CN110618277B

CN110618277B - Method for measuring fecal pancreatic elastase-1 by latex enhanced immunoturbidimetry

Info

Publication number: CN110618277B
Application number: CN201910850314.3A
Authority: CN
Inventors: 侯志波; 巴里·马歇尔; 郑敬元; 张伟
Original assignee: Shenzhen Hongmei Diagnosis Technology Co ltd
Current assignee: Shenzhen Hongmei Diagnosis Technology Co ltd
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2020-08-04
Anticipated expiration: 2039-09-10
Also published as: CN110618277A

Abstract

The invention combines the monoclonal antibody into the latex enhanced immunoturbidimetry for the first time, is used for detecting the pancreatic elastase 1 in human excrement and diagnosing the chronic pancreatitis or the pancreatic exocrine dysfunction, and compared with the prior art, the monoclonal antibody for specifically identifying the pancreatic elastase 1 is adopted, so that the detection sensitivity and specificity are obviously improved, and the method has a certain clinical application prospect.

Description

Method for measuring fecal pancreatic elastase-1 by latex enhanced immunoturbidimetry

Technical Field

The invention belongs to the technical field of medical examination and in-vitro diagnosis, relates to a kit for measuring pancreatic elastase 1 in excrement, and particularly relates to a kit for quantitatively measuring pancreatic elastase 1 in human excrement by using a latex-enhanced immunoturbidimetry method and application thereof.

Background

Fecal pancreatic elastase 1 (FE-1) is a pancreatic endoprotease secreted from the exocrine gland, and is excreted into the duodenum via the duodenal papilla, and when the concentration is lowered, it specifically suggests the occurrence of chronic pancreatitis or pancreatic exocrine insufficiency. The fecal pancreatic elastase 1 has stability in intestinal tract, and can indirectly reflect the function of pancreatic exocrine by detecting the content of the fecal elastase in the feces, and the method is safe, non-invasive and not influenced by pancreatic enzyme replacement therapy. However, the method for detecting the fecal trypsin elastase 1 by the conventional immunochromatography method has the problems of low accuracy and specificity, complex operation and inconvenient detection.

When light passes through a turbid medium solution, the light is partially absorbed due to the presence of turbid particles in the solution, the amount of absorption being proportional to the amount of turbid particles, and this method of determining the amount of light absorption is known as turbidimetric transmittance. The method is earlier than Schultre and Schuick et al reported in 1959 that the method is applied to the change of turbidity caused by the formation of a complex after the combination of plasma protein and antibody, and then transmission turbidimetry is carried out, generally adopting a transmission turbidimetry method for the quantification of antigen by antibody, which is called immunity transmission. However, classical immunoturbidimetry, with a small amount of small antigen-antibody complexes, is extremely difficult to develop turbidity unless left for a long time: if larger complexes are formed, the amount of antigen and antibody used is also larger, and obviously does not meet the requirement of micro-quantification. Latex enhanced immunoturbidimetry (PETIA) now widely used in biochemical analyzers was developed. The principle is a method of forming a complex by utilizing the specific binding between an antigen and an antibody, and quantifying the antigen or the antibody by measuring the amount of the formed complex.

A latex-enhanced immunoturbidimetric assay (L atex-enhanced immunoturbidimetric assay) is a homogeneous transmission immunoturbidimetric assay for humoral protein, which is characterized in that polyclonal antibodies are cross-linked on the surface of nano-scale polymer latex microspheres, and the microspheres, when being combined with antigens, can rapidly aggregate together in a short time, thereby changing the light transmittance of reaction liquid, and the change of the light transmittance (i.e. absorbance) of the reaction liquid has strong correlation with the concentration of the detected antigens, and the concentration of the detected antigens can be reflected in a certain range.

With the development of inspection medicine, detection of low-concentration detection substances is more and more, the concentration of the detection substances is being reduced from the level of mu g/m L to the level of ng/m L, the requirement on the sensitivity of diagnostic reagents is higher, and for the traditional latex-enhanced immunoturbidimetry, the detection sensitivity needs to be further improved to meet the detection requirement, the traditional method for improving the detection sensitivity comprises the steps of adopting antibodies with higher affinity, adopting larger particles and latex particles with better stability, selecting a better buffer solution system for promoting antigen-antibody reaction and the like, but the cost of the reagents is increased, how to improve the analysis sensitivity and precision of analytes facing the level of ng/m L is improved, and meanwhile, the development of the reagents is always challenged without increasing the cost.

In the prior art, no related kit and detection method for determining pancreatic elastase 1 in human excrement by using a latex enhanced immunoturbidimetry method exist at present. And the current latex-enhanced immunoturbidimetry is based on polyclonal antibodies, and a detection method based on monoclonal antibodies is not described.

Disclosure of Invention

Therefore, the invention aims to solve the technical problems, and provides a kit for quantitatively determining human fecal pancreatic elastase 1 by using a latex-enhanced turbidimetric immunoassay and application thereof.

In order to solve the technical problems, the technical scheme of the invention is as follows:

in one embodiment, the monoclonal antibody is a monoclonal antibody that can be used in latex turbidimetric detection, and the monoclonal antibody is prepared by analyzing the antigenicity, hydrophilicity and surface structure probability of the protein sequence of the trypsin 1 (SEQ ID NO:1) using DNASTAR software, selecting a 17 amino acid or so peptide fragment with high antigenicity, high hydrophilicity and high surface probability as the immunizing peptide fragment L PQEGAI L ANNSPCYITC (FE-1-AG, SEQ ID NO:2) with the sequence corresponding to the amino acids at position 132 and 148 of the protein and the terminal cysteine artificially added to provide free thiol groups for coupling to a carrier protein.

(1) The preparation of the antigen is carried out,

(2) animal immunization BA L B/C mice were immunized,

(3) the cells are fused and the cell is fused,

(4) screening the monoclonal antibody, and screening,

(5) and (3) preparing a monoclonal antibody.

One aspect of the invention provides a kit for quantitatively determining pancreatic elastase 1 in excrement, which comprises a reagent 1, a reagent 2 and a pancreatic elastase 1 antigen calibrator solution; wherein the reagent 1 comprises an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution; the reagent 2 comprises latex particles coated with an anti-human trypsin 1 monoclonal antibody, electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution; the pancreatic elastase 1 antigen calibrator solution comprises pancreatic elastase 1 and a stabilizer. In a specific embodiment, the anti-human trypsin 1 monoclonal antibody is coupled to the surface of the latex particle by chemical crosslinking, wherein the chemical crosslinking comprises the following steps:

s1, adding latex particles and a crosslinking surfactant into the crosslinking buffer solution to obtain a latex particle solution;

s2, dissolving the anti-human pancreatic elastase 1 monoclonal antibody in a crosslinking buffer solution until the concentration is 1-10 mu mol/m L to obtain an anti-human pancreatic elastase 1 monoclonal antibody solution;

s3, mixing the latex particle solution with the anti-human pancreatic elastase 1 monoclonal antibody solution, adding a chemical cross-linking agent, and reacting at room temperature for 1-3h to obtain the latex particles coated with the anti-human pancreatic elastase 1 monoclonal antibody.

In another embodiment, the electrolyte is at least one of sodium chloride, potassium chloride, magnesium sulfate, and is present in the reagent at a concentration of 0.1-10%.

In another embodiment, the stabilizer is at least one of casein, mannitol, chitosan, disodium ethylene diamine tetraacetate, and bovine serum albumin, and the concentration of the stabilizer in the reagent is 0.1-10%.

In another embodiment, the buffer is one of MES buffer, boric acid buffer, acetate buffer, phosphate buffer and glycine buffer, the concentration of the buffer is 10-500 mmol/L, and the pH value of the buffer is 5-9.

In another embodiment, the surfactant is at least one of tween, fatty alcohol polyglycol ether and polyoxyethylene phenyl ether, and the concentration of the surfactant in the reagent is 0.1-10%.

In another embodiment, the preservative is at least one of sodium azide, phenol, p-hydroxybenzoic acid, ethyl p-hydroxybenzoate and Proclin preservative, and the concentration of the preservative in the reagent is 0.1-10%.

In another embodiment, the chemical crosslinker is at least one of EDC, N-hydroxysuccinimide, N-hydroxythiosuccinimide, carboximides, hydrazides, potassium isocyanate; the crosslinking buffer solution is one of MES, MOPSO, MOPS, HEPES and PBS buffer solution, and the pH value of the crosslinking buffer solution is 6-9.

In another embodiment, the concentration of the pancreatic elastase 1 in the pancreatic elastase 1 antigen calibrator solution is 0.01-1600 μ g/m L.

The invention also provides application of the kit for quantitatively determining the pancreatic elastase 1 in excrement in the detection of the pancreatic exocrine function.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the kit for quantitatively determining the pancreatic elastase 1 in the excrement comprises a reagent 1, a reagent 2 and a pancreatic elastase 1 antigen calibrator solution; wherein the R1 reagent comprises electrolyte, stabilizer, surfactant, preservative and buffer; the reagent 2 comprises latex particles coated with the monoclonal antibody of the anti-human trypsin 1, electrolyte, a stabilizer, a surfactant, a preservative and buffer solution; the pancreatic elastase 1 antigen calibrator solution comprises pancreatic elastase 1 and a stabilizer. The kit is a device for determining the pancreatic elastase 1 in excrement by using a latex enhanced immunoturbidimetry, has the advantages of stability, accuracy, time saving and labor saving, can be combined with a full-automatic biochemical analyzer, realizes the effect of full-automatic rapid determination of clinical detection and analysis of samples, has detection sensitivity improved by more than 10 times compared with the traditional immunoturbidimetry reagent, is coupled with the surface of latex particles by an anti-human pancreatic elastase 1 monoclonal antibody through a chemical crosslinking method, is combined with an antibody Fc fragment through a chemical bond, and improves the combination rate of the antibody

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a graph showing the difference in absorbance when a test sample is examined by the kit for quantitatively determining pancreatic elastase 1 in feces according to the embodiment of the present invention, wherein the x-axis represents the reaction time (each point is 22.5 seconds) and the y-axis represents the absorbance value;

FIG. 2 is a graph showing a standard curve of a test trypsin 1 standard using the kit for quantitatively determining trypsin 1 in feces according to an embodiment of the present invention, in which the x-axis represents the concentration (. mu.g/ml) of trypsin 1 and the y-axis represents the difference in absorbance.

Detailed Description

EXAMPLE 1 monoclonal antibody preparation

a) Prokaryotic expression plasmid pET-28a (+) capable of expressing the antigen polypeptide is synthesized by a company, and the name of the plasmid is pET-EF-1-AG;

b) the plasmid is transferred into an escherichia coli B L21 (DE3) competent cell, the kanamycin resistance is screened, a single colony is selected for PCR identification, and the screened positive clone cell is the engineering bacterium containing the recombinant plasmid pET-EF-1-AG.

c) Expression and purification of recombinant antigen protein, recombinant plasmid pET-EF-1-AG, in E.coli B L21 (DE3) is prepared through inoculating single colony of E.coli B L21 (DE3) containing recombinant plasmid pET-EF-1-AG into 5ml L B liquid culture medium containing 50 ug/ml kanamycin, shaking culture at 37 deg.c for 9-10 hr, adding 5ml bacterial liquid into 500ml L B liquid culture medium containing 50 ug/ml kanamycin, expanding culture for 2 hr to OD600 of 0.6, adding IPTG in 0.6mmol/l concentration, shaking culture at 16 deg.c overnight, centrifuging to collect overnight induced thallus, L ysisBuffer, ultrasonic crushing for 15min after 30min, centrifuging to collect supernatant, purifying GroE L protein in supernatant with Ni-NTA, eluting with 20mmol/l recombinant imidazole-containing eluent, and re-suspending the eluted recombinant imidazole in 200mmol eluent.

(2) Animal immunization BA L B/C mouse immunization

Pre-immune mouse sera were collected via the ocular vein on day 1 as a negative control. 20 mu g of the initial immune antigen recombinant protein is mixed with equal volume of Freund's complete adjuvant, and the antigen and the adjuvant are fully emulsified by adopting a syringe method. Mice were injected intraperitoneally at multiple points subcutaneously. On day 14, the dosage of the boosting immune antigen is the initial immune dosage, and the boosting immune antigen is fully emulsified with Freund incomplete adjuvant with equal volume and is injected into the abdominal cavity at multiple points. Day 21, blood was taken intravenously and antibody titers were determined by the indirect method. On day 28, the immunization was boosted until a satisfactory immunization effect was achieved. Shock immunization, one immunization three days prior to fusion.

(3) Cell fusion

The spleens of immunized mice were aseptically placed in a plate, spleen tissues were washed with incomplete medium, the spleen envelopes were peeled with two forceps and spleen tissues were forceps-minced to free cells as much as possible and add more incomplete medium to facilitate filtration with a mesh stainless steel screen to filter splenocytes, the mesh was wetted from below before filtration, cells were filtered into a 50m L fusion tube to mix well-grown SP2/0 cells in logarithmic growth phase with splenocytes at a ratio of 1: 10.

Fusion was conducted using PEG with a molecular weight of 1500 as a mediator, cells were resuspended in HAT medium after centrifugation, and the plates were plated with 2 drops per well of the HAT medium containing the cells. The cells were cultured at 37 ℃ in a 5% CO2 incubator. Half-amount liquid change is carried out on HAT culture medium on the 3 rd day after fusion, and half-amount liquid change is carried out once on the fifth day and the seventh day respectively. HT medium was changed after two weeks.

(4) Monoclonal antibody screening

And (4) observing the growth condition of the hybridoma cells, taking the supernatant when the clone grows to 1/3-1/2 of the bottom area of the hole, detecting by using an indirect method, and screening positive clones. Hybridoma cells in positive wells were subjected to cloning culture by the limiting dilution method, and after five times, the antibody positivity of the cloned cells was 100%. The positive clone cells were further expanded. The hybridoma cells are continuously passed in vitro for more than 3 months, repeatedly frozen and restored, supernatant is periodically collected, and the antibody in the supernatant is measured by a method for screening the antibody until the cell line can stably secrete the monoclonal antibody.

(5) Preparation of monoclonal antibodies

Selecting mice over 12 weeks old, injecting liquid paraffin into abdominal cavity, collecting well-grown monoclonal hybridoma cells after one week, injecting 1 × 10 into abdominal cavity of each mouse⁶And (3) hybridoma cells. Collecting ascites after 7-10 days, mixing the ascites with the same volume with normal saline, precipitating by using a saturated ammonium sulfate precipitation method, removing supernatant, then re-suspending and dissolving the precipitate by using normal saline, then adding saturated ammonium sulfate to 33 percent, discarding supernatant, re-suspending and dissolving the precipitate by using normal saline, filling into a dialysis bag, dialyzing, and purifying by using AKTAproteina chromatographic column chromatography for later use.

Example 2 preparation of standards

a) Prokaryotic expression plasmid pET-28a (+) capable of expressing the full-length polypeptide is synthesized by the company, and the name of the plasmid is pET-EF-1;

b) the plasmid is transferred into an escherichia coli B L21 (DE3) competent cell, the kanamycin resistance is screened, a single colony is selected for PCR identification, and the screened positive clone cell is the engineering bacterium containing the recombinant plasmid pET-EF-1.

c) Expression and purification of recombinant antigen protein, recombinant plasmid pET-EF-1, in E.coliB L21 (DE3) expression and purification, E.coliB L21 (DE3) single colony containing recombinant plasmid pET-EF-1 is inoculated in 5ml L B liquid culture medium containing 50 ug/ml kanamycin, shake culture is carried out for 9-10 h at 37 ℃ at 180r/min, 5ml bacterial liquid is added into 500ml L B liquid culture medium containing 50 ug/ml kanamycin, 1: 100 expansion culture is carried out for 2h until OD600 is about 0.6, IPTG with final concentration of 0.6mmol/l is added into the bacterial liquid, shake culture is carried out at 16 ℃ overnight, overnight induced centrifugation is collected, L ysis Buffer is resuspended, ultrasonic crushing is carried out for 15min after ice bath is carried out for 30min, supernatant is centrifugally collected, recombinant GroE L mmol protein in supernatant is purified by Ni-NTA, eluent containing 20mmol imidazole is firstly used for washing off, and 200mmol of recombinant imidazole-containing eluent is eluted.

Example 3 kit preparation

This example provides a kit for quantitatively determining trypsin 1 in excrement, which comprises reagent 1, reagent 2 and trypsin 1 antigen calibrator solution.

The reagent 1 comprises 0.2% of electrolyte, 8% of stabilizer, 0.15% of surfactant, 5% of preservative and the balance of buffer solution with the concentration of 10 mmol/L, wherein the electrolyte is sodium chloride, the stabilizer is casein, the surfactant is tween, the preservative is sodium azide, the buffer solution is MED buffer solution, and the pH value of the buffer solution is 6.5.

The reagent 2 comprises 4mg/m L of latex particles coated with monoclonal antibodies against human trypsin 1, 0.2% of electrolyte, 8% of stabilizer, 0.15% of surfactant, 5% of preservative and the balance of buffer solution with the concentration of 10 mmol/L, wherein the electrolyte is potassium chloride, the stabilizer is mannitol, the surfactant is fatty alcohol polyglycol ether, the preservative is phenol, the buffer solution is phosphoric acid buffer solution, and the pH value of the buffer solution is 6.5.

In this embodiment, the anti-human pancreatic elastase 1 monoclonal antibody is coupled to the surface of the latex particle by a chemical crosslinking method, which specifically includes the following steps:

s1, 100mg of latex particles are added to 5ml of MES buffer (50mM, pH6.5) and crosslinked surfactant SDS (final concentration 0.01%) is added to obtain a latex particle solution, wherein the particle size of the latex particles is 100-500 nm.

S2, the anti-human trypsin 1 monoclonal antibody was dissolved in 5ml of MES cross-linked buffer (50mM, pH6.5) to a concentration of 1. mu. mol/m L, to obtain an anti-human trypsin 1 monoclonal antibody solution.

S3, mixing the latex particle solution with the anti-human trypsin 1 monoclonal antibody solution, adding 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) serving as a chemical cross-linking agent, and reacting at room temperature for 2 hours to obtain the latex particles coated with the anti-human trypsin 1 monoclonal antibody.

The pancreatic elastase 1 antigen calibrator solution comprises pancreatic elastase 1 and a stabilizer, wherein the concentration of the pancreatic elastase 1 is 0 [ mu ] g/m L, 100 [ mu ] g/m L, 250 [ mu ] g/m L, 500 [ mu ] g/m L, 750 [ mu ] g/m L and 1500 [ mu ] g/m L, and the stabilizer is bovine serum albumin.

Experimental example 4 application of kit

1. Preparation of standards

Human pancreatic elastase 1 purified by gene recombination technology is used as mother liquor, and the mother liquor is diluted by bovine serum albumin gradient to prepare calibrators, wherein the calibrators comprise S5, 1600 mu g/m L, S4, 800 mu g/m L, S3, 400 mu g/m L, S2, 200 mu g/m L, S1, 100 mu g/m L and S0, 0 mu g/m L, and the concentration of the bovine serum albumin is 1-9999 mu g/m L.

2. Preparation of quality control product

The method comprises the steps of taking human pancreatic elastase 1 purified by a gene recombination technology as a mother solution, and diluting the mother solution by using bovine serum albumin gradient to prepare quality control products with two concentrations of C1: 300 mug/m L and C2: 110 mug/m L, wherein the concentration of the bovine serum albumin is 1-9999 mug/m L.

3. Preparation of standard curve

The dominant wavelength was detected using a fully automated biochemical analyzer: 600nm, sub-wavelength: 750 nm.

The dosage of the reagent is as follows: 3. mu.l of sample; reagent 1: 300 mu l; reagent 2: 100 μ l.

Measurement method (two-point end-point method): mu.l of reagent 1 was added to 3. mu.l of the sample, reacted at 37 ℃ for 5 minutes, and then 100. mu.l of reagent 2 was added to start reading absorbance A1, and after 10 minutes, reading absorbance A2 again, and the absorbance difference Δ A-A2-A1 was calculated as shown in FIG. 1.

Preparing a standard curve, namely adopting the trypsin elastase 1 standard product of the invention with the concentrations of S6: 1600 mu g/m L, S5: 800 mu g/m L, S4: 400 mu g/m L, S3: 200 mu g/m L, S2: 100 mu g/m L, S1: 50 mu g/m L and S0: 0 mu g/m L, measuring the standard curve of the trypsin 1 standard product according to the steps, and as shown in figure 2, each point on the curve in figure 2 represents a content of the standard product, wherein the x axis represents the concentration of the trypsin 1 and the y axis represents the difference of absorbance.

4. Determination of the Linear Range

1600 mu g/m L of a trypsin 1 high-concentration sample close to the upper limit of a linear range is diluted by physiological saline according to the proportion of 1/2, 1/4, 1/8, 1/16 and 1/32 to prepare 6 solutions with different concentrations, in addition, the physiological saline without the trypsin 1 is used as a blank solution, the two-point endpoint method is used for detecting each concentration, the measured concentration value and the theoretical concentration are subjected to linear regression analysis, and the calculated regression equation is that y is 0.9893x-0.944, and the correlation coefficient r is 0.9998, which shows that the kit has better correlation in the linear range of 0-1600 mu g/m L.

5. Determination of accuracy

The kit disclosed by the invention is used for carrying out feces determination on 20 cases of people (Shenzhen Shang Xin Kunjing Hospital in Dapeng New district, Shenzhen) by using a full-automatic biochemical analyzer (200 μ g/m L is a normal specimen, 200 μ g/m L is a light and medium pancreatic exocrine insufficiency specimen, and <100 μ g/m L is a severe pancreatic exocrine insufficiency specimen), and the test results are shown in the following table 1, and the results show that the kit disclosed by the invention has high correlation with clinical symptoms.

TABLE 1

6. Sensitivity measurement

The test reagent of the pancreatic elastase 1 was calibrated on a fully automated biochemical analyzer using a calibration of pancreatic elastase 1, and the change in absorbance of the reagent reaction was recorded as 0.09 for the calibration (concentration of 100. mu.g/m L), i.e., the sensitivity of the reagent was 0.09 for a calibration concentration of 100. mu.g/m L.

7. Determination of in-batch precision

The same sample was measured 10 times with the kit of the present invention, the mean value and the batch precision were calculated, and the test results are shown in table 2, showing that the batch precision is 1.7%.

TABLE 2

8. Interference rejection analysis

The interferent selection formulations and test results are shown in table 3 below. The result shows that the reagent box of the invention has good anti-interference capability.

TABLE 3

It is to be understood that the above examples are illustrative only for the purpose of clarity of description and are not intended to limit the embodiments. It will be apparent to those skilled in the art that other variations and modifications can be made based on the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

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Claims

1. A kit for detecting chronic pancreatitis is characterized in that the kit is used for quantitatively determining pancreatic elastase 1 in excrement, and the kit comprises latex particles coated with an anti-human pancreatic elastase 1 monoclonal antibody;

wherein the latex particles are dissolved in a solution 2, and the solution 2 further comprises an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution;

the liquid electrolyte also comprises a solution 1 and a quality control product, wherein the solution 1 comprises an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution;

the anti-human pancreatic elastase 1 monoclonal antibody specifically recognizes an epitope L PQEGAI L ANNSPCYITC (named as FE-1-AG, SEQ ID NO:2) of human pancreatic elastase 1;

wherein the monoclonal antibody is coupled to the surface of the latex particle by chemical crosslinking, the chemical crosslinking comprising the steps of:

s3, mixing the latex particle solution with the anti-human pancreatic elastase 1 monoclonal antibody solution, adding a chemical cross-linking agent, and reacting at room temperature for 2-4h to obtain latex particles coated with the anti-human pancreatic elastase 1 monoclonal antibody;

the electrolyte is at least one of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate, and the concentration of the electrolyte in the reagent is 0.1-10%;

the stabilizer is at least one of casein, mannitol, chitosan, ethylene diamine tetraacetic acid disodium and bovine serum albumin, and the concentration of the stabilizer in the reagent is 0.1-10%;

the preservative is at least one of sodium azide, phenol, p-hydroxybenzoic acid, ethyl p-hydroxybenzoate and Proclin preservative, and the concentration of the preservative in the reagent is 0.1-10%;

in the pancreatic elastase 1 antigen calibrator solution, the concentration of the pancreatic elastase 1 is 0.01-1600 mu g/m L.

2. Use of the kit for quantitative determination of pancreatic elastase 1 in feces according to claim 1 for the preparation of a product for detecting the exocrine pancreatic function.