Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof
1. An anti-human NGAL monoclonal antibody (NG08) having a heavy chain variable region comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:3, HCDR1 as shown in sequence SEQ ID NO:4 and/or HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 5;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 6, LCDR1 shown as a sequence SEQ ID NO: 7 and/or LCDR2 as shown in SEQ ID NO: LCDR3 shown in fig. 8.
2. The anti-human NGAL monoclonal antibody of claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 11.
3. An anti-human NGAL monoclonal antibody (NG19), the heavy chain variable region of which comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 13, HCDR1 as shown in sequence SEQ ID NO: 14 and/or HCDR2 as set forth in sequence SEQ ID NO: HCDR3 shown at 15;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 16, as shown in sequence SEQ ID NO: 17 and/or LCDR2 as shown in SEQ ID NO: LCDR3 shown at 18.
4. The anti-human NGAL monoclonal antibody of claim 3, wherein the amino acid sequence is shown in SEQ ID NO. 21.
5. An anti-human NGAL monoclonal antibody (NG20) having a heavy chain variable region comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 23, HCDR1 as shown in sequence SEQ ID NO: 24 and/or HCDR2 as set forth in sequence SEQ ID NO: HCDR3 shown at 25;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 26, LCDR1 as shown in sequence SEQ ID NO: 27 and/or LCDR2 as set forth in sequence SEQ ID NO: LCDR3 shown at 28.
6. The monoclonal antibody against human NGAL of claim 5, having an amino acid sequence shown in SEQ ID NO. 31.
7. An anti-human NGAL monoclonal antibody (NG37) having a heavy chain variable region comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 33, HCDR1 as set forth in sequence SEQ ID NO: 34 and/or HCDR2 as shown in SEQ ID NO: HCDR3 shown at 35;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 36, LCDR1 as set forth in sequence SEQ ID NO: 37 and/or LCDR2 as set forth in sequence SEQ ID NO: LCDR3 shown at 38.
8. The anti-human NGAL monoclonal antibody of claim 7, wherein the amino acid sequence is shown in SEQ ID NO. 41.
9. A human NGAL latex enhanced immunoturbidimetry assay kit, comprising a R1 reagent, a R2 reagent and a standard substance, wherein the R2 reagent contains latex microspheres simultaneously labeled with the anti-human NGAL monoclonal antibody of claim 1, 3, 5 and 7.
10. The human NGAL latex-enhanced immunoturbidimetric assay kit of claim 9, wherein in the R1 reagent: the buffer solution is phosphate buffer solution (0.01M-0.05M, pH7.0-7.4); the stabilizer is 40g/L-80g/L NaCl; 5-10g/L of PEG 6000-20000 as a thickening agent, 0-10 g/L of BSA as a protective agent, 0.05-0.1g/L of Proclin300 as a preservative, and 0.1-0.2% of Tween 80 or Tritonx100 as a surfactant. (ii) a
In the R2 reagent: the buffer solution is 0.01M-0.05M phosphate buffer solution or ethanesulfonic acid buffer solution with pH5.9-7.4; the preservative is 0.1-0.2g/L of Proclin 300; the stabilizer is 10g/L-20g/L NaCl, 50 g/L-100 g/L glucose or sucrose, the protective agent is 10g/L-20g/L BSA, and 0-0.1% Tween 20; in addition, there are latex microballoons (the mass concentration is 0.125% -0.33%) marked with 4 kinds of recombinant mouse anti-human NGAL monoclonal antibodies;
the standard comprises 5 solutions of recombinant human NGAL at different concentrations, wherein: the buffer solution is 0.01M-0.05M phosphate buffer solution with pH7.0-7.4; the protective agent is 0g/L-10g/L BSA, 0-2 g/L PEG8000 or 0-20 g/L sorbitol, and the stabilizer is 0-13g/L NaCl.
Background
neutropHil gelatinase-associated lipocalin (NGAL), also known as human lipocalin 2(lipocalin 2), has three forms of protein, a monomer with a molecular weight of 25kD, a self-polymerized 46kD homodimer, and a 135kD heterodimer with MMP-9. In recent years, NGAL has been used as a new marker of acute kidney injury, when acute renal function injury (AKI) is diagnosed at an early stage, the NGAL concentration in blood and urine generally rises rapidly, 2 hours is the most obvious, and the traditional indexes such as serum creatinine, urease and the like need to rise obviously after 24-72 hours. NGAL is also a potential novel marker reflecting chronic damage to the kidney, and in patients with Chronic Kidney Disease (CKD), NGAL can reflect the extent of renal damage with certainty, and is a strong and independent risk indicator of CKD progression. In conclusion, the quantitative detection product of NGAL has important clinical significance and strong market demand.
At present, enzyme-linked immunosorbent assay, chemiluminescence assay, immunoturbidimetry and the like are used for detecting NGAL. The basic principle of the latex immunoturbidimetry is as follows: NGAL protein in clinical samples reacts with specific anti-human NGAL antibodies coated to latex microspheres to form immune complexes of antigen antibodies, so that the latex microspheres form a reticular polymer, the turbidity of a detection system is increased, and the concentration of the NGAL protein is positively correlated with the turbidity within a certain range. With the increase of clinical detection demand and the popularization of full-automatic biochemical analyzers, the biochemical latex turbidimetry is the mainstream detection method in clinic due to the advantages of short reaction time, good precision, easy automation and the like.
Disclosure of Invention
The invention provides a recombinant neutrophil gelatinase-associated lipocalin (NGAL for short), the amino acid sequence of which is shown in SEQ ID NO:1, the optimized codon sequence is shown as SEQ ID NO:2, respectively.
The invention also provides 4 kinds of mouse anti-human NGAL monoclonal antibodies, a first anti-human NGAL monoclonal antibody (NG08), the heavy chain variable region of which contains the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:3, HCDR1 as shown in sequence SEQ ID NO:4 and/or HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 5;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 6, LCDR1 shown as a sequence SEQ ID NO: 7 and/or LCDR2 as shown in SEQ ID NO: LCDR3 shown in fig. 8.
Preferably, the amino acid sequence of the heavy chain variable region of the NG08 antibody of the present invention is shown in SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10.
The amino acid sequences of the preferred NG08 monoclonal antibody are respectively shown as SEQ ID NO. 11; the nucleotide sequences of the monoclonal antibodies are respectively shown as SEQ ID NO. 12.
A second anti-human NGAL monoclonal antibody (NG19) having a heavy chain variable region comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 13, HCDR1 as shown in sequence SEQ ID NO: 14 and/or HCDR2 as set forth in sequence SEQ ID NO: HCDR3 shown at 15;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 16, as shown in sequence SEQ ID NO: 17 and/or LCDR2 as shown in SEQ ID NO: LCDR3 shown at 18.
Preferably, the amino acid sequence of the heavy chain variable region of the NG19 antibody of the present invention is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20.
The amino acid sequences of the preferred NG19 monoclonal antibody are respectively shown as SEQ ID NO. 21; the nucleotide sequences of the monoclonal antibodies are respectively shown as SEQ ID NO. 22.
A third anti-human NGAL monoclonal antibody (NG20) having a heavy chain variable region comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 23, HCDR1 as shown in sequence SEQ ID NO: 24 and/or HCDR2 as set forth in sequence SEQ ID NO: HCDR3 shown at 25;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 26, LCDR1 as shown in sequence SEQ ID NO: 27 and/or LCDR2 as set forth in sequence SEQ ID NO: LCDR3 shown at 28.
Preferably, the amino acid sequence of the heavy chain variable region of the NG20 antibody of the present invention is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30.
The amino acid sequences of the preferred NG20 monoclonal antibody are respectively shown as SEQ ID NO. 31; the nucleotide sequences of the monoclonal antibodies are respectively shown as SEQ ID NO. 32.
A fourth anti-human NGAL monoclonal antibody (NG37), the heavy chain variable region of which comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 33, HCDR1 as set forth in sequence SEQ ID NO: 34 and/or HCDR2 as shown in SEQ ID NO: HCDR3 shown at 35;
and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 36, LCDR1 as set forth in sequence SEQ ID NO: 37 and/or LCDR2 as set forth in sequence SEQ ID NO: LCDR3 shown at 38.
Preferably, the amino acid sequence of the heavy chain variable region of the NG37 antibody of the present invention is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40.
The amino acid sequences of the preferred NG37 monoclonal antibody are respectively shown as SEQ ID NO. 41; the nucleotide sequences of the monoclonal antibodies are respectively shown as SEQ ID NO. 42.
The invention also provides a latex enhanced immunoturbidimetry detection kit prepared from the recombinant anti-human NGAL monoclonal antibody mixture. The NGAL protein immune latex enhanced turbidimetric assay kit comprises an R1 reagent, an R2 reagent and a standard substance, wherein the R1 reagent comprises: buffer solution 1, stabilizer, coagulant, protective agent, preservative and surfactant; the R2 reagent includes: buffer solution 2, preservative, stabilizer and protective agent; the standard product comprises: buffer 3, protective agent and stabilizing agent.
Preferably, in the R1 reagent: the buffer solution 1 is phosphate buffer solution (0.01M-0.05M, pH7.0-7.4); the stabilizer is 40g/L-80g/L NaCl; 5-10g/L of PEG 6000-20000 as a thickening agent, 0-10 g/L of BSA as a protective agent, 0.05-0.1g/L of Proclin300 as a preservative, and 0.1-0.2% of Tween 80 or Tritonx100 as a surfactant.
Preferably, in the R2 reagent: the buffer solution 2 is 0.01M-0.05M phosphate buffer solution or ethanesulfonic acid buffer solution with pH5.9-7.4; the preservative is 0.1-0.2g/L of Proclin 300; the stabilizer is 10g/L-20g/L NaCl, 50 g/L-100 g/L glucose or sucrose, the protective agent is 10g/L-20g/L BSA, and 0-0.1% Tween 20; in addition, there are four kinds of latex microspheres (0.125-0.33 wt% concentration) labeled with recombinant mouse anti-human NGAL monoclonal antibody.
Preferably, the standard comprises 5 solutions of recombinant human NGAL at different concentrations, wherein: the buffer solution 3 is a phosphate buffer solution with the pH value of 7.0-7.4 and the M of 0.01-0.05; the protective agent is 0g/L-10g/L BSA, 0-2 g/L PEG8000 or 0-20 g/L sorbitol, and the stabilizer is 0-13g/L NaCl.
Preferably, the preparation method of the latex microsphere marked with NGAL protein antibody comprises the following steps:
diluting the equal mass mixture of 4 recombinant mouse anti-human NGAL monoclonal antibodies to 0.5-4mg/mL by using phosphate buffer solution to prepare an antibody diluent; diluting latex microspheres (polystyrene latex, purchased from JSR company, with the particle size of 100-.
The invention adopts the escherichia coli as an expression system to prepare the recombinant human NGAL, and the method has the characteristics of short period, large expression quantity and low cost. The prepared NGAL protein can be used as a raw material of a calibrator and a quality control product in a detection kit, can be used for preparing a mouse monoclonal antibody as an effective immunogen, is subjected to recombinant expression on the basis of obtaining a mouse antibody sequence, and the prepared single-chain antibody is coated on the surface of a latex microsphere and is used as an important raw material of an R2 reagent in the NGAL quantitative detection kit.
The invention prepares various antibodies, and performs pairing screening to obtain an antibody combination with sensitivity and specificity meeting the requirements; the antibody is convenient for mass production, and can meet the requirement of large-scale clinical application in the future; meanwhile, the antibody combination from the monoclonal antibody is convenient for the quality control of the raw materials of the kit, and can meet the requirement of stable performance of the kit. The antibody combination is debugged and optimized to obtain the quantitative detection reagent for latex immunoturbidimetry, which is simple and convenient to operate, and has sensitivity, specificity and related detection performance meeting the requirements of human clinical sample detection.
Drawings
The standard curve of the kit of fig. 1 is plotted, with the abscissa of the plot being the NGAL calibrator concentration and the ordinate being the absorbance Δ a.
Detailed Description
The present invention will be described in further detail below by way of examples, but the present invention is not limited to these examples.
EXAMPLE 1 preparation of murine mAb
1. Animal immunization
BALB/c female mice (purchased from Kyoto Kavens laboratory animals Co., Ltd., Changzhou) were immunized with NGAL protein (amino acid sequence shown in SEQ ID NO:1, codon sequence after optimization shown in SEQ ID NO: 2) according to a general immunization program. For specific immunization, see "guidelines for antibody preparation and use". And tracking the serum titer of the immune mice by adopting an indirect ELISA method, selecting the immune mice with the highest serum titer, and performing fusion experiments on the spleen cells and myeloma cells of the mice.
2. Cell fusion
(1) Preparation of spleen cells
Immunized mice, eyeballs are picked and blood is taken, after cervical vertebra is cut off, the immunized mice are placed in 75% (v/v) alcohol for soaking for 10 minutes, spleens of the immunized mice are taken out from a sterile operating platform, the spleens are placed in a cell screen, cells are fully ground, the cells are screened, the spleen is centrifugally washed for a plurality of times by using sterile 1640 culture medium (purchased from Gibco company), and then the cells are resuspended to prepare single cell suspension, and the single cell suspension is counted for standby.
(2) Preparation of feeder cells
Taking one female BALB/c mouse 8-10 weeks old, picking an eyeball to obtain negative serum, and immersing the negative serum in 75% (v/v) alcohol for 10 minutes after the cervical vertebra is cut off; the abdominal skin was aseptically peeled, the peritoneum was exposed, and about 10mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of the mouse with a syringe, and the abdomen was gently massaged and air-blown several times. Sucking the culture medium containing the macrophages and injecting the culture medium into 20% 1640HAT culture medium for later use;
taking one female BALB/c mouse with the age of 2-3 weeks, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies after cervical vertebra breakage; aseptically placing thymus into a cell screen, grinding, sieving to obtain thymocytes, and placing the thymocytes into the 20% 1640HAT culture medium containing macrophages for later use.
(3) Cell fusion
Mouse myeloma cell line SP2/0 was selected at the logarithmic growth phase and collected and counted. Get about 108The above spleen cells were combined with 2X 107Each of the above SP2/0 cell lines was mixed in a fusion tube, centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded (discarded as clean as possible), and the fusion tube was gently rubbed back and forth on the palm of the hand to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added slowly and quickly over 60 seconds, 30mL of 1640HT medium was added and stopped, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the precipitate was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.
Mixing the HAT culture medium, subpackaging at 200 μ L/well into 96-well cell culture plate, standing at 37 deg.C and 5% CO2Cultured in a cell culture box. After one week, 20% 1640HAT medium was replaced with 10% 1640HT medium, and after 3 days, the supernatant was examined.
3. Screening of anti-human NGAL specific hybridoma
(1) Preparation of the test plate: diluting the recombinant human NGAL protein to 1 mu g/mL by using CB coating solution, coating a 96-hole ELISA plate, coating the ELISA plate at 100 mu L/hole at 2-8 ℃ overnight, washing and patting the plate dry at one time; PBST buffer containing 2% bovine serum albumin was blocked (200 ul/well) for 2 hours at 37 ℃; patting dry for later use.
(2) Screening of positive clones: adding 100 mu L/well of cell culture supernatant to be detected into the detection plate, acting at 37 ℃ for 30 minutes, washing and drying, adding 100 mu L/well of HRP-labeled goat anti-mouse IgG, acting at 37 ℃ for 30 minutes, washing and drying, adding 100 mu L/well of TMB color development solution, developing in the dark at 37 ℃ for 15 minutes, adding 100 mu L/well of TMB color development solution into each well50 μ L of 2M H2SO4The reaction was stopped and the value read at OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1. Selecting positive clone strains to carry out cell cloning screening. After three to four rounds of cloning screening, the positive rate of the monoclonal cell strain is determined to be a stable cell strain with 100 percent of positive rate, and the cell strain is determined. The hybridoma cell strains NGAL08, NGAL19, NGAL20 and NGAL37 all have higher titer, and then the hybridoma cell strains are further subjected to antibody variable region sequence sequencing analysis.
Example 3 recombinant expression and purification of Single chain antibodies
According to the sequencing result in example 2, a connecting peptide (GGGGS)3 is added between the heavy chain and light chain variable regions of the antibodies of hybridoma cell strains NG08, NG19, NG20 and NG37, six histidines are introduced, the whole genes are directly fused, and the recombinant expression of the single-chain antibody is carried out by using a pichia pastoris expression system. The expressed single-chain antibodies were named NG08, NG19, NG20 and NG37, respectively. The recombinant expression of the single-chain antibody is specifically as follows:
a) construction of Single chain antibody Gene expression vector
The gene sequence of the single-chain antibody NG08 is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 11; the gene sequence of the single-chain antibody NG19 is shown as SEQ ID NO. 22, and the amino acid sequence is shown as SEQ ID NO. 21; the gene sequence of the single-chain antibody NG20 is shown as SEQ ID NO. 32, and the amino acid sequence is shown as SEQ ID NO. 31; the gene sequence of the single-chain antibody NG37 is shown in SEQ ID NO. 42, and the amino acid sequence is shown in SEQ ID NO. 41. Introducing XhoI restriction sites and XhoI sequences in a pPICZ alpha A vector into the upstream of gene fragments of single-chain antibodies NG08, NG19, NG20 and NG37, introducing histidine tags and XbaI restriction sites into the downstream of the gene fragments, carrying out whole-gene synthesis, and constructing into a pUC57 plasmid (purchased from Nanjing King Shirui Biotechnology Co., Ltd.), thereby obtaining a long-term storage plasmid, wherein the plasmids are named as pUC57-NG08-scFv, pUC57-NG19-scFv, pUC57-NG20-scFv and pUC57-NG 37-scFv. Performing PCR amplification, wherein
The upstream primer P1 is TGT AAA ACG ACG GCC AGT;
the downstream primer P2 is CAG GAA ACA GCT ATG AC.
After a conventional PCR procedure, agarose gel electrophoresis analysis revealed that the product size was consistent with the expected size.
After recovery and purification of the PCR-derived gene products, they were digested simultaneously with XhoI (# R0146S, available from New England Biolabs) and XbaI (# R0145V, available from New England Biolabs), ligated to pPICZ. alpha.A (V19520, available from Invitrogen) plasmid using T4 ligase, transformed into DH 5. alpha. competent cells, and cultured overnight at 37 ℃ in LB plates containing Zeocin (R250-01, available from Invitrogen). And screening positive clone bacteria for sequencing the next day, comparing the sequences, and obtaining a single-chain antibody expression vector which is marked as pPICZ alpha-NG 08-scFv, pPICZ alpha-NG 19-scFv, pPICZ alpha-NG 20-scFv and pPICZ alpha-NG 37-scFv, wherein the sequences are completely consistent with expected sequences.
b) Construction, screening and expression of single-chain antibody gene in pichia host engineering strain
YPDS solid medium preparation: refer to the Invitrogen company EasySelect Pichia Expression Kit Specification; pichia competent cells: refer to the easy select Pichia Expression Kit Specification; preparing a BMGY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen; preparing a BMMY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen.
The plasmids pPICZ alpha-NG 08-scFv, pPICZ alpha-NG 19-scFv, pPICZ alpha-NG 20-scFv and pPICZ alpha-NG 37-scFv were linearized by restriction with SacI restriction enzymes, respectively. After ethanol precipitation, the linearized vector is electrically transformed into X-33 competent yeast cells, spread on YPDS solid medium containing Zeocin and cultured at 30 ℃ for 3-5 days, and then positive clones are generated.
Inoculating the recombinant single-chain antibody genetic engineering strain obtained in the above step into BMGY culture medium, culturing at 30 deg.C and 220rpm until the thallus density reaches OD600Methanol was added every 24 hours to a final concentration of 1.0% (v/v) 2.0 to 6.0. After one week, the fermentation broth was collected.
c) Single chain antibody purification
The single-chain antibodies NG08, NG19, NG20 and NG37 are purified by adopting a histidine-tag affinity column, and HisTrap HP is selected as a pre-packed column, and the specific steps are as follows:
(1) impurity removal pretreatment of fermentation liquor: the supernatants of the fermentation broths of the single-chain antibodies NG08, NG19, NG20 and NG37 obtained by the above expression were centrifuged to collect the supernatants, and a binding buffer was added thereto so that the final concentration of the supernatants was 300mM NaCl, 20mM NaH2PO4 and 10mM Imidazole, adjusted to pH7.5, and filtered through a 0.45 μm filter.
(2) HisTrap HP affinity column purification: the pretreated single-chain antibodies NG08, NG19, NG20 and NG37 fermentation broth were subjected to affinity purification using a fully automated intelligent protein purification system (AKTA avant150, available from GE healthcare), and the column was HisTrap HP (17-5248-02, available from GE healthcare). The binding buffer was 300mM NaCl, 20mM NaH2PO410mM Imidazole, pH7.5, and elution buffer 300mM NaCl, 20mM NaH2PO4500mM Imidazole, pH 7.5. Linear elution was performed during elution and the individual elution peaks were collected. The purity of the purified protein reaches more than 95 percent; the collection tubes meeting the requirements are combined, the buffer solution is changed into a PBS solution, ultrafiltration concentration (1mg/ml) is carried out, and the mixture is filtered and sterilized and stored at the temperature of minus 20 ℃ for standby.
Example 3 preparation and methods of use of the assay kit
The detection kit is a liquid double reagent, and comprises an R1 reagent, an R2 reagent and a calibrator as follows:
1. r1 reagent: a stabilizing agent (such as NaCl), a thickening agent (such as PEG10000), a protective agent (such as BSA), a preservative (such as Proclin300) and a surfactant (such as Tritonx100) are added into the buffer solution 1, and the mixture is stirred and mixed uniformly to obtain the R1 reagent.
2. R2 reagent: diluting the equal mass mixture of 4 recombinant anti-human NGAL monoclonal antibodies to 1.0mg/mL by using a phosphate buffer solution to prepare an antibody diluent; diluting latex microspheres (polystyrene latex, purchased from JSR company, Japan, with a particle size of 220nm) to 1% (mass concentration) with ethanesulfonic acid buffer solution, adding 0.04 (mass ratio of EDC/latex microspheres) of activator EDC, stirring and reacting at 30 ℃ for 30min, removing unreacted EDC by centrifugation after the reaction is finished, adding 0.05 antibody/latex microspheres mass ratio) of the diluted solution of the recombinant anti-human NGAL monoclonal antibody mixture, stirring and reacting at 37 ℃ for 60min, removing unreacted antibody by centrifugation after the reaction is finished, adding a reaction terminating solution to terminate the reaction, centrifuging, and finally adding buffer solution 2 and a preservative (for example: proclin300), stabilizers (e.g.: NaCl, sucrose), protective agents (e.g.: BSA and Tween20) to obtain the R2 reagent (wherein the mass concentration of the latex microspheres marked with NGAL antibody is 0.125-0.33%).
3. Calibration products: NGAL standards were individually added to buffers consisting of buffer 3, a preservative (e.g., BSA) stabilizer (e.g., NaCl), and a preservative (e.g., Proclin300) to prepare a set of calibrators at final concentrations of 4524, 2162, 1075, 237, and 0 ng/mL.
Preferably, in order to improve the accuracy of the kit, different R1 and R2 and calibrator reagents are prepared for performance detection and comparison of accuracy (recovery rate) and analytical sensitivity, and specific evaluation results are shown in table 1 below. The kit prepared by selecting various buffers, stabilizers, protective agents, preservatives, chelating agents and the like has better detection performance.
Analysis sensitivity: taking a sample with the known content of the neutrophil gelatinase-associated lipocalin (or NGAL for short) of (100-300) ng/mL as a detection sample, repeatedly measuring for 3 times, and recording the absorbance difference of the kit under the specified parameters. The difference in absorbance was converted to 200ng/mL NGAL.
Accuracy (add recovery): selecting human serum sample (conventional sample) with proper concentration, adding calibrator (NGAL with theoretical concentration of C)Theory of the invention) Preparing recovery sample, adding deionized water into conventional sample to obtain base sample, wherein the amount of the calibrator does not exceed 1/10, detecting each recovery sample and base sample for 3 times, and determining average concentration as C1And C0. According to the calculation formula: recovery rate is (C)1-C0)/CTheory of the inventionRespective recoveries were calculated at 100%.
TABLE 1 preparation comparison of the kit
EXAMPLE 4 Performance test of the assay kit
Control kit: the recombinant antibody used in the reagent R2 is replaced by a commercial rabbit anti-human NGAL polyclonal antibody of A manufacturer, and other reagents and experimental schemes are the same as those of the preparation example of the example 3;
the kit of the invention comprises: see specifically the NGAL detection kit prepared in the preparation example of example 3;
commercial kits: clinical NGAL latex enhanced turbidimetric kit purchased from well-known C company;
the key performance of the kit is compared.
1. Linear range
And (3) measuring the absorbance of 4 NGAL standard substances with different concentrations at 540-600 nm by adopting a Beckmann full-automatic biochemical analyzer AU480, and drawing a working curve of the calibrator (see figure 1). The result shows that the detection range of the kit has wider linear range than that of a contrast kit, and the kit is more favorable for directly detecting a high-concentration pathological sample without diluting the sample, and is simple and easy to operate. In addition, the linear relationship is also better than that of the commercial kit.
Since the linear range of the control kit can not meet the clinical application requirements, further research on the control kit is not carried out. The following performance evaluations were carried out only for the kit of the present invention and commercial kits.
2. Precision degree
Repeatability: the kit provided by the invention is used for detecting clinical serum samples with NGAL concentrations of 100-200 ng/mL and 400-600 ng/mL, the test is repeated for 10 times, and the Coefficient of Variation (CV) is calculated respectively. As seen in the detection results, the repeatability of the kit and the commercialized kit completely reaches CV less than or equal to 10%, but the CV value of the kit is lower, which shows that the uniformity is better than that of the commercialized kit (see Table 2).
Inter-batch difference: the detection reagents of the invention with four different batches are respectively used, the NGAL concentration is 100-200 ng/mL and the clinical serum sample with 400-600 ng/mL, each batch is tested repeatedly for at least 3 times, and the reagent (kit) batch-to-batch difference R is calculated. The commercial kit is marked by 10 percent, and the detection result shows that the kit has small batch difference and stable process.
3. Accuracy of
Adding and recovering: selecting human serum sample (conventional sample) with proper concentration, adding calibrator (NGAL with theoretical concentration of C)Theory of the invention) Preparing recovery sample, adding deionized water into conventional sample to obtain base sample, wherein the amount of the calibrator does not exceed 1/10, detecting each recovery sample and base sample for 3 times, and determining average concentration as C1And C0. According to the calculation formula: recovery rate is (C)1-C0)/CTheory of the inventionRespective recoveries were calculated at 100%. The results show that the recovery rate of the kit can meet the requirement of 90-110%, and the accuracy (high concentration sample) of the commercial kit exceeds 110%, which shows that the kit has better specificity and accuracy (see table 2).
TABLE 2 evaluation results of accuracy and precision Properties
EXAMPLE 5 applicability of the test kit
The detection kit is applied to a full-automatic biochemical analyzer, but is not limited to the full-automatic biochemical analyzer, wherein the main parameter setting and detection method comprises the following steps (for example, Beckman full-automatic biochemical analyzer AU 5800):
detection wavelength: the main wavelength is 540-600 nm, and the auxiliary wavelength is none; detecting the temperature: 37 ℃;
the detection method comprises the following steps: and (3) uniformly mixing 180 mu L of reagent R1 with 1.5 mu L of sample, incubating for 5min, adding 45 mu L of reagent R2, immediately reading absorbance A1, incubating for 5min, reading absorbance A2, and calculating the change of absorbance delta A-A2-A1. And (3) drawing a working curve of the calibrator by using multipoint nonlinear calibration, wherein the NGAL content in the sample can be obtained by calculation on the working curve according to the delta A change value.
The kit disclosed by the invention is used for evaluating the performances such as precision, accuracy and the like on a Beckmann AU5800 biochemical analyzer (specifically see example 4, wherein the accuracy adopts a relative deviation method), and the results are shown in Table 3. The result shows that the precision and the accuracy of the kit meet the requirements, and the kit can also be applied to other types of Beckmann biochemical analyzers and has wider applicability.
TABLE 3 Performance of the kits of the invention in AU5800
Sequence listing
<110> Jiangsu Zhonghong bioengineering institute of drug creation Limited
<120> neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof
<130> neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof
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Met Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys
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Tyr Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp
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Pro Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
50 55 60
Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp
65 70 75 80
Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly
85 90 95
Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val
100 105 110
Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser
115 120 125
Gln Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu
130 135 140
Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu
145 150 155 160
Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys
165 170 175
Ile Asp Gly
<210> 2
<211> 543
<212> DNA
<213> Homo sapiens
<400> 2
atgcaggata gcacctctga cctgattccg gcaccgccgc tgagcaaagt gccgctgcag 60
caaaactttc aagataatca gttccaaggc aaatggtatg tggttggcct ggcgggtaac 120
gccattctgc gtgaagataa agacccgcag aaaatgtatg ctaccatcta cgaactgaaa 180
gaagataaat catacaacgt tacgtcggtc ctgtttcgta agaaaaaatg cgactactgg 240
attcgcacct ttgttccggg ctgtcagccg ggtgaattta cgctgggcaa catcaaaagc 300
tatccgggtc tgacctctta cctggtccgt gtcgtgagta cgaactacaa tcagcatgcg 360
atggtgtttt tcaagaaagt gagccagaat cgtgaatatt tcaaaatcac cctgtacggt 420
cgcaccaaag aactgacgag cgaactgaaa gaaaacttca tccgcttcag taaatccctg 480
ggcctgccgg aaaatcatat cgtgtttccg gttccgattg atcagtgcat cgacggttaa 540
tga 543
<210> 3
<211> 7
<212> PRT
<213> Mus musculus
<400> 3
Ile Asn Thr Ser Gly Ser Thr
1 5
<210> 4
<211> 9
<212> PRT
<213> Mus musculus
<400> 4
Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr
1 5
<210> 5
<211> 15
<212> PRT
<213> Mus musculus
<400> 5
Ala Arg Trp Ala Ile Tyr Tyr Gly Asn Phe Trp Tyr Phe Asp Val
1 5 10 15
<210> 6
<211> 5
<212> PRT
<213> Mus musculus
<400> 6
Ser Ser Val Ser Tyr
1 5
<210> 7
<211> 3
<212> PRT
<213> Mus musculus
<400> 7
Asn Thr Ser
1
<210> 8
<211> 9
<212> PRT
<213> Mus musculus
<400> 8
Gln Gln Arg Ser Ser Tyr Pro Phe Thr
1 5
<210> 9
<211> 123
<212> PRT
<213> Mus musculus
<400> 9
Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser
1 5 10 15
Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
20 25 30
Gly Tyr Tyr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu
35 40 45
Trp Met Gly Tyr Ile Asn Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Trp Ala Ile Tyr Tyr Gly Asn Phe Trp Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 10
<211> 106
<212> PRT
<213> Mus musculus
<400> 10
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Asn Thr Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 11
<211> 244
<212> PRT
<213> Mus musculus
<400> 11
Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser
1 5 10 15
Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
20 25 30
Gly Tyr Tyr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu
35 40 45
Trp Met Gly Tyr Ile Asn Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Trp Ala Ile Tyr Tyr Gly Asn Phe Trp Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln
130 135 140
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr
145 150 155 160
Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys
165 170 175
Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr Asn Thr Ser Asn Leu Pro
180 185 190
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr
195 200 205
Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
210 215 220
Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys
<210> 12
<211> 735
<212> DNA
<213> Mus musculus
<400> 12
tctgatgtgc agcttcagga gtcaggacct gacctggtga aaccttctca gtcactttca 60
ctcacctgca ctgtcactgg ctactccatc accagtggtt attactggca ctggatccgg 120
cagtttccag gaaacacact ggaatggatg ggctatataa ataccagtgg tagcactaac 180
tacaacccat ctctcaaaag tcgaatctct atcactcgag acacatccaa gaaccagttc 240
ttcctgcagt tgaattctgt gactactgag gacacagcca catattactg tgcaagatgg 300
gctatctact atggtaactt ctggtacttc gatgtctggg gcgcagggac cacggtcacc 360
gtctcctcag gtggtggtgg atccggaggt ggtggttctg gtggtggtgg ttctcaaatt 420
gttctcaccc agtctccagc aatcatgtct gcatctccag gggagaaggt caccataacc 480
tgcagtgcca gctcaagtgt aagttacata cactggttcc agcagaagcc aggcacttct 540
cccaaactct ggatttataa cacatccaac ctgccttctg gagtccctgc tcgcttcagt 600
ggcagtggat ctgggacctc ttactctctc acaatcagcc gaatggaggc tgaagatgct 660
gccacttatt actgccagca aaggagtagt tacccattca cgttcggctc ggggacaaag 720
ttggaaataa aataa 735
<210> 13
<211> 9
<212> PRT
<213> Mus musculus
<400> 13
Asp Ser Glu Val Phe Pro Ile Ala Tyr
1 5
<210> 14
<211> 8
<212> PRT
<213> Mus musculus
<400> 14
Ile Leu Pro Ser Ile Gly Arg Thr
1 5
<210> 15
<211> 11
<212> PRT
<213> Mus musculus
<400> 15
Ala Arg Ser Arg Tyr Glu Asp Tyr Phe Asp Tyr
1 5 10
<210> 16
<211> 6
<212> PRT
<213> Mus musculus
<400> 16
Gln Asp Val Ala Thr Ala
1 5
<210> 17
<211> 3
<212> PRT
<213> Mus musculus
<400> 17
Trp Ala Ser
1
<210> 18
<211> 9
<212> PRT
<213> Mus musculus
<400> 18
His Gln Tyr Ser Phe Tyr Pro Tyr Thr
1 5
<210> 19
<211> 118
<212> PRT
<213> Mus musculus
<400> 19
Asn Ser Ile Met Ala Trp Val Trp Asn Leu Leu Ile Pro Gly Ser Ser
1 5 10 15
Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Ile Ala
20 25 30
Tyr Ile Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp Ile
35 40 45
Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Asn Tyr Gly Glu Lys Phe
50 55 60
Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala Tyr
65 70 75 80
Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ser Arg Tyr Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Ser
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 20
<211> 108
<212> PRT
<213> Mus musculus
<400> 20
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ala Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys His Gln Tyr Ser Phe Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 21
<211> 240
<212> PRT
<213> Mus musculus
<400> 21
Asn Ser Ile Met Ala Trp Val Trp Asn Leu Leu Ile Pro Gly Ser Ser
1 5 10 15
Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Ile Ala
20 25 30
Tyr Ile Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp Ile
35 40 45
Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Asn Tyr Gly Glu Lys Phe
50 55 60
Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala Tyr
65 70 75 80
Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ser Arg Tyr Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Ser
100 105 110
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys Phe Met
130 135 140
Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
145 150 155 160
Asp Val Ala Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
165 170 175
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
180 185 190
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
195 200 205
Ser Ser Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys His Gln Tyr
210 215 220
Ser Phe Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
<210> 22
<211> 723
<212> DNA
<213> Mus musculus
<400> 22
aattcgatta tggcttgggt gtggaacttg cttattcctg ggtcttcagt aaagctttca 60
tgcaaggatt ttgattcaga agtcttccct attgcttata taagttggat tagacagaag 120
cctggccatg gatttgaatg gattggagac atactcccaa gtattggtag aacaaactat 180
ggagagaagt ttgaggacaa ggccacactg gatgcagaca cagtgtccaa cacagcctac 240
ttggagctca acagtctgac atctgaagac tctgctattt actattgtgc aaggagcagg 300
tacgaggact actttgacta ctggggccaa ggctccactc tcacagtctc ctcaggtggt 360
ggtggatccg gaggtggtgg ttctggtggt ggtggttctg acattgtgat gacccagtct 420
cacaaattca tgtccacatc agttggagac agggtcagca tcacctgcaa ggccagtcag 480
gatgtggcta ctgctgttgc ctggtatcaa cagaagccag gtcaatctcc taaacttctg 540
atttactggg catccacccg gcacactgga gtccctgatc gcttcacagg cagtggatct 600
gggacagatt tcactctcac cattagcagt gtgcagtctg aagacttggc agattatttc 660
tgtcaccaat atagcttcta tccgtacacg ttcggagggg ggaccaagct ggaaataaaa 720
taa 723
<210> 23
<211> 8
<212> PRT
<213> Mus musculus
<400> 23
Gly Phe Asn Ile Lys Tyr Asn Tyr
1 5
<210> 24
<211> 8
<212> PRT
<213> Mus musculus
<400> 24
Ile Asp Pro Ala Asn Gly Asn Thr
1 5
<210> 25
<211> 12
<212> PRT
<213> Mus musculus
<400> 25
Ala Arg Glu Gly Gly Arg Tyr Asp Gly Phe Ala Asn
1 5 10
<210> 26
<211> 12
<212> PRT
<213> Mus musculus
<400> 26
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr
1 5 10
<210> 27
<211> 3
<212> PRT
<213> Mus musculus
<400> 27
Trp Ala Ser
1
<210> 28
<211> 9
<212> PRT
<213> Mus musculus
<400> 28
Gln Asn Asp Tyr Ser Phe Pro Phe Thr
1 5
<210> 29
<211> 119
<212> PRT
<213> Mus musculus
<400> 29
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Leu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Tyr Asn
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Pro Ala Asp Ile Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Gly Arg Tyr Asp Gly Phe Ala Asn Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 30
<211> 113
<212> PRT
<213> Mus musculus
<400> 30
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 31
<211> 247
<212> PRT
<213> Mus musculus
<400> 31
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Leu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Tyr Asn
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Pro Ala Asp Ile Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Gly Arg Tyr Asp Gly Phe Ala Asn Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser
130 135 140
Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser
145 150 155 160
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser
180 185 190
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
210 215 220
Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Phe Pro Phe Thr Phe Gly Ser
225 230 235 240
Gly Thr Lys Leu Glu Ile Lys
245
<210> 32
<211> 744
<212> DNA
<213> Mus musculus
<400> 32
gaggttcagc tgcagcagtc tggggcagaa cttttgaagc caggggcctc agtcaagttg 60
tcctgcacag cttctggctt caacattaaa tacaactata tacactgggt gaaacagagg 120
cctgaacagg gcctggactg gattggaagg attgatcctg cgaatggtaa tactaaatat 180
gacccgaagt tccagggcaa ggccactatg ccagcagaca tatcctccaa cacagcctac 240
ctgcacctca gcagcctgac atctgaggac actgccgtct attactgtgc tagagagggg 300
gggaggtacg acggttttgc taactggggc caagggactc tggtcactgt ctctgcaggt 360
ggtggtggat ccggaggtgg tggttctggt ggtggtggtt ctgacattgt gatgacacag 420
tctccatcct ccctgactgt gacagcagga gagaaggtca ctatgagctg caagtccagt 480
cagagtctgt taaacagtgg aaatcaaaag aactacttga cctggtacca gcagaaacca 540
gggcagcctc ctaaattgtt gatctactgg gcatccacta gggaatctgg ggtccctgat 600
cgcttcacag gcagtggatc tggaacagat ttcactctca ccatcagcag tgtgcaggct 660
gaagacctgg cagtttatta ctgtcagaat gattatagtt ttccattcac gttcggctcg 720
gggacaaagt tggaaataaa ataa 744
<210> 33
<211> 8
<212> PRT
<213> Mus musculus
<400> 33
Gly Phe Ser Leu Thr Ser Tyr Gly
1 5
<210> 34
<211> 7
<212> PRT
<213> Mus musculus
<400> 34
Ile Trp Ser Asp Gly Ser Thr
1 5
<210> 35
<211> 10
<212> PRT
<213> Mus musculus
<400> 35
Ala Arg His Gly Tyr Tyr Ala Met Asp Tyr
1 5 10
<210> 36
<211> 6
<212> PRT
<213> Mus musculus
<400> 36
Gln Ser Ile Ser Asp Tyr
1 5
<210> 37
<211> 3
<212> PRT
<213> Mus musculus
<400> 37
Tyr Ala Ser
1
<210> 38
<211> 9
<212> PRT
<213> Mus musculus
<400> 38
Gln Asn Gly His Ser Phe Pro Trp Thr
1 5
<210> 39
<211> 116
<212> PRT
<213> Mus musculus
<400> 39
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Val Val Ile Trp Ser Asp Gly Ser Thr Thr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Arg His Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
115
<210> 40
<211> 107
<212> PRT
<213> Mus musculus
<400> 40
Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
65 70 75 80
Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 41
<211> 238
<212> PRT
<213> Mus musculus
<400> 41
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Val Val Ile Trp Ser Asp Gly Ser Thr Thr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Arg His Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
130 135 140
Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile
145 150 155 160
Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg
165 170 175
Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg
180 185 190
Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser
195 200 205
Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser
210 215 220
Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235
<210> 42
<211> 723
<212> DNA
<213> Mus musculus
<400> 42
aattcgatta tggcttgggt gtggaacttg cttattcctg ggtcttcagt aaagctttca 60
tgcaaggatt ttgattcaga agtcttccct attgcttata taagttggat tagacagaag 120
cctggccatg gatttgaatg gattggagac atactcccaa gtattggtag aacaaactat 180
ggagagaagt ttgaggacaa ggccacactg gatgcagaca cagtgtccaa cacagcctac 240
ttggagctca acagtctgac atctgaagac tctgctattt actattgtgc aaggagcagg 300
tacgaggact actttgacta ctggggccaa ggctccactc tcacagtctc ctcaggtggt 360
ggtggatccg gaggtggtgg ttctggtggt ggtggttctg acattgtgat gacccagtct 420
cacaaattca tgtccacatc agttggagac agggtcagca tcacctgcaa ggccagtcag 480
gatgtggcta ctgctgttgc ctggtatcaa cagaagccag gtcaatctcc taaacttctg 540
atttactggg catccacccg gcacactgga gtccctgatc gcttcacagg cagtggatct 600
gggacagatt tcactctcac cattagcagt gtgcagtctg aagacttggc agattatttc 660
tgtcaccaat atagcttcta tccgtacacg ttcggagggg ggaccaagct ggaaataaaa 720
taa 723
