TRPM8 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-1 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-3 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-6 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-9 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-15 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive) T-18 Normal human 100% (15 human 100% (15 human beings beings negative) beings negative) CP/CPPS 86.67% (13 human 86.67% (13 human patient beings positive) beings positive)

Embodiment 5

[0171] A Test Strip for Diagnosis of Chronic Prostatitis/Chronic Pelvic Pain Syndromes (CP/CPPS)

[0172] In a homogeneous immunological method, for example, specific methods are: respectively pasting an NC film, an absorbent pad, a conjugate pad sprayed with fluorescent-labeled antibody and glass fiber which is infiltrated in developing solvent and then dried, in such a manner that an immuno-chromatographic strip is assembled. TRPM8 protein or TRPM8 related polypeptide fragments with a concentration of 10 ng/kg are evenly sprayed on the NC film by a non-contact micro-sprinkler on a three-dimensional spraying platform to form 2 lines with a moderate thickness in parallel including a non-contact micro-sprinkler T line for coating antigen and a C line of goat anti human/rat with a concentration of 10 ng/kg and a dosage of 100 ul;

[0173] putting the immuno-chromatographic strip in an incubator to keep 37° C. for 1 h and taking out; cutting the immuno-chromatographic strip into a plurality of paper slips with a size at a range of 4 mm±2 mm, and sending the plurality of paper slips to an aluminum foil bag with desiccant for a sealed preservation;

[0174] dropping liquid to be detected on an appropriate position of the test trip, performing chromatography, shooting and detecting under a characteristic excitation wave length of 320 nm and an emission wavelength of 620 nm;

[0175] performing sensitivity, specificity and simulation of positive samples according to the method mentioned above;

[0176] wherein when the samples to be detected is siphoned from bottom to top along the paper slip by capillary action, according to chromatographic theory, the samples to be detected is moved from a detecting end of the paper slip to the other end of the paper slip, wherein the samples to be detected respectively passes through the conjugate pad, the NC film, the T line and the C line to reach the absorbent pad.

[0177] After the chromatography, if the C line is not colored, the paper strip is considered invalid; if the C line develops color, the paper strip is considered valid; if the C line develops colors but the T line is not colored, the test result is positive; if both the T line and the C line develop color, the test result is negative.

TABLE-US-00010 TABLE 6 TRPM8 protein humor TRPM8 antibody humor Morbidity situation detection results detection result Patients of chronic Positive (both C line Positive (both C line prostatitis or and T line are and T line are chronic pelvic pain developed) developed) syndrome Normal human Negative (C line Negative (C line beings developed, T line developed, T line not developed) not developed)

[0178] Invalid: ribbon does not appear on the quality control region (C), indicating that operation procedure is incorrect or the reagent strip has gone corrupt, re-testing is performed.

[0179] Note: The test strip control line and test line ribbon may appear light or dark color due to the contents of TRPM8 or TRPM8 antibody in body fluids, the result is determined according to the above criteria. For insurance purposes, the test is performed again after three days.

Embodiment 6

[0180] Therapeutic Effect of TRPM8 Protein and TRPM8 Related Polypeptide Antibodies in Treatment of Chronic Prostatitis/Chronic Pelvic Pain Syndromes (CP/CPPS).

[0181] Implementation

[0182] Respectively prepare monoclonal antibody of TRPM8 protein and TRPM8 related peptide, perform intravenous subcutaneous injection by 1-30000 IU for once a day, and 14 days are a course of treatment, wherein the relationships between the using dosage and the clinical manifestation are as shown in Table. 7.

TABLE-US-00011 TABLE 7 Condition of chronic Application prostatitis/chronic monoclonal pelvic pain antibody of TRPM8 syndromes, and TRPM8 related Clinical improvement CP/CPPS peptide TRPM8 (IU) indicators of patients Mild 1000~10000 IU, once a Symptoms improved; day, intravenous TRPM8 humoral antibody injection detection value <5 Moderate 2000~20000 IU, once a ng/ml; TRPM8 protein day, intravenous in body fluids to detect injection the value <10 ng/ml; Severe 3000~30000 IU or prostatic fluid above, once a leukocytes <5/HPF; day, intravenous Ultrasonic B-show injection normal prostate volume and structure.

[0183] The results are shown in Table 8; the results show that TRPM8 and TRPM8 related polypeptide monoclonal antibodies may be effective in curing or alleviating chronic prostatitis or chronic pelvic pain syndrome.

TABLE-US-00012 TABLE 8 Patient Cure Efficiency Effective Ineffective Dosage and number in rate rate rate rate administration group (%) (%) (%) (%) Negative 2000 IU, once 100 0 0 0 100 control: a day, hepgarin intravenous injection Positive Four pills at a 100 60 10 70 30 control time, 3 times a medicine: day, by oral Qianliekang administration TRPM8 10000 IU, 100 62 10 72 28 monoclonal once a day, antibody intravenous injection T-1 15000 IU, 100 61 20 82 18 monoclonal once a day, antibody intravenous injection T-6 13000 IU, 100 62 30 92 8 monoclonal once a day, antibody intravenous injection T-9 6000 IU, once 100 92 5 97 3 monoclonal a day, antibody intravenous injection T-12 8000 IU, once 100 83 12 95 5 monoclonal a day, antibody intravenous injection T-15 9000 IU, once 100 81 10 91 9 monoclonal a day, antibody intravenous injection T-18 9600 IU, once 100 88 5 93 7 monoclonal a day, antibody intravenous injection

Embodiment 7

[0184] The Therapeutic Action of TRPM8 Protein and TRPM8 Related Polypeptides Desensitization Therapy for Chronic Prostatitis or Chronic Pelvic Pain Syndrome

[0185] TRPM8 protein and TRPM8 related polypeptide are respectively prepared. By injecting 1-30000 units by intravenous or subcutaneous once a day, each course comprises 14 days. The relationships between the application dosage and the clinical manifestation are as shown in Table. 9.

TABLE-US-00013 TABLE 9 Condition of chronic Application of prostatitis/chronic monoclonal pelvic pain antibody of TRPM8 syndromes, and TRPM8 related Clinical improvement CP/CPPS peptide TRPM8 (IU) indicators of patients Mild 1~10000 IU, once a Symptoms improved; day, intravenous TRPM8 humoral antibody injection detection value <5 Moderate 2000~20000 IU, once ng/ml; TRPM8 protein a day, intravenous in body fluids to detect injection the value <10 ng/ml; Severe 3000~30000 IU or prostatic fluid above, once a leukocytes <5/HPF; day, intravenous Ultrasonic B-show injection normal prostate volume and structure.

[0186] The results are shown in Table 10. The results show that TRPM8 and TRPM8 polypeptide associated desensitization therapy can be effective in curing or alleviating chronic prostatitis or chronic pelvic pain syndrome.

TABLE-US-00014 TABLE 10 Patient Cure Effective Dosage and number in rate Efficiency rate Ineffective administration group (%) rate (%) (%) rate (%) Negative 2000 IU, once 100 0 0 0 100 control: a day, hepgarin intravenous injection Positive Four pills at a 100 60 10 70 30 control time, 3 times a medicine: day, by oral Qianliekang administration TRPM8 10000 IU, 100 62 10 72 28 monoclonal once a day, antibody intravenous injection T-1 15000 IU, 100 61 20 82 18 monoclonal once a day, antibody intravenous injection T-6 13000 IU, 100 62 30 92 8 monoclonal once a day, antibody intravenous injection T-9 6000 IU, once 100 92 5 97 3 monoclonal a day, antibody intravenous injection T-12 8000 IU, once 100 83 12 95 5 monoclonal a day, antibody intravenous injection T-15 9000 IU, once 100 81 10 91 9 monoclonal a day, antibody intravenous injection T-18 9600 IU, once 100 88 5 93 7 monoclonal a day, antibody intravenous injection

[0187] One skilled in the art will understand that the embodiment of the present invention as shown in the drawings and described above only is exemplary and not intended to be limiting.

[0188] It will thus be seen that the objects of the present invention have been fully and effectively accomplished. Its embodiments have been shown and described for the purposes of illustrating the functional and structural principles of the present invention and is subject to change without departure from such principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the following claims.