Antibody A4D7 targeting procalcitonin and its application

Abstract

The present invention provides an antibody A4D7 targeting procalcitonin and its application. The amino acid sequences of the light chain and heavy chain of the antibody A4D7 are shown in SEQ ID NO: 1 and 2, respectively. A lateral flow immunochromatography detection reagent is developed using the antibody. The antibody has high sensitivity and good specificity and has broad application prospects in the field of immunodiagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to an antibody A4D7 targeting procalcitonin and an application thereof. Background Art

[0002] Procalcitonin (PCT) is a non-hormonal active glycoprotein composed of 116 amino acids and is the precursor of calcitonin (CT). PCT was discovered in the 1980s in the study of tumor markers. In the 1990s, in the study of acute lung injury markers, it was found that the serum PCT index of patients with sepsis and septic shock would increase sharply. This topic was introduced to discuss whether there is a relationship between PCT and infection. Subsequent studies have shown that PCT can be used as a serum marker for severe bacterial infections, especially sepsis. It is an important indicator for early auxiliary differential diagnosis of systemic inflammatory response and can be used as an important indicator for judging the severity of the disease, treatment efficacy and prognosis.

[0003] PCT participates in immune regulation and is used as an indicator of inflammation in clinical applications:

[0004] As an immunomodulatory protein, PCT is one of the most commonly used markers for detecting inflammation. PCT induction is strictly regulated by the body, and its activation steps are highly correlated in time and function, and this correlation may be used to reflect the severity of the disease. Under physiological conditions, PCT is synthesized by parafollicular cells of the thyroid gland. In the presence of bacterial infection, cells in other parts of the body (such as monocytes and neuroendocrine cells) activate the induced expression of the CALC-I gene corresponding to procalcitonin under cytokine stimulation, which leads to the continuous expression and release of PCT. In addition, PCT is very stable in vivo and in vitro, easy to detect, and its elevated concentration is not easily affected by the body's immunosuppressive state. For this reason, PCT is considered to be a biomarker that plays an important role in the early detection of systemic inflammatory responses, and is therefore widely used in the differential diagnosis of a variety of clinical symptoms, including bacterial infectious diseases, the judgment of the condition, and the assessment of prognosis, including but not limited to major trauma (accidental trauma, surgery, burns), multiple organ dysfunction, severe pancreatitis or severe liver disease or kidney disease, and early neonatal birth. In addition, non-infectious PCT elevation may be related to factors such as severe tissue damage, elevated plasma pro-inflammatory cytokine levels, severe organ environmental disorders, or paraneoplastic syndromes. PCT has high sensitivity and specificity in distinguishing infectious from non-infectious fever (such as tumor fever, drug fever). Lymphoma patients are prone to serious infections after chemotherapy due to immune dysfunction and granulocytosis. Studies have shown that PCT levels are significantly elevated in patients with granulocytopenia accompanied by obvious infection and fever, while PCT levels in patients with focal infection or unexplained fever do not change significantly, which provides a reference for the early diagnosis of bacterial bloodstream infection and avoiding excessive use of antibiotics.

[0005] Standardized PCT detection method:

[0006] PCT has a half-life of 25-30 hours, good in vitro stability, and is easy to detect. After 24 hours of blood collection at room temperature, the PCT concentration decreases by about 12%. When stored at 4°C, it only decreases by 6%. Therefore, if the blood cannot be tested immediately after collection, the sample must be stored at low temperature. At present, the main clinical detection methods for PCT include chemiluminescence immunoassay, radioimmunoassay, colloidal gold method, transmission immunoturbidimetry, time-resolved fluorescence immunoassay technology, etc., and its basic principle is the immune binding reaction between antigen and antibody.

[0007] The detection method of the lateral flow rapid diagnostic kit can usually only provide qualitative results and is not suitable for specific pathogens (such as certain bacteria, mycoplasmas, viruses, etc.) that require precise quantitative results to diagnose the infection. The lateral flow rapid diagnostic kit based on time-resolved fluorescence immunoassay technology, under the premise of using specific instruments and using the above-mentioned (such as new designs such as rare earth element chelates) specific antibody chemical labeling, has the characteristics of high sensitivity, convenient operation, high degree of automation, wide standard curve, no interference from natural sample fluorescence, simple and stable preparation of markers, and no radioactive pollution, and can be widely used for rapid detection or diagnosis. Through the immune response, it is possible to consider using quantitative or semi-quantitative methods to estimate the concentration of the labeled antibody and deduce the concentration of the bound target antigen, so that theoretically rapid diagnosis and precise (semi-) quantification of specific pathogenic microorganisms can be achieved.

[0008] In any case, the lateral flow rapid diagnostic kit meets the general standards of sensitivity, stability, economy and ease of use, and usually does not require the equipment required for rapid diagnosis, so it has important and extensive practical value. These advantages of lateral flow immunochromatography rapid diagnostic kits are particularly important for units and regions that lack professional equipment and untrained technicians, while for units and regions with professional equipment and trained technicians, the new design of the corresponding product can still meet the requirements of fine quantitative or semi-quantitative. Summary of the invention

[0009] The purpose of the present invention is to provide an antibody A4D7 targeting procalcitonin and an application thereof.

[0010] To achieve the purpose of the present invention, in the first aspect, the present invention provides an antibody A4D7 targeting procalcitonin, wherein the amino acid sequences of the light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody are RSSDSLLHSNGITYLF, QMSALAS and AQNLHLPWT, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody M1301 are SDYSWE, YIDFRGSTKYNPSLRS and RGSFRY, respectively.

[0011] The amino acid sequences of the light chain and heavy chain of the antibody are shown in SEQ ID NO: 1 and 2, respectively.

[0012] In a second aspect, the present invention provides use of the antibody in preparing a procalcitonin detection reagent or kit.

[0013] In a third aspect, the present invention provides a procalcitonin detection reagent or kit prepared from the antibody.

[0014] In a fourth aspect, the present invention provides use of the antibody in preparing a procalcitonin immunochromatographic test strip or a kit.

[0015] In a fifth aspect, the present invention provides use of the antibody in the preparation of a procalcitonin lateral flow immunochromatography diagnostic kit.

[0016] In a sixth aspect, the present invention provides a procalcitonin immunochromatographic test strip, comprising a sample pad, a conjugation pad, an NC membrane, a water absorbent pad and a base plate, wherein the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the conjugation pad, the NC membrane and the water absorbent pad are sequentially pasted on the base plate.

[0017] The conjugate pad is coated with the antibody F4B4 targeting procalcitonin coupled with immune (fluorescent) microspheres; the detection line is coated with the antibody A4D7; and the quality control line is coated with the sheep anti-mouse polyclonal antibody.

[0018] The amino acid sequences of the light chain and heavy chain of antibody F4B4 are shown in SEQ ID NOs: 3 and 4, respectively.

[0019] Preferably, the concentration of the antibody F4B4 coated on the conjugate pad is 10-15 μg / mL.

[0020] Preferably, the concentration of the antibody A4D7 coated on the detection line is 1.5-2 mg / mL.

[0021] Preferably, the sample pad and the absorbent pad are made of cellulose, and the binding pad is made of glass fiber.

[0022] In the seventh aspect, the present invention provides a qualitative and (semi-) quantitative rapid detection method for procalcitonin (including non-diagnostic purposes), wherein the detection method is a double antibody sandwich method, wherein the first antibody used is the antibody F4B4 targeting procalcitonin (the amino acid sequences of its light chain and heavy chain are shown in SEQ ID NO: 3 and 4, respectively), and the second antibody used is the antibody A4D7.

[0023] In the present invention, antibodies F4B4 and A4D7 bind to different epitopes of procalcitonin, respectively.

[0024] In the present invention, the corresponding antigen amino acid sequence is an amino acid sequence that has been adjusted or modified accordingly, and the modified material includes but is not limited to one or more of nanomaterials, fluorescent materials, enzymes, biotin and proteins. It is beneficial to apply the adjusted or modified amino acid sequence to the detection of procalcitonin, the design of procalcitonin immune antigens, etc.

[0025] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0026] (I) The present invention provides an antibody A4D7 that targets and recognizes procalcitonin, and uses it to construct a lateral flow immunochromatography rapid diagnostic kit. The antibody has high specificity and high affinity characteristics, and one or more of the amino acid sequences are used as the core. It is applied to the design of therapeutic targets for procalcitonin, and a therapeutic preparation for procalcitonin can be designed. The therapeutic target design includes but is not limited to the target design of therapeutic antibodies.

[0027] (ii) The antibody can not only be effectively used for the quantitative and / or qualitative detection of procalcitonin, but can also be used for a variety of detection methods such as ELISA detection, immunochemiluminescence detection and immunofluorescence detection, with a wide range of applications and strong applicability.

[0028] (III) The advantages of the lateral flow immunochromatography rapid diagnostic kit of the present invention are low detection limit and high sensitivity; and the selected antibodies specifically target antigen epitopes with highly conserved amino acid sequences, which reduces detection costs, shortens detection time, and improves detection efficiency. The lateral flow immunochromatography rapid diagnostic kit is a detection technology with important application value, which can achieve the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness and no equipment required. These advantages of the lateral flow immunochromatography rapid diagnostic kit are of great significance, especially for units or regions that lack professional equipment and well-trained technicians. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of protein electrophoresis using purified recombinant PCT protein in a preferred embodiment of the present invention.

[0030] Figure 2 The graphs are the binding curves of the antibody A4D7 in the preferred embodiment of the present invention with the recombinant procalcitonin protein antigen at gradient concentrations. Among them, 0.5 μg ( / mL) is the binding curve of 50 ng of antigen coated per well (100 μL per well), and 1 μg ( / mL) is the binding curve of 100 ng of antigen coated per well (100 μL per well).

[0031] Figure 3 The results of the pairing screening experiment of the monoclonal antibodies in the preferred embodiment of the present invention; wherein A is A4D7 paired with F4B4, B is A4D7 paired with A8E5, and C is A4D7 paired with C6H1.

[0032] Figure 4 The figure shows the stability test result of antibody A4D7 in the preferred embodiment of the present invention.

[0033] Figure 5 and Figure 6The results of using the procalcitonin solution standard substance to detect the lateral flow immunochromatography rapid test reagent developed based on the antibody of the present invention (different dilutions) in the preferred embodiment of the present invention; wherein, Figure 5 For analysis results without batch separation, Figure 6 The results of batch analysis. DETAILED DESCRIPTION

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. Example 1 Preparation of hybridomas producing monoclonal antibodies

[0035] 1. Design, preparation and carrier coupling of procalcitonin protein antigen

[0036] According to the relevant information of the procalcitonin gene on GenBank (NM_001741.3, SEQ ID NO: 5), the amino acid sequence of procalcitonin (SEQ ID NO: 6) was obtained. After immunogenicity, hydrophilicity and surface accessibility analysis, the peptide sequence of 141 amino acids in the CDS region of procalcitonin was finally selected as the antigen ( Figure 1 ), the antigen procalcitonin was synthesized by self-expression (the full-length gene was cloned into the expression vector pET20b).

[0037] 2. Immunization of mice

[0038] The synthetic antigen procalcitonin protein was mixed with Freund's complete adjuvant at a ratio of 1:1 and emulsified into 500 μL. The mixture was then subcutaneously injected into 3 female Balb / c mice aged 6-8 weeks. The antigen vaccination dose for each mouse was 50 μg. Three weeks later, the antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and emulsified into 500 μL. The antigen vaccination dose for each mouse was 50 μg. After an interval of three weeks, 50 μg was intraperitoneally injected without adjuvant. A total of 4 immunizations were performed. The results are shown in Table 1:

[0039] Table 1 Results of the immunization of mice with synthetic antigen procalcitonin

[0040]

[0041] 3. Immune serum titer determination

[0042] The indirect ELISA method was used to determine the titer of immune serum. 50 μg of synthetic antigen procalcitonin protein was dissolved in 10 mL 0.05 M pH 9.6 phosphate buffer and coated on a polystyrene 96-well plate, 100 μL / well, overnight at 4°C. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), blocked with 100 μL / well of 10 mM PBS containing 1% BSA blocking solution at 37°C for 2 h, and washed three times with PBS (containing 0.05% v / v Tween-20). Blood was collected from the tail vein of mice 10 days after the third immunization, and the mouse immune serum was stained with 10 mM PBS containing 1% BSA for 10 -2 ~10 -8 The plate was diluted 1:10000 and added to a 96-well plate at 100 μL / well at 37°C for 1 hour. The plate was washed three times with PBS (containing 0.05% v / v Tween-20) and then 100 μL / well of 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) was added at 37°C for 30 minutes. The plate was washed as above and then developed with TMB at 100 μL / well at room temperature in the dark for 10 minutes. 50 μL / well of 2M H 2 SO 4 The reaction was terminated, and the absorbance at 450 nm was measured. The serum of mice before immunization was used as a negative control, and the ratio of the measured value to the control value ≥ 2.1 was used as a positive judgment value to determine the titer of the immune serum.

[0043] 4. Preparation of hybridomas

[0044] Take serum with a titer greater than 1:10 5For mice, 3 days before fusion, take the synthesized antigen procalcitonin protein and mix it with an equal volume of PBS, then inject it intraperitoneally into BALB / c mice to be fused at a volume of 50μg / 500μL per mouse for booster immunization. Aseptically remove the spleen of the mouse, make a spleen cell suspension and mix it with the mouse myeloma cell line SP2 / 0 in the logarithmic growth phase in a 1:1 ratio, centrifuge at 1000g for 5 minutes at room temperature, discard the supernatant, flick the bottom of the centrifuge tube with your fingers to loosen the precipitate, place the centrifuge tube in a 37℃ water bath, and add 50% polyethylene glycol (PEG, MW4000, Sigma) kept in a 37℃ water bath drop by drop into the centrifuge tube with a dropper, shake the centrifuge tube while dropping, and complete the drop within 1 minute. After the drop is completed, After standing for 2 minutes, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 10 mL of serum-free 1640 medium preheated at 37°C were added every 1 minute to terminate the effect of polyethylene glycol. The cell mixture was centrifuged at 1000g for 5 minutes at room temperature, the supernatant was discarded, and HAT culture medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) was added to gently resuspend the cells, and the cells were divided into 96-well plates, 200 μL per well. After three days of culture, the cell fusion was observed, and half of the HAT culture medium was replaced for several consecutive days until clones were formed. Seven days after fusion, HT culture medium (hypoxanthine (H) and thymidine (T) (HT, Sigma)) was replaced for culture.

[0045] 5. Screening of hybridoma cells secreting anti-procalcitonin monoclonal antibodies

[0046] The cell culture supernatant was screened by indirect ELISA, and the positive cloned hybridoma cells with higher titers were selected for subcloning (Table 2), and cloned 2-3 times continuously by limiting dilution method until the cell positive rate reached 100%, and finally a cell line that stably secreted anti-procalcitonin protein monoclonal antibody was obtained, which was labeled as A4D7, etc. The cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

[0047] Table 2 Monoclonal antibody screening results

[0048]

[0049]

[0050] 6. Preparation and purification of ascites

[0051] The hybridoma cell line A4D7 was cultured at 1×10 6 / mouse was injected into the peritoneal cavity of 8-10 week old BALB / c female mice pretreated with liquid paraffin, and the abdominal fluid was extracted when the abdomen of the mice was swollen after 10-14 days of feeding and observation. Monoclonal antibodies were purified by affinity chromatography Protein G Sepharose Fast Flow, and the purity of monoclonal antibodies was determined by SDS-PAGE, and the purity reached more than 90%.

[0052] Example 2 Characterization of monoclonal antibody A4D7

[0053] 1. Determination of antibody concentration: The ascites prepared by hybridoma cells A4D7 and the like was purified to obtain the anti-procalcitonin monoclonal antibody A4D7 and the like, and the concentration was determined by Nanodrop nucleic acid protein assay produced by Thermofisher, and the concentration was >1 mg / ml.

[0054] 2. Antibody subtype identification: The Thermofisher mouse monoclonal antibody subtype identification kit was used to identify the subtype of the hybridoma cell line. The subtype of the antibody secreted by A4D7 was IgG1, and the light chain was κ chain.

[0055] 3. Identification of the potency of purified antibodies: 50 μg of synthetic procalcitonin protein was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), blocked with 10 mM PBS containing 1% BSA (150 μL / well), blocked at 37°C for 2 h, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL of purified antibody was added to each well, incubated at 37°C for 1 h, washed three times with PBS (containing 0.05% v / v Tween-20), horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was added as secondary antibody, incubated at 37°C for 30 min, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL of TMB was added to each well, incubated at 37°C for 15 min, and 2M H 2 SO 4 The solution terminated the reaction and the absorbance was detected by an enzyme marker at 450 nm.

[0056] 4. Antibody binding test:

[0057] The procalcitonin protein was diluted to 0.5 μg / mL and 1 μg / mL with 1×CB, and added to the wells of the ELISA plate at 100 μL / well, and duplicate wells were made, and placed at 4°C overnight or 37°C for adsorption for 2 hours. The coated microplate was dried, washed once according to the operating program (AFP program) set by the plate washer, and the blocking solution was added at 200 μL / well, placed in a 37°C incubator for 2 hours, and then placed at 4°C overnight. Before use, the blocked microplate was taken out from 4°C, dried, and the cleaning solution (1×PBS-T) was added to wet the ELISA plate; the monoclonal antibody A4D7 of the present invention was pre-diluted to 30 μg / mL with 1×PBS, and the pre-dilution multiple m was recorded, and then diluted 10 times, that is, 3 μg / mL as the highest concentration (S1), and then diluted in a 1:3 gradient (diluted in a 96-deep well plate), a total of 8 dilution gradients (S1-S8).

[0058] Add 100 μL of diluted antibody to a 96-well microplate that has been tapped clean on absorbent paper and incubate at 37°C for 30 min. After incubation, spin dry the plate and tap dry on absorbent paper. Wash the plate three times with a plate washer. Add 100 μL of 1×PBS to each well in columns 1-4. Add 200 μL of urea treatment solution to each well in columns 5 and 6 and incubate at 37°C for 30 min. After incubation, spin dry the plate and tap dry on absorbent paper. , wash the ELISA plate 3 times with a plate washer, add 100μL of GAM-HRP enzyme-labeled secondary antibody diluted 10,000 times with secondary antibody diluent to each well, and incubate at 37°C for 30 minutes; after incubation, spin dry the ELISA plate, pat dry on absorbent paper, wash the ELISA plate 3 times with a plate washer, take TMB color development solution, add 100μL color development solution to each well, and incubate at 37°C for 5-10 minutes; after color development, add 50μL stop solution to each well. Set the ELISA reader to read at 450nm / 630nm.

[0059] The ELISA antigen-antibody binding experiment was tested under the conditions of 0.5μg / mL and 1μg / mL antigen coating, and the data were obtained by testing the absorbance value OD ( Figure 2 ), and the corresponding polynomial curve was fitted. The corresponding binding showed obvious gradient with the change of antibody dilution ratio. At the same time, the affinity binding reflected by the two curves under the conditions of 0.5μg / mL and 1μg / mL antigen coating reflected the specificity of binding and reflected that the concentration corresponding to the antibody binding force reached 6.56E-10mol / L (under the condition of 0.5μg / mL antigen coating, the highest OD reading was 50%, that is, the concentration of antigen-antibody complex accounted for half of the total concentration, and the antibody concentration value K 0.5 ) and 1.28E-09mol / L (1μg / mL antigen coating conditions, the highest OD reading is 50%, that is, the concentration of the antigen-antibody complex accounts for half of the total concentration, the antibody concentration value K 1).

[0060] Binding strength calculation: Calculate the average OD readings of the duplicate wells of S1-S8 antibody concentrations before and after urea treatment under 0.5μg / mL and 1μg / mL antigen coating conditions. Substitute the readings into the binding strength calculation formula:

[0061]

[0062] 5. Monoclonal antibody pairing screening experiment:

[0063] The purified A4D7 monoclonal antibody was used to complete the coupling work. It was matched with three other procalcitonin monoclonal antibodies, the clone numbers of which were F4B4, A8E5, and C6H1. 49 clinical samples were tested using the matched products and the test results were obtained. The results were analyzed by Bland-Altman curve as follows:

[0064] Through curve analysis, it can be seen that when A4D7 is combined with F4B4 for sample testing, the test results have the highest correlation with the background value of the serum sample. Therefore, A4D7 and F4B4 are the best combination ( Figure 3 , A~C).

[0065] 6. Antibody stability verification:

[0066] The experimental operation method is as follows:

[0067] 6.1 Take 2 mg of antibody A4D7, divide it into 4 vials of 0.5 mg each, and seal them with sealing film.

[0068] 6.2 Take one of the tubes for processing and coupling to make a reagent. Store the reagent.

[0069] 6.3 Randomly take one of the tubes and store it in the freezer, and store the other tube in a 37°C incubator for 2 days (48 hours).

[0070] 6.4 After storage, return the antibody to room temperature.

[0071] 6.5 Prepare the antibodies according to the process. After the preparation is completed, use the samples to test the three experimental groups.

[0072] The test results under control storage, frozen storage and 37°C storage were compared. The stability of the antibody and the stability of the reagent prepared using the antibody showed good results ( Figure 4 ).

[0073] 7. Monoclonal antibody A4D7 sequencing

[0074] The monoclonal antibody A4D7 of procalcitonin prepared by the purified hybridoma cell A4D7 was obtained. The light chain variable region and the heavy chain variable region were sequenced to show that the amino acid sequences of the light chain and the heavy chain of the antibody A4D7 are shown in SEQ ID NO: 1 and 2, respectively. The light chain variable region sequences of the above-mentioned procalcitonin targeting antibody are CDR-L1 (RSSDSLLHSNGITYLF), CDR-L2 (QMSALAS) and CDR-L3 (AQNLHLPWT), and the heavy chain variable region sequences are CDR-H1 (SDYSWE), CDR-H2 (YIDFRGSTKYNPSLRS) and CDR-H3 (RGSFRY). Antibodies with corresponding variable region sequences have high affinity and good specificity; therefore, the corresponding antibody variable regions can be used for the development of recombinant antibodies, single-chain antibodies, bispecific antibodies, and for the development of related products for diagnostic or therapeutic purposes.

[0075] Example 3 Performance test of procalcitonin lateral flow immunochromatography reagent

[0076] Immuno(fluorescent) microspheres are one of the labeling materials used in immunochromatographic test kits. They can covalently bind to biomacromolecules such as proteins with amino groups by modifying their surface carboxyl terminals. This mild coupling can maximize the activity of biomacromolecules. The above advantages make immuno(fluorescent) microspheres a widely used labeling material in commercial immunochromatographic test strips on the market.

[0077] The lateral flow immunochromatography rapid diagnostic kit is a test paper-based method for reporting results suitable for immediate testing. Its core component is the lateral flow immunochromatography test strip, which is generally a narrow strip, usually 4-6 mm wide and no more than 6-7 cm long. A standard lateral flow immunochromatography test strip should include four main parts, namely: the main material of the sample pad area is made of cellulose, and the sample pad area is fully immersed in the test object when in use; the main material of the binding pad area is made of glass fiber, and the binding pad area generally contains (coupled) antigens or (coupled) antibodies; the main material of the detection area is made of nitrocellulose membrane (NC membrane), and there are mainly detection lines and quality control lines in the detection area, and the detection lines and quality control lines contain corresponding antibodies respectively; the main material of the absorbent pad area is made of cellulose.

[0078] When the lateral flow test paper is inserted into the solution to be tested, the solution will flow from the sample pad area into the binding pad area. When the solution contains the substance to be tested, the substance to be tested will bind to the (coupled) antigen or (coupled) antibody in the binding pad area. The (coupled) antibody or (coupled) antigen rehydrated by the solution or the complex formed with the sample to be tested will pass through the detection area (NC membrane) and the absorbent pad area in turn under the action of capillary force. At this time, the lateral flow test paper will have two result display models, namely the standard model and the competitive model:

[0079] When the lateral flow immunochromatographic test paper adopts the standard model (detecting PCT protein antigen), the test line (including the specific antibody targeting PCT protein antigen) can only capture the complex of the antigen to be tested and the (coupled) primary antibody: if the antigen to be tested exists in the solution, the test line can capture the complex formed by the antigen to be tested and the (coupled) primary antibody, and because the (coupled) primary antibody itself will always be captured by the quality control line (including the corresponding secondary antibody), the color of both the test line and the quality control line will change at this time, and the result is judged as a positive reaction. If the antigen to be tested does not exist in the solution, only the quality control line (including the corresponding secondary antibody) can capture the colloidal gold (coupled) primary antibody and change color. At this time, the quality control line will be colored but the test line will not be colored, and the result is judged as a negative reaction.

[0080] When the lateral flow test paper adopts a competitive model (detecting IgG antibodies targeting PCT protein antigens), the test antibody binds to the (coupled) PCT protein antigen coated on the binding pad area, and the test antibody and the colloidal gold (coupled) PCT protein antigen can each be captured by the test line (containing anti-IgG antibodies): when the test antibody is present, it can be captured by the test line while the (coupled) antigen cannot be captured by the test line (because of the competitive blocking of the test antibody), and thus no color change can occur, but the quality control line (containing the PCT protein antigen paired antibody) can still capture the (coupled) antigen, at which time the test line does not show color but the quality control line does, and the result is determined to be a positive reaction; if the test antibody does not exist in the solution, both the test line and the quality control line can capture the (coupled) antigen, at which time both the test line and the quality control line will change color, and the result is determined to be a negative reaction.

[0081] After the antibodies (A4D7 and F4B4) are paired, the above method is used to implement the (semi) quantitative relationship evaluation of the antibody targeting the purified procalcitonin protein: the quality control products of the antibody targeting the procalcitonin protein produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. and the (Roche) antigen products are used to detect the lateral flow immunochromatography rapid test kit developed by the antibody of the present invention. Under the premise of using a specific instrument (FIC-Q1) and specific chemical labeling of antibodies (antibodies targeting procalcitonin), the concentration of the labeled antibody is estimated relatively accurately within a certain concentration range by means of time-resolved fluorescent immunoassay (and compared with the quantitative accuracy of other companies' homogenized products for estimating the concentration of the labeled antibody). Since the target antigen PCT and the paired antibodies are both purified protein products developed by the company, they can be used to evaluate the accuracy of the quantitative relationship within a certain concentration range. As can be seen from the comparison in Table 3, by comparing the relationship between the antibody concentration estimated by the curve fitting of the quality control product (QC-product-based Ab-concentration estimation, or "QC-based Ab-con") and T / C, the relationship between the antibody concentration estimated by the directly measured antigen (Roche) background (Ag-background-based Ab-concentrationestimation, or "Ag-based Ab-con") and T / C, and the relationship between the antibody concentration estimated by the fitting curve obtained by the fixed parameters recommended by the finished product kit (Product-fixed-model-based Ab-concentration estimation, or "Product-based Ab-con") and T / C, the results indicate that the antibody concentration estimation method obtained by the fixed parameters recommended by the finished product kit and the estimation method of the antibody concentration by the quality control product can maintain a stable linear relationship in a larger concentration range, compared with the method of estimating the antibody concentration by directly measuring the antigen background, thereby achieving relatively more accurate quantification (for the analysis results regardless of batches, see Figure 5 The analysis results of the batches are shown in Figure 6 ).

[0082] Table 3 Correlation analysis table

[0083]

[0084] * p<.05, ＊＊ p<.01, ＊＊＊ p<.001

[0085] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Antibody A4D7 targeting procalcitonin, characterized in that: The amino acid sequences of the light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody are RSSDSLLHSNGITYLF, QMSALAS and AQNLHLPWT, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody are SDYSWE, YIDFRGSTKYNPSLRS and RGSFRY, respectively.

2. The antibody according to claim 1, characterized in that The amino acid sequences of the light chain and heavy chain of the antibody are shown in SEQ ID NO: 1 and 2, respectively.

3. Use of the antibody according to claim 1 or 2 in the preparation of a procalcitonin detection reagent or kit.

4. A procalcitonin detection reagent or kit prepared from the antibody according to claim 1 or 2.

5. Use of the antibody according to claim 1 or 2 in the preparation of a procalcitonin immunochromatographic test strip or kit.

6. Use of the antibody according to claim 1 or 2 in the preparation of a procalcitonin lateral flow immunochromatographic diagnostic kit.

7. A procalcitonin immunochromatographic test strip, characterized in that: It includes a sample pad, a conjugation pad, an NC membrane, a water absorbent pad and a bottom plate, wherein the NC membrane is provided with a detection line and a quality control line, and the sample pad, the conjugation pad, the NC membrane and the water absorbent pad are sequentially pasted on the bottom plate; Wherein, the conjugate pad is coated with the antibody F4B4 targeting procalcitonin coupled with immune microspheres; the detection line is coated with the antibody according to claim 1 or 2, and the quality control line is coated with sheep anti-mouse polyclonal antibody; The amino acid sequences of the light chain and heavy chain of antibody F4B4 are shown in SEQ ID NOs: 3 and 4, respectively.

8. The test strip according to claim 7, characterized in that: The concentration of the antibody A4D7 coated on the conjugate pad is 10-15 μg / mL; The concentration of the antibody A4D7 coated on the test line is 1.5-2 mg / mL.

9. The test strip according to claim 7 or 8, characterized in that: The sample pad and the water absorbent pad are made of cellulose, and the binding pad is made of glass fiber.

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