CN109991409B

CN109991409B - Pepsinogen I/pepsinogen II detection kit

Info

Publication number: CN109991409B
Application number: CN201811462911.0A
Authority: CN
Inventors: 邓川; 郭亚楠; 张守涛; 陶新博; 杨永芳
Original assignee: Zhejiang Jukang Biotech Ltd
Current assignee: Zhejiang Jukang Biotech Ltd
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2022-03-04
Anticipated expiration: 2038-12-03
Also published as: CN109991409A

Abstract

The invention provides a pepsinogen I/pepsinogen II detection kit, which belongs to the technical field of fluorescence immunochromatography in medical immunology, is a detection kit of a fluorescence quantitative immunochromatography, comprises a detection card and is characterized in that: the detection card is provided with from bottom to top in sequence: PVC board, sample pad, combined pad, chromatographic film and absorbent paper; the chromatographic membrane comprises a detection area and a quality control area, wherein the detection area is coated with a detection area of an anti-PG I monoclonal antibody and a detection area of a PG II monoclonal antibody, and the quality control area is coated with a goat anti-mouse IgG antibody. The test sample of the kit is a human serum, plasma or whole blood sample, and the kit has good detection specificity, higher sensitivity, simplicity and convenience in operation and stable fluorescent marker, so that the detection accuracy is ensured.

Description

Pepsinogen I/pepsinogen II detection kit

Technical Field

The invention belongs to the technical field of fluorescence immunochromatography in medical immunology, and particularly relates to a pepsinogen I/pepsinogen II detection kit.

Background

Pepsinogen (PG), a digestive enzyme precursor secreted from the stomach, is an inactive precursor of pepsin in gastric juice and can be divided into two subtypes PG I and PG II, depending on distribution and immunogenicity. PG I and PG II are good diagnostic indicators of Helicobacter Pylori (HP) infection and atrophic gastritis, and the content thereof can reflect the secretory function of gastric mucosa. PG I mainly reacts on the mucous membrane state of the gastric fundus and the gastric corpus, the secretion of gastric acid is increased, the PG I of gastritis and peptic ulcer is increased, the secretion of gastric acid is reduced or the PG I of atrophy of gastric mucosal glands is reduced; when atrophic gastritis occurs, the gastric mucosa main cells are lost, the gastric mucosa secretory capacity is reduced, and the PG I level is obviously reduced; PG II has a large correlation with the pathological changes of the gastric fundus mucosa (relative to the gastric antral mucosa), and the increase of PG II is related to atrophy of the gastric fundus ducts, intestinal metaplasia or pseudopyloric metaplasia and dysplasia; PG II is the most sensitive index of Helicobacter Pylori (HP) infection, the raising amplitude of PG II is more obvious than that of PG I, and the ratio of PG I to PG II is reduced; therefore, the detection results of PG I, PG II and PG I/PG II can be used as the index for diagnosis and identification of stomach diseases.

CN 104698174B discloses a pepsinogen I and II joint detection kit, which comprises an enzyme linked plate coated with anti-PG I and anti-PG II antibodies, wherein the anti-PG I antibody is obtained by respectively cloning and expressing partial gene sequences PG I-A and PG I-B of a PG I antigen through gene engineering to obtain recombinant proteins PG I-A and PG I-B, and immunizing a mouse to prepare a monoclonal antibody; the nucleotide sequence of PG I-A is shown in SEQ ID No.1 in the sequence table, and the nucleotide sequence of PG I-B is shown in SEQ ID No.2 in the sequence table; the anti-PG II antibody is prepared by cloning and expressing a PG II antigen gene sequence through genetic engineering to obtain PG II recombinant protein and immunizing a mouse with the PG II recombinant protein to obtain a monoclonal antibody, wherein the nucleotide sequence of the PG II antigen is shown in SEQ ID No.3 in a sequence table. The kit can be used for simultaneously detecting PG I and PG II, and is simple to operate and rapid in detection. However, the kit can only be used for detecting the content of PG I and PG II in serum.

Disclosure of Invention

The invention aims to provide a pepsinogen I/pepsinogen II detection kit, wherein a test sample is a human serum, plasma or whole blood sample, and the kit has good detection specificity, higher sensitivity, simplicity and convenience in operation and ensures the detection accuracy by a stable fluorescent marker.

The technical scheme adopted by the invention for realizing the purpose is as follows:

pepsinogen I/pepsinogen II detect reagent box, it is the detect reagent box of fluorescence quantitative immunochromatography, including the detection card, the detection card is equipped with by lower supreme in proper order: PVC board, sample pad, combined pad, chromatographic film and absorbent paper;

wherein the chromatographic membrane comprises a detection zone and a quality control zone; the detection area is coated with the detection areas of the anti-PG I monoclonal antibody and the anti-PG II monoclonal antibody and is used for capturing antigens; the quality control region is coated with goat anti-mouse IgG antibody for providing signal detection.

Preferably, the anti-PG I monoclonal antibody is obtained by immunizing human anti-PG I, and has a sequence shown as SEQ ID NO. 1.

Preferably, the anti-PG II monoclonal antibody is obtained by immunizing human anti-PG II, and has a sequence shown as SEQ ID NO. 2.

Preferably, the chromatographic membrane is a nitrocellulose membrane.

Preferably, the anti-PG I monoclonal antibody and PG II monoclonal antibody-microsphere coupled compound marked by the rare earth fluorescent microsphere is adsorbed on the binding pad.

Further preferably, the diameter of the fluorescent microsphere is 100-250nm, and the fluorescent microsphere is a rare earth fluorescent microsphere containing one or more rare earth lanthanide elements; the monoclonal antibody is a purified and mixed monoclonal antibody.

Preferably, the kit further comprises an IC card and a diluent.

Preferably, the test sample of the kit is human serum, plasma or whole blood. The kit is only used for detecting human serum, plasma or whole blood samples, other body fluid samples are not applicable, the serum, plasma and whole blood samples are collected according to a conventional method, sodium citrate, EDTA or heparin anticoagulant plasma can be used as an anticoagulant, the serum or plasma is required to be detected within 4 hours after being separated, the serum or plasma can be stored for 5 days at the temperature of 2-8 ℃, the serum or plasma is required to be stored for more than 5 days at-20 ℃, the detection is finished within 1 hour after the sample is restored to the room temperature before the detection, and repeated freeze thawing is not needed; the whole blood sample can be stored for 3 days at the temperature of 2-8 ℃ without being frozen.

The kit of the invention adopts an immunofluorescence double-antibody sandwich method to quantitatively detect the content of pepsinogen I (PG I) and pepsinogen II (PG II) in human serum, plasma or whole blood. After a sample is dripped into a sample adding hole of the detection card, PG I and PG II in the sample are combined with an anti-PG I monoclonal antibody and a PG II monoclonal antibody marked by fluorescent substances in the combination pad to form a reaction compound, the reaction compound moves forwards along the nitrocellulose membrane along with the chromatography action and is captured by the corresponding monoclonal antibody on a detection line of the nitrocellulose membrane, the capturing amount of PG I and PG II in the sample is positively correlated with the signal intensity of the detection area, and the content of PG I and PG II in the sample is quantitatively detected through a fluorescence analyzer.

The invention also provides application of the pepsinogen I/pepsinogen II detection kit, and application of the pepsinogen I/pepsinogen II detection kit in-vitro quantitative detection of the content of pepsinogen I and pepsinogen II in human serum, plasma or whole blood samples. The method is mainly used for early screening of gastric cancer; screening gastric ulcer, atrophic gastritis, Helicobacter Pylori (HP) infection; evaluation of therapeutic effect of Helicobacter Pylori (HP); evaluating recurrence and curative effect of peptic ulcer; evaluation of recurrence after gastric cancer resection; dynamic monitoring of individual gastric mucosal function.

The invention also provides a using method of the pepsinogen I/pepsinogen II detection kit, which comprises the steps of sucking 150 mu L of whole blood sample, adding one drop of diluent or serum and 100 mu L of plasma sample into a sample adding hole of a test card, inserting the test card into a clamping groove of an applicable instrument after 10 minutes, and carrying out quantitative interpretation on a result; the normal reference interval is: PG I >70 ng/mL; PG II <20 ng/mL.

Compared with the prior art, the invention has the beneficial effects that: 1) the test sample of the kit is a human serum, plasma or whole blood sample, and the kit has good detection specificity, higher sensitivity, simplicity and convenience in operation and stable fluorescent marker, so that the detection accuracy is ensured; 2) the kit can simultaneously quantitatively detect the content of pepsinogen I and pepsinogen II in a sample, has a synergistic and complementary effect on the diagnosis and identification of stomach diseases, can provide accurate and reliable diagnosis, danger stratification and prognosis information for patients with stomach diseases, and has the advantages of rapidness, effectiveness, accuracy and the like; 3) compared with the prior art, the two monoclonal antibody-microsphere coupled compounds prepared by the method have the advantages of long fluorescence life, large Stokes shift, no overlap of excitation spectrum and emission spectrum and no mutual interference phenomenon.

The pepsinogen I/pepsinogen II detection kit provided by the invention adopts the technical scheme, overcomes the defects of the prior art, and is reasonable in design and convenient to operate.

Drawings

Fig. 1 is a schematic structural diagram of a test paper card in the invention.

Reference numerals: PVC board; 2. a sample pad; 3. a bonding pad; 4. a chromatographic membrane; 5. an absorbent pad.

Detailed Description

The following further describes embodiments of the present invention with reference to specific examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. In addition, the term "coating" in the present invention is a term in the field of immunization, and includes adsorption and immobilization.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1:

as shown in fig. 1, the pepsinogen I/pepsinogen II detection kit is a detection kit of a fluorescence quantitative immunochromatography, and comprises a detection card, wherein the detection card is sequentially provided with: a PVC plate 1, a sample pad 2, a combination pad 3, a chromatographic membrane 4 and absorbent paper 5; wherein the chromatographic membrane comprises a detection zone and a quality control zone; the detection area is coated with the detection areas of the anti-PG I monoclonal antibody and the anti-PG II monoclonal antibody and is used for capturing antigens; the quality control region is coated with goat anti-mouse IgG antibody for providing signal detection. The chromatographic membrane 4 is a nitrocellulose membrane and is prepared by the following steps:

1) adopting cell strains different from the anti-PG I and anti-PG II monoclonal antibody cell strains used on the combination pad 3, preparing and purifying three mouse-derived monoclonal antibodies such as an anti-human cardiac troponin I antibody, an anti-myoglobin antibody and an anti-creatine kinase isozyme antibody by adopting a standard ascites production process, obtaining a monoclonal antibody matched with the labeled antibody, subpackaging and storing at-20 ℃ for later use;

2) and (3) adjusting the concentrations of the three mouse-derived monoclonal antibodies and the goat anti-mouse IgG antibody to 1-3mg/ml by using coating diluent respectively, wherein the membrane solution amount is 1-3ul/cm, the three mouse-derived monoclonal antibodies and the goat anti-mouse IgG antibody are respectively used as a detection line and a quality control line and are parallelly sprayed on a nitrocellulose membrane for coating, the space between the detection line and the quality control line is 3-7mm, and then the nitrocellulose membrane is placed in an oven and dried for 2 hours at 37 ℃.

The above sample pad 2 was prepared by the following steps: the glass fiber membrane was immersed in a treatment solution containing 1.0% Triton X-100, 2.5% BSA, 0.15M Tris buffer, pH7.5, at 4 ℃ for 4 hours, and then placed in an oven and dried at 37 ℃ for 2 hours.

The anti-PG I monoclonal antibody used above is obtained by immunizing human anti-PG I and has a sequence as VDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDACTNHNRFNPEDSSTYTGSSN LWVPSVYCSSLQSTSETVSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETE PGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQDLFSVYLSADDQS GSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEAIACAEGCQAIV DTGTSLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPV PPSAYILQSEGSCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLA PVA; the anti-PG II monoclonal antibody was obtained by immunizing human anti-PG II and had a sequence as MAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMA YMDAAYFGEISIGTPPQNFLVLFDTGSSNLWVPSVYCQSQACTSHSRFNPSESS TYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQ FDGIMGLAYPALSVDEATTAMQGMVQEGALTSPVFSVYLSNQQGSSGGAVVF GGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTS LLTVPQQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSY ILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA GGGGSLVLESSGLGPLLTPSRAAPPSSTLQLPEKPLEQTWNILTPFTKTLPVSNL SRKVTSWAGVGIPVTCLPEAGSGGERRAECGLGVPTTRGPPRSQHHSGA.

The combination pad 3 is adsorbed with anti-PG I monoclonal antibody and PG II monoclonal antibody-microsphere coupling compound marked by rare earth fluorescent microsphere, the diameter of the fluorescent microsphere is 100-400 nm, the fluorescent microsphere is rare earth fluorescent microsphere containing one or more rare earth lanthanide elements, which is stable under the ground state, and emits fluorescence with the wavelength range of 550-650mm under the action of excitation light source of 300-400 nm; the monoclonal antibodies are purified and mixed monoclonal antibodies, and are respectively derived from monoclonal antibody cell strains aiming at 2-6 different anti-PG I monoclonal antibodies and anti-PG II monoclonal antibody epitopes.

Wherein, the bonding pad 3 is prepared by the following steps: the glass fiber membrane was soaked in 150mM Tris-HCl treatment solution (containing 1.0% Triton X-100, 2.5% BSA, pH7.4) at 4 ℃ for 2 hours, and then taken out of the oven at 37 ℃ and dried for 4 hours for use. The glass fiber membrane is placed on a three-dimensional spraying point platform, two coupling compounds of an anti-PG I monoclonal antibody and an anti-PG II monoclonal antibody marked by rare earth fluorescent microspheres are uniformly mixed by a non-contact type micro quantitative nozzle and then sprayed on the glass fiber membrane, and the glass fiber membrane is dried for 1 hour at 37 ℃ to obtain the product.

The anti-PG I monoclonal antibody and PG II monoclonal antibody-microsphere coupling compound marked by the rare earth fluorescent microspheres adsorbed on the combined pad 3 is prepared by the following steps:

1) obtaining of monoclonal antibody cell strain: respectively immunizing a mouse with pure human anti-PG I and anti-PG II products, preparing a monoclonal antibody cell strain with high specificity and high affinity by adopting a standard monoclonal antibody preparation method, carrying out pairing screening on the obtained monoclonal antibody cell strain, and preferably selecting the monoclonal antibody cell strain for the kit according to a pairing result and affinity data;

2) preparation of monoclonal antibody: preparing and purifying two mouse-derived monoclonal antibodies of anti-PG I and anti-PG II antibodies by adopting a standard ascites production process, subpackaging and storing at-20 ℃ for later use;

3) aldehyde group of the rare earth fluorescent microsphere: taking 5mg rare earth fluorescent microspheres, washing the microspheres for 3 times by using 20mM carbonate buffer solution with pH of 9.5 by using a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending the microspheres in 100ul of the carbonate buffer solution, adding 500ul of aldehyde-group-converted glucan, uniformly mixing the mixture, carrying out dark reaction for 4 hours at room temperature, washing and suspending the microspheres in 100ul of the carbonate buffer solution by using the same centrifugal method, and placing the microspheres at 4 ℃ for later use;

4) preparing two coupling compounds of anti-human anti-PG I and anti-PG II antibodies marked by rare earth fluorescent microspheres: the two antibody-microsphere coupling complexes were separately coupled, as follows: selecting anti-PG I from 2 monoclonal cell strains with different antigen epitopes, dialyzing 2mg of human anti-PG II with the carbonate buffer solution at 4 ℃ overnight according to the mass ratio of 1:1, mixing with the aldehyde-based rare earth fluorescent microspheres, and reacting at 4 ℃ overnight; then, sodium borohydride was added to a final concentration of 5mM, and the reaction was carried out at 4 ℃ for 4 hours; adding equal volume of blocking solution (50mM Tris-HCl, pH7.4, containing 2.5% BSA, 5% sucrose), and blocking at 4 deg.C overnight; then, the cells were washed 3 times with 50mM Tris-HCl, pH7.4 buffer by centrifugation, resuspended in 100. mu.l of 50mM Tris-HCl buffer (containing 1.2% NaCl, 0.5% BSA, 0.1% Tween 20), and stored at 4 ℃ in the dark for further use.

The kit also comprises 1 IC card and diluent, wherein 10/25 persons/box are mixed with 1X 5 mL/piece of diluent, and 40 persons/box are mixed with 2X 5 mL/piece of diluent. The dilution was 0.05% PBS-T solution (pH 7.2).

The test sample of the kit is human serum, plasma or whole blood. The kit is only used for detecting human serum, plasma or whole blood samples, other body fluid samples are not applicable, the serum, plasma and whole blood samples are collected according to a conventional method, sodium citrate, EDTA or heparin anticoagulant plasma can be used as an anticoagulant, the serum or plasma is required to be detected within 4 hours after being separated, the serum or plasma can be stored for 5 days at the temperature of 2-8 ℃, the serum or plasma is required to be stored for more than 5 days at-20 ℃, the detection is finished within 1 hour after the sample is restored to the room temperature before the detection, and repeated freeze thawing is not needed; the whole blood sample can be stored for 3 days at the temperature of 2-8 ℃ without being frozen.

The kit adopts an immunofluorescence double-antibody sandwich method to quantitatively detect the content of pepsinogen I (PG I) and pepsinogen II (PG II) in human serum, plasma or whole blood. After a sample is dripped into a sample adding hole of the detection card, PG I and PG II in the sample are combined with an anti-PG I monoclonal antibody and a PG II monoclonal antibody marked by fluorescent substances in the combination pad to form a reaction compound, the reaction compound moves forwards along the nitrocellulose membrane along with the chromatography action and is captured by the corresponding monoclonal antibody on a detection line of the nitrocellulose membrane, the capturing amount of PG I and PG II in the sample is positively correlated with the signal intensity of the detection area, and the content of PG I and PG II in the sample is quantitatively detected through a fluorescence analyzer.

The detection card of the kit is stored at 4-30 ℃, the validity period is 18 months, and the detection card is valid within 2 hours after the aluminum foil bag of the reagent is opened.

The above kit uses the following instruments: NP007 dry fluoroimmunoassay analyzer of wiener biotechnology limited, AFS1000 dry fluoroimmunoassay analyzer of cantonese blue brib biotechnology limited. Each test kit reaction plate has an internal quality control process to ensure the daily quality control requirements when in use. The quality control procedure is performed simultaneously for each patient sample test. Once the reaction plate is properly inserted into the fluorescence analyzer, the instrument will be fully recognized. Incorrect empty information will show up as false information on the fluorescence analyzer and will require retesting.

The performance indexes of the kit are as follows:

1) linear range:

PGI is within the range of 10.00 ng/mL-160.00 ng/mL, and the linear correlation coefficient r is more than or equal to 0.9900;

PGII is in the range of 6.25 ng/mL-1000.00 ng/mL, and the linear correlation coefficient r is more than or equal to 0.9900.

2) Repeatability:

the PGI variation Coefficient (CV) is less than or equal to 15 percent;

the PGII Coefficient of Variation (CV) is less than or equal to 15 percent.

3) Inter-batch difference:

the relative range (R) of PGI is less than or equal to 20 percent;

the relative range (R) of PGII is less than or equal to 20 percent.

4) Accuracy:

the PGI takes an internal reference product as a detection sample, and the deviation between a measured value and a marked value is less than or equal to 20 percent;

the PGII takes an internal reference product as a detection sample, and the deviation between a measured value and a marked value is less than or equal to 20 percent.

5) The lowest detection limit is:

PGI: not higher than 10.00 ng/mL;

PGII: not higher than 6.25 ng/mL.

The notes on the above kit:

1. the product is a disposable in vitro diagnostic reagent, and can be used repeatedly within the effective period.

2. The test card and assembly are only suitable for use with an associated fluorescence analyzer.

3. The product is not required to be opened before use, and the package is obviously damaged.

4. Reagent components of different batches cannot be used in a mixed mode, and the IC card and the detection card cannot be used in a mixed mode.

5. Appropriate protective measures should be taken during collection, disposal, storage, blending of the sample and detection.

6. The aluminum foil bag is filled with a drying agent, and the food cannot be eaten.

7. The test card stored at low temperature needs to be opened after being restored to room temperature so as not to absorb moisture.

8. The detection card and the fluorescence analyzer should avoid vibration and electromagnetic environment when in use; in normal use, the instrument generates vibration and is normal; the detection is performed without pulling out the IC card.

9. It is recommended to use a fresh sample, which if there is significant hemolysis or blood clotting in the sample will interfere with the test and lead to erroneous results, which are never used.

10. The detection result of the reagent is only used for clinical reference, and the clinical diagnosis and treatment of patients should be comprehensively considered by combining the symptoms, physical signs, medical history, other laboratory examinations, treatment response and other conditions.

11. All specimens, waste liquids, tubes and the like are treated in an infectious pollutant treatment mode. After detection, the used detection card is discarded in a corresponding biohazard container and is treated according to biohazard.

12. If there is a problem or suggestion in using the reagent, please contact the manufacturer.

The pepsinogen I/pepsinogen II detection kit is used for in vitro quantitative detection of the content of pepsinogen I and pepsinogen II in human serum, plasma or whole blood samples. The method is mainly used for early screening of gastric cancer; screening gastric ulcer, atrophic gastritis, Helicobacter Pylori (HP) infection; evaluation of therapeutic effect of Helicobacter Pylori (HP); evaluating recurrence and curative effect of peptic ulcer; evaluation of recurrence after gastric cancer resection; dynamic monitoring of individual gastric mucosal function.

The using method of the pepsinogen I/pepsinogen II detection kit specifically comprises the following steps:

1) preparing: taking out the detection card from the aluminum foil packaging bag, and placing the detection card on a horizontal and dry plane (for example, the detection card taken out of a refrigerator needs to be balanced to room temperature and then is taken out of the sealed aluminum foil bag for use, otherwise, the experimental result is influenced);

2) calibration: confirming that the batch numbers of the IC card and the detection card are matched, and calibrating the IC card;

3) sample adding: sucking 150 microliter of whole blood, adding one drop of diluent or serum and 100 microliter of plasma into a sample adding hole of the test card;

4) and (3) detection: inserting the detection card into a card slot of a suitable instrument after 10 minutes, and carrying out quantitative interpretation on the result; the normal reference interval is: PGI >70 ng/mL; PGII <20 ng/mL.

Example 2:

in order to improve the stability and sensitivity of the kit, the optimization measures adopted further comprise:

the anti-PG I monoclonal antibody and PG II monoclonal antibody-microsphere coupling compound marked by the rare earth fluorescent microsphere adsorbed on the combined pad 3 is prepared by the following steps:

the method comprises the following steps of: taking 5mg rare earth fluorescent microspheres, washing for 3 times by using 20mM carbonate buffer solution with pH of 9.5 by using a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending the microspheres in 100ul of the carbonate buffer solution containing 1.5ul of monoethanolamine thioglycolate, adding 500ul of aldehyde dextran and 0.2 mu g of 2-aminoethanesulfonic acid, uniformly mixing, carrying out dark reaction for 4 hours at room temperature, washing and suspending the microspheres in 100ul of the carbonate buffer solution by using the same centrifugal method, and placing the microspheres at 4 ℃ for later use. The existence of the thioglycolic acid monoethanolamine and the 2-aminoethanesulfonic acid can improve the hydroformylation degree of the rare earth fluorescent microsphere, provide a coupling site for covalent combination of the anti-PG I monoclonal antibody and the PG II monoclonal antibody, improve the binding force of the rare earth fluorescent microsphere with the anti-PG I monoclonal antibody and the PG II monoclonal antibody, and increase the water solubility due to the existence of free aldehyde groups, thereby further increasing the chance of collision between the rare earth fluorescent microsphere and the anti-PG I monoclonal antibody and the PG II monoclonal antibody.

Example 3:

stability test

Detecting the stability of the card: the detection card in the kit is usually stored in a dry and cool environment at room temperature. The test card is placed in a 50 ℃ oven for one month for accelerated destruction stability test, which is equivalent to the effectiveness of the test card stored for 1 year at normal temperature. And (3) placing the detection card in a 50 ℃ oven for 1 week, 2 weeks, 3 weeks and 4 weeks respectively, taking out the detection card, placing the detection card in a drying room, comparing the detection card with a reagent card placed in the drying room at normal temperature, storing the sample diluent at 4 ℃, and detecting the stability of the card. The reaction curves of the test card of example 2 in the case of the standard test after the test of the standard sample is placed in a 50 ℃ oven for accelerated destruction for 1 week, 2 weeks, 3 weeks and 4 weeks are basically consistent with the test card stored at normal temperature, the T/C values of the fluorescent signals of low, medium and high concentration are basically unchanged, and the stability of the fluorescent quantitative immunochromatography test card is good after the accelerated destruction test. The coincidence degree of the reaction curve of the detection card in the example 1 and the detection card stored at normal temperature under the condition that the detection card is placed in a 50 ℃ oven to be accelerated and damaged for 1 week, 2 weeks, 3 weeks and 4 weeks under the test of the standard substance is worse than that of the detection card in the example 2, which shows that the stability of the detection card in the example 2 is better than that of the detection card in the example 1, and further shows that the existence of the monoethanolamine thioglycolate and the 2-aminoethanesulfonic acid can improve the binding force of the rare earth fluorescent microsphere and the anti-PG I monoclonal antibody and the anti-PG II monoclonal antibody, and further improve the stability of the detection card.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Sequence listing

<120> pepsinogen I/pepsinogen II detection kit

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<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Asp Glu Gln Pro Leu Glu Asn Tyr Leu Asp Met Glu Tyr Phe Gly

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Thr Ile Gly Ile Gly Thr Pro Ala Gln Asp Phe Thr Val Val Phe Asp

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Ala Cys Thr Asn His Asn Arg Phe Asn Pro Glu Asp Ser Ser Thr Tyr

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Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Val Tyr Cys Ser Ser Leu

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Gln Ser Thr Ser Glu Thr Val Ser Ile Thr Tyr Gly Thr Gly Ser Met

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Thr Gly Ile Leu Gly Tyr Asp Thr Val Gln Val Gly Gly Ile Ser Asp

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Thr Asn Gln Ile Phe Gly Leu Ser Glu Thr Glu Pro Gly Ser Phe Leu

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Tyr Tyr Ala Pro Phe Asp Gly Ile Leu Gly Leu Ala Tyr Pro Ser Ile

115 120 125

Ser Ser Ser Gly Ala Thr Pro Val Phe Asp Asn Ile Trp Asn Gln Gly

130 135 140

Leu Val Ser Gln Asp Leu Phe Ser Val Tyr Leu Ser Ala Asp Asp Gln

145 150 155 160

Ser Gly Ser Val Val Ile Phe Gly Gly Ile Asp Ser Ser Tyr Tyr Thr

165 170 175

Gly Ser Leu Asn Trp Val Pro Val Thr Val Glu Gly Tyr Trp Gln Ile

180 185 190

Thr Val Asp Ser Ile Thr Met Asn Gly Glu Ala Ile Ala Cys Ala Glu

195 200 205

Gly Cys Gln Ala Ile Val Asp Thr Gly Thr Ser Leu Leu Thr Gly Pro

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Thr Ser Pro Ile Ala Asn Ile Gln Ser Asp Ile Gly Ala Ser Glu Asn

225 230 235 240

Ser Asp Gly Asp Met Val Val Ser Cys Ser Ala Ile Ser Ser Leu Pro

245 250 255

Asp Ile Val Phe Thr Ile Asn Gly Val Gln Tyr Pro Val Pro Pro Ser

260 265 270

Ala Tyr Ile Leu Gln Ser Glu Gly Ser Cys Ile Ser Gly Phe Gln Gly

275 280 285

Met Asn Leu Pro Thr Glu Ser Gly Glu Leu Trp Ile Leu Gly Asp Val

290 295 300

Phe Ile Arg Gln Tyr Phe Thr Val Phe Asp Arg Ala Asn Asn Gln Val

305 310 315 320

Gly Leu Ala Pro Val Ala

325

<210> 2

<211> 477

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Ala Val Val Lys Val Pro Leu Lys Lys Phe Lys Ser Ile Arg Glu

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Thr Met Lys Glu Lys Gly Leu Leu Gly Glu Phe Leu Arg Thr His Lys

20 25 30

Tyr Asp Pro Ala Trp Lys Tyr Arg Phe Gly Asp Leu Ser Val Thr Tyr

35 40 45

Glu Pro Met Ala Tyr Met Asp Ala Ala Tyr Phe Gly Glu Ile Ser Ile

50 55 60

Gly Thr Pro Pro Gln Asn Phe Leu Val Leu Phe Asp Thr Gly Ser Ser

65 70 75 80

Asn Leu Trp Val Pro Ser Val Tyr Cys Gln Ser Gln Ala Cys Thr Ser

85 90 95

His Ser Arg Phe Asn Pro Ser Glu Ser Ser Thr Tyr Ser Thr Asn Gly

100 105 110

Gln Thr Phe Ser Leu Gln Tyr Gly Ser Gly Ser Leu Thr Gly Phe Phe

115 120 125

Gly Tyr Asp Thr Leu Thr Val Gln Ser Ile Gln Val Pro Asn Gln Glu

130 135 140

Phe Gly Leu Ser Glu Asn Glu Pro Gly Thr Asn Phe Val Tyr Ala Gln

145 150 155 160

Phe Asp Gly Ile Met Gly Leu Ala Tyr Pro Ala Leu Ser Val Asp Glu

165 170 175

Ala Thr Thr Ala Met Gln Gly Met Val Gln Glu Gly Ala Leu Thr Ser

180 185 190

Pro Val Phe Ser Val Tyr Leu Ser Asn Gln Gln Gly Ser Ser Gly Gly

195 200 205

Ala Val Val Phe Gly Gly Val Asp Ser Ser Leu Tyr Thr Gly Gln Ile

210 215 220

Tyr Trp Ala Pro Val Thr Gln Glu Leu Tyr Trp Gln Ile Gly Ile Glu

225 230 235 240

Glu Phe Leu Ile Gly Gly Gln Ala Ser Gly Trp Cys Ser Glu Gly Cys

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Gln Ala Ile Val Asp Thr Gly Thr Ser Leu Leu Thr Val Pro Gln Gln

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Tyr Met Ser Ala Leu Leu Gln Ala Thr Gly Ala Gln Glu Asp Glu Tyr

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Gly Gln Phe Leu Val Asn Cys Asn Ser Ile Gln Asn Leu Pro Ser Leu

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Thr Phe Ile Ile Asn Gly Val Glu Phe Pro Leu Pro Pro Ser Ser Tyr

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Ile Leu Ser Asn Asn Gly Tyr Cys Thr Val Gly Val Glu Pro Thr Tyr

325 330 335

Leu Ser Ser Gln Asn Gly Gln Pro Leu Trp Ile Leu Gly Asp Val Phe

340 345 350

Leu Arg Ser Tyr Tyr Ser Val Tyr Asp Leu Gly Asn Asn Arg Val Gly

355 360 365

Phe Ala Thr Ala Ala Gly Gly Gly Gly Ser Leu Val Leu Glu Ser Ser

370 375 380

Gly Leu Gly Pro Leu Leu Thr Pro Ser Arg Ala Ala Pro Pro Ser Ser

385 390 395 400

Thr Leu Gln Leu Pro Glu Lys Pro Leu Glu Gln Thr Trp Asn Ile Leu

405 410 415

Thr Pro Phe Thr Lys Thr Leu Pro Val Ser Asn Leu Ser Arg Lys Val

420 425 430

Thr Ser Trp Ala Gly Val Gly Ile Pro Val Thr Cys Leu Pro Glu Ala

435 440 445

Gly Ser Gly Gly Glu Arg Arg Ala Glu Cys Gly Leu Gly Val Pro Thr

450 455 460

Thr Arg Gly Pro Pro Arg Ser Gln His His Ser Gly Ala

465 470 475

Claims

1. Pepsinogen I/pepsinogen II detect reagent box, it is the detect reagent box of fluorescence quantitative immunochromatography, including detection card, IC card and diluent, its characterized in that: the detection card is sequentially provided with from bottom to top: PVC board, sample pad, combined pad, chromatographic film and absorbent paper; the chromatographic membrane comprises a detection area and a quality control area, wherein the detection area is coated with a detection area of an anti-PG I monoclonal antibody and a detection area of a PG II monoclonal antibody, and the quality control area is coated with a goat anti-mouse IgG antibody; the anti-PG I monoclonal antibody and PG II monoclonal antibody-microsphere coupling compound marked by rare earth fluorescent microspheres is adsorbed on the binding pad; wherein, the anti-PG I monoclonal antibody is obtained by immunizing human PG I and has a sequence shown as SEQ ID NO. 1; the anti-PG II monoclonal antibody is obtained by immunizing human PG II and has a sequence shown as SEQ ID NO. 2;

the rare earth fluorescent microspheres are aldehyde rare earth fluorescent microspheres, and the preparation method of the aldehyde rare earth fluorescent microspheres comprises the following steps: taking 5mg rare earth fluorescent microspheres, washing for 3 times by using 20mM carbonate buffer solution with pH of 9.5 by using a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending the microspheres in 100ul of the carbonate buffer solution containing 1.5ul of monoethanolamine thioglycolate, adding 500ul of aldehyde dextran and 0.2 mu g of 2-aminoethanesulfonic acid, uniformly mixing, carrying out dark reaction for 4 hours at room temperature, washing and suspending the microspheres in 100ul of the carbonate buffer solution by using the same centrifugal method, and placing the microspheres at 4 ℃ for later use.

2. The pepsinogen I/pepsinogen II detection kit according to claim 1, characterized in that: the chromatographic membrane is a nitrocellulose membrane.

3. The pepsinogen I/pepsinogen II detection kit according to claim 1, characterized in that: the diameter of the fluorescent microsphere is 100-250nm, and the fluorescent microsphere is a rare earth fluorescent microsphere containing one or more rare earth lanthanide elements; the monoclonal antibody is a purified and mixed monoclonal antibody.

4. The pepsinogen I/pepsinogen II detection kit according to claim 1, characterized in that: the test sample of the kit is human serum, plasma or whole blood.