CN113402606B

CN113402606B - Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof

Info

Publication number: CN113402606B
Application number: CN202010185664.5A
Authority: CN
Inventors: 马永; 杨芸; 赵利利
Original assignee: ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: ZONHON BIOPHARMA INSTITUTE Inc
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2023-06-06
Anticipated expiration: 2040-03-17
Also published as: CN113402606A

Abstract

The invention relates to four anti-human neutrophil gelatinase-associated lipocalin (NGAL) antibodies and application thereof in a detection kit for detecting the content of neutrophil gelatinase-associated lipocalin in a blood sample. In particular, the invention provides 4 kinds of mouse anti-human NGAL monoclonal antibodies and application thereof in a latex enhanced turbidimetric immunoassay kit. The invention prepares a plurality of antibodies, and performs pairing screening to obtain antibody combinations with sensitivity and specificity meeting requirements; meanwhile, the monoclonal antibody source antibody combination 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 latex immunoturbidimetric quantitative detection reagent which is easy and convenient to operate, and has the sensitivity, the specificity and the related detection performance can meet the requirements of human clinical sample detection is obtained by carrying out debugging and optimizing operation of a detection system on the antibody combination.

Description

Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof

Technical Field

The invention belongs to the field of medical immune in-vitro diagnosis, and in particular relates to recombinant neutrophil gelatinase-associated lipocalin and application thereof in a detection kit for detecting the content of the neutrophil gelatinase-associated lipocalin (neutropHil gelatinaseassociated lipocalin, NGAL) in a blood sample.

Background

Neutrophil gelatinase-associated lipocalin (neutropHil gelatinaseassociated lipocalin, NGAL), also known as human lipocalin 2 (lipocalin 2), has three forms of protein, monomer with a molecular weight of 25kD, self-polymerizing 46kD homodimer, and forming 135kD heterodimer with MMP-9 polymer. In recent years, NGAL has been used as a new marker of acute kidney injury, and when Acute Kidney Injury (AKI) is diagnosed early, NGAL concentration in blood and urine is usually increased rapidly, and 2h is most obvious, while serum creatinine, urease and the like which are traditional indexes need to be increased obviously after 24-72 h. NGAL is also a potential new marker reflecting chronic damage to the kidney, and in Chronic Kidney Disease (CKD) patients NGAL accurately reflects the extent of kidney damage, a powerful and independent risk indicator for CKD progression. In summary, the quantitative detection product of NGAL will have important clinical significance and strong market demand.

The detection methods of NGAL at present include enzyme-linked immunosorbent assay, chemiluminescence method, immunoturbidimetry and the like. The basic principle of the latex immunoturbidimetry is as follows: the NGAL protein in the clinical sample reacts with the specific anti-human NGAL antibody coated on the latex microsphere to form an immune complex of the antigen antibody, so that the latex microsphere forms a netty 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 demands and the popularization of full-automatic biochemical analyzers, the biochemical latex turbidimetry has the advantages of short reaction time, good precision, easy automation and the like, and becomes a main flow detection method in clinic.

Disclosure of Invention

The invention provides a recombinant neutrophil gelatinase related lipocalin (NGAL) with an amino acid sequence shown in SEQ ID NO:1, the codon sequence after optimization is shown as SEQ ID NO: 2.

The invention also provides 4 murine anti-human NGAL monoclonal antibodies, a first anti-human NGAL monoclonal antibody (NG 08), the heavy chain variable region of which comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:3, HCDR1 as set forth in SEQ ID NO:4 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown in 5;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:6, LCDR1 as set forth in SEQ ID NO:7 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in fig. 8.

Preferably, the amino acid sequence of the heavy chain variable region of the NG08 antibody is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10.

The amino acid sequence of the preferred NG08 monoclonal antibody is shown as SEQ ID NO. 11; the nucleotide sequences for encoding the monoclonal antibodies are respectively shown as SEQ ID NO. 12.

A second anti-human NGAL monoclonal antibody (NG 19) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:13, HCDR1 as set forth in SEQ ID NO:14 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 15;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:16, LCDR1 as set forth in SEQ ID NO:17 and/or LCDR2 as set forth in SEQ ID NO:18, LCDR3.

Preferably, the amino acid sequence of the heavy chain variable region of the NG19 antibody is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20.

The amino acid sequence of the preferred NG19 monoclonal antibody is shown as SEQ ID NO. 21; the nucleotide sequences for encoding the monoclonal antibodies are respectively shown as SEQ ID NO. 22.

A third anti-human NGAL monoclonal antibody (NG 20) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:23, HCDR1 as set forth in SEQ ID NO:24 and/or HCDR2 as set forth in SEQ ID NO:25, HCDR3;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:26, LCDR1 as set forth in SEQ ID NO:27 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 28.

Preferably, the amino acid sequence of the heavy chain variable region of the NG20 antibody is shown as SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 30.

The amino acid sequence of the preferred NG20 monoclonal antibody is shown as SEQ ID NO. 31; the nucleotide sequences for encoding the monoclonal antibodies are respectively shown as SEQ ID NO. 32.

A fourth anti-human NGAL monoclonal antibody (NG 37) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:33, HCDR1 as set forth in SEQ ID NO:34 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 35;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:36, LCDR1 as set forth in SEQ ID NO:37 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 38.

Preferably, the amino acid sequence of the heavy chain variable region of the NG37 antibody is shown as SEQ ID NO. 39, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 40.

The amino acid sequence of the preferred NG37 monoclonal antibody is shown as SEQ ID NO. 41; the nucleotide sequences for encoding the monoclonal antibodies are respectively shown as SEQ ID NO. 42.

The invention also provides a latex-enhanced turbidimetric immunoassay kit prepared from the recombinant anti-human NGAL monoclonal antibody mixture. The invention discloses an NGAL protein immune latex enhanced turbidimetric detection kit, which 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 comprises: buffer solution 3, protective agent and stabilizer.

Preferably, in the R1 reagent: phosphate buffer (0.01M-0.05M, pH 7.0-7.4) as buffer 1; the stabilizer is 40g/L-80g/L NaCl; 5-10g/L PEG 6000-20000, 0-10 g/L BSA as protecting agent, 0.05-0.1g/L Proclin300 as preservative, 0.1-0.2% Tween 80 or Tritonx100 as surfactant.

Preferably, in the R2 reagent: buffer 2 is phosphate buffer or ethanesulfonic acid buffer with pH of 5.9-7.4 and 0.01M-0.05M; the preservative is Proclin300 with the concentration of 0.1-0.2 g/L; the stabilizer is 10g/L-20g/L NaCl, 50 g/L-100 g/L glucose or sucrose, the protecting agent is 10g/L-20g/L BSA, 0-0.1% Tween 20; in addition, there are latex microspheres (0.125% -0.33% by mass concentration) labeled with four recombinant murine anti-human NGAL monoclonal antibodies.

Preferably, the standard comprises 5 solutions of recombinant human NGAL at different concentrations, wherein: buffer 3 is phosphate buffer with pH7.0-7.4 and 0.01-0.05M; the protecting agent is BSA of 0g/L-10g/L, PEG8000 of 0-2 g/L or sorbitol of 0-20 g/L, and NaCl of 0-13 g/L.

Preferably, the preparation method of the latex microsphere marked with the NGAL protein antibody comprises the following steps:

diluting the mass mixture of 4 recombinant mouse anti-human NGAL monoclonal antibodies to 0.5-4mg/mL by using phosphate buffer solution to prepare antibody diluent; diluting latex microspheres (polystyrene latex, purchased from JSR corporation, with the particle size of 100-400 nm) to 1% (mass concentration) by using MES buffer solution, adding EDC with the mass ratio of 0.02-0.08 (EDC/latex microspheres), stirring and reacting for 30min at 30 ℃, removing unreacted EDC through centrifugation after the reaction is finished, adding rabbit anti-human NGAL polyclonal antibody diluent, stirring and reacting for 30-120min at 25-37 ℃, removing unreacted antibody through centrifugation after the reaction is finished, and adding termination reaction solution to terminate the reaction.

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 calibrator and quality control material in a detection kit, can be used as an effective immunogen for preparing a mouse monoclonal antibody, can be subjected to recombinant expression on the basis of obtaining a mouse anti-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 a plurality of antibodies, and performs pairing screening to obtain antibody combinations with sensitivity and specificity meeting requirements; the antibody is convenient for mass production, and can meet the requirement of large-scale clinical application in the future; meanwhile, the monoclonal antibody source antibody combination 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 latex immunoturbidimetric quantitative detection reagent which is easy and convenient to operate, and has the sensitivity, the specificity and the related detection performance can meet the requirements of human clinical sample detection is obtained by carrying out debugging and optimizing operation of a detection system on the antibody combination.

Drawings

The standard curve of the kit of FIG. 1 is plotted with the concentration of NGAL calibrator on the abscissa and absorbance DeltaA on the ordinate.

Detailed Description

The present invention will be described in further detail by way of examples, but the present invention is not limited to these examples.

EXAMPLE 1 preparation of murine monoclonal antibody

1. Immunization of animals

BALB/c female mice (purchased from Kwanas laboratory animal Co., ltd.) were immunized with NGAL protein (amino acid sequence shown as SEQ ID NO:1, codon sequence shown as SEQ ID NO:2 after optimization) according to a general immunization program. Specific immunization conditions are described in the guidelines for antibody preparation and use. The serum titer of the immunized mice is tracked by adopting an indirect ELISA method, and the immunized mice with the highest serum titer are selected to perform fusion experiments on spleen cells and myeloma cells of the mice.

2. Cell fusion

(1) Preparation of spleen cells

Mice were immunized, blood was collected from the eyeballs, sacrificed by cervical spine breakage, immersed in 75% (v/v) alcohol for 10 minutes, the spleens were removed in a sterile operating table, placed in a cell screen, the cells were ground thoroughly, the screen was washed by centrifugation with sterile 1640 medium (available from Gibco company) for several times, and the cells were resuspended to make a single cell suspension and counted for use.

(2) Preparation of feeder cells

Taking one female BALB/c mouse of 8-10 weeks old, picking eyeball to obtain negative serum, killing by cervical vertebra and soaking in 75% (v/v) alcohol for 10 min; the abdominal skin was aseptically uncovered, the peritoneum was exposed, and about 10ml of 1640ht medium (purchased from SIGMA) was injected into the abdominal cavity of the mice using a syringe, the abdomen was gently massaged and blown several times. Sucking the medium containing the macrophages and injecting the medium into 20%1640HAT medium for later use;

taking one female BALB/c mouse with 2-3 weeks of age, killing the female BALB/c mouse by cervical vertebra fracture, and soaking the female BALB/c mouse in 75% (v/v) alcohol for 10 minutes; taking thymus in a cell screen in a sterile manner, grinding, screening to obtain thymus cells, and placing the thymus cells in the 20%1640HAT medium containing macrophages for later use.

(3) Cell fusion

Mouse myeloma cell line SP2/0 in log phase was selected, collected and counted. Get about 10 ⁸ Spleen cells as described above and 2X 10 ⁷ Each of the above SP2/0 cell lines was added to the fusion tube and mixed, the supernatant was discarded (discarded as much as possible) after centrifugation at 1000rpm, and the fusion tube was gently rubbed back and forth over the palm to loosen the pellet. Slowly and then quickly adding within 60 seconds1mL of pre-heated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added to stop 30mL of 1640HT medium, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the pellet was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.

Mixing the HAT culture medium, sub-packaging into 96-well cell culture plate at 200 μl/well, and placing at 37deg.C and 5% CO ₂ Is cultured in a cell culture incubator. After one week, 20%1640hat medium was replaced with 10%1640ht medium, and the supernatant was taken after 3 days for detection.

3. Screening of anti-human NGAL specific hybridoma strains

(1) Preparation of the detection plate: diluting recombinant human NGAL protein to 1 mug/mL by using CB coating liquid, coating a 96-hole ELISA enzyme label plate, coating 100 mug/hole, coating overnight at 2-8 ℃, washing and beating to dryness once; blocking (200 ul/well) with 2% bovine serum albumin in PBST buffer, blocking for 2 hours at 37 ℃; and (5) drying the mixture for standby.

(2) Screening of positive clones: adding 100 μL/well of cell culture supernatant to be detected into the detection plate, washing and drying after 30min at 37 ℃, adding 100 μL/well of HRP-labeled goat anti-mouse IgG, washing and drying after 30min at 37 ℃, adding 100 μL/well of TMB color development liquid, developing for 15 min at 37 ℃ in a dark place, and adding 50 μL of 2M H per well ₂ SO ₄ The reaction was terminated and the values were read at OD 450. Positive well determination principle: OD450 value/negative control value is more than or equal to 2.1. And selecting positive clone strains for cell cloning screening. After three to four rounds of cloning and screening, determining that the positive rate of the monoclonal cell strain is 100 percent as a stable cell strain, and determining the cell strain. The hybridoma cell strains NGAL08, NGAL19, NGAL20 and NGAL37 have higher titers, and then the antibody variable region sequence of the hybridoma cell strains is further analyzed.

EXAMPLE 3 recombinant expression and purification of Single chain antibodies

According to the sequencing results in example 2, the hybridoma cell lines NG08, NG19, NG20 and NG37 were added with a connecting peptide (GGGGS) 3 between their heavy and light chain variable regions, six histidines were introduced, and their whole genes were directly fused and recombinant expression of single chain antibodies was performed using pichia pastoris expression system. The expressed single chain antibodies were designated 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 as SEQ ID NO. 42, and the amino acid sequence is shown as SEQ ID NO. 41. The single-chain antibodies NG08, NG19, NG20 and NG37 gene fragments were introduced with XhoI cleavage site upstream and XhoI sequence in pPICZ alpha A vector, histidine tag and XbaI cleavage site downstream, subjected to total gene synthesis and constructed into pUC57 plasmid (purchased from Nanjing Jinsri Biotechnology Co., ltd.) to obtain a long-term preservation plasmid, which was designated pUC57-NG08-scFv, pUC57-NG19-scFv, pUC57-NG20-scFv and pUC57-NG37-scFv. Performing PCR amplification in which

The upstream primer P1 is TGT AAA ACG ACG GCC AGT;

the downstream primer P2 was CAG GAA ACA GCT ATG AC.

After conventional PCR procedures, agarose gel electrophoresis analysis showed that the product size was consistent with the expected size.

After the PCR-derived gene products were recovered and purified, they were digested with XhoI (#R0146S, available from New England Biolabs) and XbaI (#R0145V, available from New England Biolabs), ligated into pPICZαA (V19520, available from Invitrogen) plasmid with T4 ligase, transformed into DH5 α competent cells, and cultured overnight at 37℃in LB plates containing Zeocin (R250-01, available from Invitrogen). The positive clone bacteria are screened for sequencing and comparison in the next day, and the single-chain antibody expression vector is obtained after the sequence is completely consistent with the expected sequence, and the single-chain antibody expression vector is marked as pPICZ alpha-NG 08-scFv, pPICZ alpha-NG 19-scFv, pPICZ alpha-NG 20-scFv and pPICZ alpha-NG 37-scFv.

b) Construction, screening and expression of single-chain antibody gene in pichia pastoris host engineering strain

Preparation of YPDS solid medium: refer to Invitrogen company EasySelect Pichia Expression Kit specification; pichia pastoris competent cells: reference is made to EasySelect Pichia Expression Kit description; BMGY medium preparation: refer to Invitrogen company Multi-Copy Pichia Expression Kit Specification; preparing BMMY culture medium: refer to Invitrogen company Multi-Copy Pichia Expression Kit Specification.

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 enzyme digestion with SacI, respectively. After ethanol precipitation, the linearized vector was electrotransformed into X-33 competent yeast cells, plated onto Zeocin-containing YPDS solid medium, and cultured at 30℃for 3-5 days, and positive clones were generated.

Inoculating the recombinant single-chain antibody gene engineering strain obtained in the above to BMGY culture medium, culturing at 30deg.C and 220rpm until the bacterial density reaches OD ₆₀₀ =2.0 to 6.0, methanol was added every 24 hours to a final concentration of 1.0% (v/v). After one week, the fermentation broth was collected.

c) Single chain antibody purification

Purifying single-chain antibodies NG08, NG19, NG20 and NG37 by using histidine tag affinity columns, and selecting a pre-packed column as a HisTrap HP, wherein the specific steps are as follows:

(1) Impurity removal pretreatment of fermentation liquor: the single-chain antibodies NG08, NG19, NG20 and NG37 were obtained by the above expression, and the supernatant was collected by centrifugation and filtered with a 0.45 μm filter membrane by adding a binding buffer so that the final concentration of the supernatant was 300mM NaCl,20mM NaH2PO4, 10mM Imidazole, pH7.5.

(2) Purification by HisTrap HP affinity column: single chain antibodies NG08, NG19, NG20 and NG37 fermentation broth obtained from the pretreatment were affinity purified using a fully automatic intelligent protein purification system (AKTA avant150, available from GE healthcare Co.), and the column was HisTrap HP (17-5248-02, available from GE healthcare Co.). The binding buffer was 300mM NaCl,20mM NaH ₂ PO ₄ 10mM Imidazole,pH7.5 elution buffer 300mM NaCl,20mM NaH ₂ PO ₄ 500mM Imidazole,pH7.5. The elution was performed linearly and the individual elution peaks were collected. PurifiedThe purity of the protein reaches more than 95 percent; combining collecting pipes meeting the requirements, changing buffer solution into PBS solution, ultrafiltering and concentrating (1 mg/ml), filtering and sterilizing, and preserving at-20deg.C for use.

Example 3 preparation and use of detection kit

The detection kit of the invention is a liquid double reagent, which comprises an R1 reagent, an R2 reagent and a calibrator, and is specifically shown as follows:

1. r1 reagent: adding a stabilizer (such as NaCl), a coagulant enhancer (such as PEG 10000), a protective agent (such as BSA), a preservative (such as Proclin 300) and a surfactant (such as Triton X100) into the buffer solution 1, and stirring and mixing 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 phosphate buffer solution to prepare antibody diluent; latex microspheres (polystyrene latex, available from JSR corporation, particle size 220 nm) were diluted to 1% (mass concentration) with ethanesulfonic acid buffer, then 0.04 (EDC/latex microsphere mass ratio) activator EDC was added, stirred at 30 ℃ for 30min, after the reaction was completed, unreacted EDC was removed by centrifugation, then 0.05 antibody/latex microsphere mass ratio) recombinant anti-human NGAL monoclonal antibody mixture dilution was added, stirred at 37 ℃ for 60min, after the reaction was completed, unreacted antibody was removed by centrifugation, then termination reaction was terminated by adding termination reaction solution, centrifugation, and finally a buffer 2, preservative (e.g.: proclin 300), stabilizers (e.g.: naCl, sucrose), protectants (e.g.: BSA, tween 20) to obtain an R2 reagent (wherein the mass concentration of the latex microsphere marked with NGAL antibody is 0.125-0.33%).

3. Calibration material: the NGAL standard substance is respectively added into a buffer solution composed of a buffer solution 3, a protective agent (such as BSA), a stabilizer (such as NaCl) and a preservative (such as Proclin 300), and a group of calibration substances with final concentrations of 4524, 2162, 1075, 237 and 0ng/mL are prepared.

Preferably, in order to improve the accuracy of the kit, different R1, R2 and calibrator reagents are prepared for accuracy (recovery rate) and performance detection and comparison of analysis sensitivity, and specific evaluation results are shown in the following table 1. The kit prepared by selecting various buffers, stabilizers, protective agents, preservatives, chelating agents and the like has better detection performance.

Analytical 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 value of the kit under the specified parameters. Converted to 200ng/mL of the absorbance difference caused by NGAL.

Accuracy (addition recovery): adding a certain volume of calibrator (theoretical concentration of NGAL is C) into a human serum sample (conventional sample for short) with proper concentration _{Theory of} ) Preparing a recovered sample, adding deionized water with the same volume into a conventional sample to prepare a basic sample, wherein the added calibrator is not more than 1/10 of the total volume, detecting each recovered sample and the basic sample for 3 times, and determining that the average concentration corresponds to C ₁ And C ₀ . According to the calculation formula: recovery = (C ₁ -C ₀ )/C _{Theory of} * The respective recovery rates were calculated at 100%.

Table 1 preparation of the kit and comparison results

Example 4 Performance test of detection 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 embodiment 3;

the kit comprises: specifically, the NGAL detection kit prepared in the preparation example of the example 3;

commercial kit: clinical NGAL latex-enhanced turbidimetric kits purchased from well-known company C;

the key performances of the above kits were compared.

1. Linear range

The absorbance of 4 NGAL standard substances with different concentrations at 540-600 nm is measured by adopting a Beckmann full-automatic biochemical analyzer AU480, and a working curve of the calibrator is drawn (see figure 1). As can be seen from the results, the detection range of the kit provided by the invention has a wider linear range than that of a control kit, is more beneficial to directly detecting a high-concentration pathological sample, does not need to dilute the sample, and is easy to operate. In addition, the linear relationship is also better than commercial kits.

The linear range of the control kit cannot meet the clinical application requirements, and further research is not performed. The following performance evaluations were only developed for the kits of the present invention and commercial kits.

2. Precision of

Repeatability: clinical serum samples with NGAL concentration of 100-200 ng/mL and 400-600 ng/mL are detected by the kit, the test is repeated 10 times, and the Coefficient of Variation (CV) is calculated respectively. The detection result shows that the repeatability of the kit and the commercial kit completely reaches CV less than or equal to 10 percent, but the CV value of the kit is lower, which shows that the uniformity is superior to that of the commercial kit (see Table 2).

Batch-to-batch difference: the detection reagent of the invention with four different lot numbers is used for detecting clinical serum samples with the concentration of NGAL of 100-200 ng/mL and 400-600 ng/mL, each lot number is tested repeatedly for at least 3 times, and the difference R between the reagent (kit) lots is calculated. The detection result shows that the kit has small batch-to-batch difference and stable process as shown by 10% of the labeling of the commercial kit.

3. Accuracy of

And (3) adding and recycling: adding a certain volume of calibrator (theoretical concentration of NGAL is C) into a human serum sample (conventional sample for short) with proper concentration _{Theory of} ) Preparing a recovered sample, adding deionized water with the same volume into a conventional sample to prepare a basic sample, wherein the added calibrator is not more than 1/10 of the total volume, detecting each recovered sample and the basic sample for 3 times, and determining that the average concentration corresponds to C ₁ And C ₀ . According to the calculation formula: recovery = (C ₁ -C ₀ )/C _{Theory of} * The respective recovery rates were calculated at 100%. The result shows that the recovery rate of the kit can meet the requirement of 90-110 percent, and the quotientThe accuracy (high concentration samples) of the standardized kit exceeded 110%, showing that the kit of the present invention had 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 of the invention 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 is as follows (for example, beckmann full-automatic biochemical analyzer AU 5800):

detection wavelength: the main wavelength is 540-600 nm, and the auxiliary wavelength is absent; detecting the temperature: 37 ℃;

the detection method comprises the following steps: 180 mu L of the reagent R1 and 1.5 mu L of the sample are evenly mixed, 45 mu L of the reagent R2 is added after incubation for 5min, the absorbance A1 is immediately read, the absorbance A2 is read after incubation for 5min, and the absorbance change DeltaA=A2-A1 is calculated. And drawing a working curve of the calibrator by using multipoint nonlinear calibration, wherein the NGAL content in the sample can be calculated on the working curve according to the delta A variation value.

The performance of the kit of the invention is evaluated on a Beckmann AU5800 biochemical analyzer for precision, accuracy and the like (see in particular example 4, wherein the accuracy adopts a relative deviation method), and the results are shown in Table 3. The results show that the precision and the accuracy of the kit meet the requirements, and the kit can be applied to other models of Beckmann biochemical analyzers and has wider applicability.

TABLE 3 Performance of the inventive kits on AU5800

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<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

Claims

1. An anti-human NGAL monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:3, HCDR1 as set forth in SEQ ID NO:4 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown in 5;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:6, LCDR1 as set forth in SEQ ID NO:7 and LCDR2 as set forth in SEQ ID NO: LCDR3 as 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 whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:13, HCDR1 as set forth in SEQ ID NO:14 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 15;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:16, LCDR1 as set forth in SEQ ID NO:17 and LCDR2 as set forth in SEQ ID NO:18, LCDR3.

4. The anti-human NGAL monoclonal antibody according to claim 3, wherein the amino acid sequence is shown in SEQ ID NO. 21.

5. An anti-human NGAL monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:23, HCDR1 as set forth in SEQ ID NO:24 and HCDR2 as set forth in SEQ ID NO:25, HCDR3;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:26, LCDR1 as set forth in SEQ ID NO:27 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 28.

6. The anti-human NGAL monoclonal antibody according to claim 5, wherein the amino acid sequence is shown in SEQ ID NO. 31.

7. An anti-human NGAL monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:33, HCDR1 as set forth in SEQ ID NO:34 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 35;

and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:36, LCDR1 as set forth in SEQ ID NO:37 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 38.

8. The anti-human NGAL monoclonal antibody according to claim 7, wherein the amino acid sequence is shown in SEQ ID NO. 41.

9. A human NGAL latex-enhanced turbidimetric immunoassay kit comprising an R1 reagent, an R2 reagent and a standard, wherein the R2 reagent comprises latex microspheres simultaneously labeled with the anti-human NGAL monoclonal antibodies of claims 1, 3, 5 and 7.

10. The human NGAL latex-enhanced turbidimetric immunoassay kit of claim 9, wherein the R1 reagent: the buffer solution is phosphate buffer solution with the concentration of 0.01M-0.05M and the pH value of 7.0-7.4; the stabilizer is 40g/L-80g/L NaCl; PEG 6000-20000 with 5-10g/L of coagulant, BSA with 0-10 g/L of protective agent, proclin300 with 0.05-0.1g/L of preservative, tween 80 or Tritonx100 with 0.1-0.2% of surfactant;

among the R2 reagents: the buffer solution is phosphate buffer solution or ethanesulfonic acid buffer solution with the concentration of 0.01M-0.05M and the pH of 5.9-7.4; the preservative is Proclin300 with the concentration of 0.1-0.2 g/L; the stabilizer is 10g/L-20g/L NaCl, 50 g/L-100 g/L glucose or sucrose, the protecting agent is 10g/L-20g/L BSA, and 0-0.1% Tween 20; in addition, there are latex microballoons marked with 4 kinds of recombinant mouse anti-human NGAL monoclonal antibodies, the mass concentration is 0.125% -0.33%;

the standard comprises 5 solutions of recombinant human NGAL at different concentrations, wherein: the buffer solution is phosphate buffer solution with the concentration of 0.01M-0.05M and the pH value of 7.0-7.4; the protecting 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.