PCT protein antibody, latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein and preparation thereof

Abstract

The present invention relates to the field of biological detection technology, and provides a PCT protein antibody, including anti-human PCT protein monoclonal antibody I and / or anti-human PCT protein monoclonal antibody II; the anti-human PCT protein monoclonal antibody I includes a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2; the anti-human PCT protein monoclonal antibody II is formed by fusion of nano antibody VHH and Fc of human IgG1, and the sequence after fusion is shown in SEQ ID NO.3. The present invention also provides a latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein and a preparation method thereof, and the kit includes the above-mentioned PCT protein antibody. The advantages of the present invention are: the antibody has high affinity and good specificity, and the kit based on the antibody has high sensitivity, good specificity, and strong anti-interference ability.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a procalcitonin (PCT) protein antibody, a latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein, and a preparation method thereof. Background Art

[0002] Procalcitonin (PCT) is a precursor protein encoded by a gene located on human chromosome 11. The gene consists of 6 exons and 5 introns, with a total length of 7600bp, of which the coding region is 2800bp. In the rough endoplasmic reticulum of the parafollicular cells of the thyroid, the transcription of this gene produces the procalcitonin precursor, which is then modified and processed by endogenous peptidases to eventually generate the final product consisting of 116 amino acids, namely PCT.

[0003] PCT has important application value in the medical field, especially in the diagnosis and monitoring of infectious diseases and inflammatory conditions. PCT is mainly produced under the stimulation of bacterial toxins and inflammatory cytokines. In non-infectious inflammatory conditions, PCT in serum usually does not increase, while in infectious inflammatory processes, PCT is generated very rapidly. After being stimulated by endotoxin, the level of PCT rises significantly within 2 to 6 hours, and in the case of severe systemic infection, the blood concentration of PCT may be as high as 1000 times the normal level within 24 hours. Therefore, PCT can not only identify bacterial infection early, but also be an early warning and diagnostic indicator for sepsis. In addition, PCT is positively correlated with the severity of bacterial infection, and its upward or downward trend directly reflects the deterioration or improvement of the disease. Therefore, the detection of PCT has important reference value in the differential diagnosis, prognosis evaluation, treatment effect monitoring and rational use of antimicrobial drugs of bacterial infection, sepsis, and multiple organ failure syndrome (MODS).

[0004] The detection method of PCT has been approved by the U.S. Food and Drug Administration (FDA). In the early days, PCT was mainly detected using gel chromatography and high-performance liquid chromatography, which are complex, time-consuming, and difficult to automate. However, in recent years, the detection method of PCT has turned to immunological methods, which have the advantages of strong specificity, high sensitivity, and easy operation. Immunological methods include double antibody sandwich immunochemistry, colloidal gold method, and radioimmunoassay. The premise of these methods is to prepare monoclonal antibodies with high specificity for PCT, because the content of PCT in the plasma of healthy people is very low, usually less than 0.2ng / mL. Therefore, the preparation of PCT-specific antibodies is of key significance for immunological detection, which not only expands the scope of application of PCT in clinical practice, but also helps to reduce medical costs.

[0005] At present, most of the monoclonal antibodies used in the preparation of PCT protein latex turbidimetric kits in China are mouse or rabbit antibodies, which have some defects in affinity and specificity. The latex turbidimetric kits prepared with such antibodies have low detection sensitivity and serious nonspecific interference, which seriously affects the application of the kits in clinical testing.

[0006] Based on this, there is still much room for improvement in the preparation process of PCT protein antibodies and detection kits, and there is still a strong demand in this field for better PCT monoclonal antibody and kit preparation processes. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a PCT protein antibody, a latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein and the preparation thereof. The antibody of the present invention has high affinity and good specificity, and the kit based on the antibody has high sensitivity, good specificity and strong anti-interference ability.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A PCT protein antibody, including anti-human PCT protein monoclonal antibody I and / or anti-human PCT protein monoclonal antibody II;

[0010] The anti-human PCT protein monoclonal antibody I comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2;

[0011] The anti-human PCT protein monoclonal antibody II is formed by fusion of nano antibody VHH and Fc of human IgG1, and the sequence after fusion is shown in SEQ ID NO.3.

[0012] As one of the preferred embodiments of the present invention, the anti-human PCT protein monoclonal antibody I and the anti-human PCT protein monoclonal antibody II are also fused with Strep II tags at their C-termini, respectively. The amino acid sequence of the Strep II tag is shown in SEQ ID NO.4.

[0013] As one of the preferred embodiments of the present invention, the Strep II tag does not need to be biotinylated and can be directly bound to streptavidin.

[0014] A latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein, characterized in that it comprises the above-mentioned PCT protein antibody.

[0015] As one of the preferred embodiments of the present invention, the latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein specifically comprises reagent R1, reagent R2 and a calibrator;

[0016] The reagent R1 includes: a stabilizer, a coagulant, a blocking agent, a preservative, and a buffer;

[0017] The reagent R2 includes: a stabilizer, a preservative, a surfactant, a buffer, and latex particles coated with PCT protein antibodies;

[0018] The calibrator comprises: a preservative, a stabilizer, a PCT antigen, and a buffer;

[0019] Among them, the "latex particles coated with PCT protein antibodies" of the reagent R2 specifically use two antibodies, anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II, and the two antibodies are respectively connected to the latex particles through "streptavidin-Strep-II".

[0020] As one of the preferred embodiments of the present invention, in the reagent R1:

[0021] The stabilizer is 10 mg / mL trehalose;

[0022] The coagulant was PEG6000 with a final concentration of 0.5%;

[0023] The blocking agent was a rheumatoid factor blocking agent with a final concentration of 100ug / mL;

[0024] The preservative is 0.05% ProClin300;

[0025] The buffer included 50 mM Tris-HCl buffer and 2% NaCl, buffer pH 7.5.

[0026] As one of the preferred embodiments of the present invention, in the reagent R2:

[0027] Stabilizers included 5 mg / mL bovine serum albumin and 15 mg / mL trehalose;

[0028] The preservative is 0.05% ProClin300;

[0029] The surfactant was 0.01% Tween 20;

[0030] The buffer was 50 mM Hepes buffer, pH 7.5;

[0031] Latex particles coated with PCT protein antibody, final concentration 0.15%.

[0032] As one of the preferred embodiments of the present invention, in the calibrator:

[0033] The preservative is 0.05% sodium azide;

[0034] Stabilizers included 15 mg / mL bovine serum albumin and 100 mg / mL trehalose;

[0035] PCT antigen is a PCT protein antigen with a concentration of 0 to 100 ng / mL;

[0036] The buffer was a Tris-HCl buffer with a concentration of 50 mM and a pH of 7.2.

[0037] Among them, PCT protein antigen can be directly selected from commercially available products, for example, the "recombinant human PCT antigen" produced by Anhui Qiancheng Biotechnology Co., Ltd. is derived from Ecoli, human, purity ≥95%, product number: AG00402; recombinant PCT protein expressed and purified by other in vitro systems currently on the market can also be used.

[0038] As one of the preferred embodiments of the present invention, the "latex particles coated with PCT protein antibodies" are obtained by the following method:

[0039] (1) Washing and activation of latex particles

[0040] ① Add the latex particles to MES washing solution and centrifuge, discard the supernatant, and repeat the washing several times;

[0041] ② Resuspend the precipitate in MES coupling buffer and add EDC for activation;

[0042] ③ The activated latex particles were washed with MES coupling buffer and then resuspended;

[0043] (2) Streptavidin coupling

[0044] ① Take the activated washed latex solution and add streptavidin; mix well and couple on a shaker;

[0045] ② Wash the microsphere particles with Hepes buffer and repeat several times;

[0046] ③Add sealing agent to seal;

[0047] (3) Ligation of PCT protein antibodies with streptavidin-coated particles

[0048] The latex particle solution prepared in step (2) is mixed with the two pre-mixed PCT antibodies and incubated in a shaker to form a "latex particle-streptavidin-PCT protein antibody complex", i.e., the desired "latex particle coated with PCT protein antibody".

[0049] As one of the preferred embodiments of the present invention, in the "latex particles coated with PCT protein antibodies" of the reagent R2, the mixing mass ratio between the anti-human PCT protein monoclonal antibody I and the anti-human PCT protein monoclonal antibody II is 2:1.

[0050] A method for preparing the above-mentioned latex enhanced immunoturbidimetric kit for quantitatively detecting PCT protein comprises the following steps:

[0051] (1) Prepare reagent R1:

[0052] The stabilizer, coagulant, blocking agent, preservative and buffer of reagent R1 are mixed in the same container and mixed evenly to obtain reagent R1;

[0053] (2) Prepare dilution buffer of reagent R2:

[0054] Mixing the stabilizer, preservative, surfactant and buffer of reagent R2 to obtain a dilution buffer of reagent R2;

[0055] (3) Prepare reagent R2:

[0056] ① Washing and activation of latex particles

[0057] Add MES washing solution to the latex particles and centrifuge, discard the supernatant, and repeat the washing several times;

[0058] The pellet was resuspended in MES coupling buffer and activated by adding EDC;

[0059] The activated latex particles were washed with MES coupling buffer and then resuspended;

[0060] ②Streptavidin coupling

[0061] Take the activated washed latex solution, add streptavidin, mix well and couple on a shaker;

[0062] Wash the microsphere particles with Hepes buffer and repeat several times;

[0063] Add sealing agent to seal;

[0064] ③ Ligation of PCT protein antibody and streptavidin-coated particles

[0065] The latex particle solution prepared in step ② is mixed with the two antibodies "anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II", and incubated in a shaker to form a "latex particle-streptavidin-PCT protein antibody complex", and then diluted with the dilution buffer prepared in step (2) to the target concentration;

[0066] (4) Preparation of calibration materials:

[0067] The preservative, stabilizer, PCT antigen and buffer of the calibrator are mixed to obtain the calibrator.

[0068] As one of the preferred embodiments of the present invention, the kit of the present invention can be used for quantitatively detecting the content of PCT protein in human blood on a semi-automatic or fully-automatic biochemical analyzer, wherein the blood includes whole blood, serum and plasma.

[0069] The advantages of the present invention compared to the prior art are:

[0070] (1) The antibodies of the present invention are self-developed antibodies, and three aspects of improvements have been made: ① The antibody sequences were screened to obtain two antibodies with high affinity and good specificity; ② The Fc of one of the antibodies was transformed into a human Fc, thereby greatly reducing the detection interference caused by heterophagic antibodies; ③ Strep-II tags were added to the C-termini of the two antibodies to facilitate the coupling of the antibodies to the latex microspheres through the "streptavidin-Strep-II connection system"; the kit of the present invention based on the above self-developed antibodies has high sensitivity, good specificity, and strong anti-interference ability;

[0071] (2) The latex particles in the reagent R2 of the present invention and the PCT protein antibody form an indirect connection system, and the indirect connection system is a "streptavidin-Strep-II connection system"; the indirect connection antibody can retain the antibody activity to the greatest extent, so that the antibody can play a more precise role and greatly improve the detection sensitivity;

[0072] (3) The PCT protein detection results of the kit of the present invention and the existing kit for the same sample were statistically analyzed and the results were well correlated, accurate and reliable, with no significant difference. The kit can replace the existing products in clinical use and can significantly reduce the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a graph showing the PCT protein binding antibody affinity ELISA test results in Example 3;

[0074] Figure 2 is a linear range correlation diagram of the detection kit in Experimental Example 1;

[0075] Figure 3 This is a correlation comparison chart of the detection of clinical samples by the kit of the present invention and the comparative kit in Experimental Example 1. DETAILED DESCRIPTION

[0076] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0077] The reagents and experimental methods used in the following examples are conventional reagents or methods in the art unless otherwise specified and will not be described in detail.

[0078] Example 1

[0079] The anti-human PCT protein monoclonal antibody I of this embodiment comprises a light chain and a heavy chain; wherein the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2;

[0080] At the same time, the antibody C-terminus of the anti-human PCT protein monoclonal antibody I is also fused with a Strep II tag; the Strep II tag does not need to be biotinylated and can directly bind to streptavidin, and the amino acid sequence is shown in SEQ ID NO.4.

[0081] Example 2

[0082] An anti-human PCT protein monoclonal antibody II of this example is formed by fusion of nano antibody VHH and Fc of human IgG1, and the sequence after fusion is shown in SEQ ID NO.3.

[0083] At the same time, the antibody C-terminus of the anti-human PCT protein monoclonal antibody II is also fused with a Strep II tag; the Strep II tag does not need to be biotinylated and can directly bind to streptavidin, and the amino acid sequence is shown in SEQ ID NO.4.

[0084] Example 3

[0085] A method for obtaining the above two PCT protein antibodies (anti-human PCT protein monoclonal antibodies I and II) in this embodiment comprises the following steps:

[0086] (1) Construction of CHO-K1-PCT protein antibody stable cell line

[0087] Cell recovery: Take a tube of liquid nitrogen frozen cells (about 1x10E7 cells), thaw quickly in a 37°C water bath, centrifuge at 300g for 5 minutes. Wipe the cryotube with alcohol cotton and place it on a clean bench. Aspirate the supernatant, resuspend it with 20mL pre-warmed CHO complete medium, and culture it in a 125mL shake flask at 37°C, 5% CO 2 , rotation speed 120~130rpm.

[0088] Plasmid extraction: inoculate in advance "DH5a strain containing anti-human PCT protein monoclonal antibody I / antibody II stable carrier" (gene synthesis by a biological company, the sequence of anti-human PCT protein monoclonal antibody I is shown in Example 1, and the antibody C-terminus is fused with a Strep-II tag; the sequence of PCT protein monoclonal antibody II is shown in Example 2, and the antibody C-terminus is fused with a Strep-II tag), culture overnight at 37°C, and use a commercial plasmid extraction kit to extract the pXC17.4-PCT anti-human PCT protein monoclonal antibody I / pXC17.4-PCT antibody II stable carrier plasmid.

[0089] Cell transfection: When the cell viability is greater than 95%, there is no obvious cell aggregation, and the density is between 2 and 3x 10E6, take 4x 10E7 cells, centrifuge at 300g for 5min. Remove the supernatant and resuspend the cells with 400uL of Celetrix commercial electroporation solution. Use 25ug of anti-human PCT protein monoclonal antibody I / II plasmid, consuming 2x10E7 cells respectively. Perform electroporation at 1250V using Celetrix cell electroporator.

[0090] Cell recovery and pressurization: The transfected cells were resuspended in CHO complete medium and allowed to recover for 24 hours in a 37°C, 5% CO2 incubator. The recovered cells were centrifuged at 300g for 5 minutes, the medium was removed, CHO medium and 25uM MSX were added, and the cell density was 1x10E6. 37°C, 5% CO 2 The cells were placed in an incubator for 8 to 10 days until the cell viability was greater than 30%.

[0091] Monoclonal screening: Dilute the cell pool with cell viability greater than 30% to 2.5 cell / mL with Kangsheng commercial monoclonal culture medium, and spread 200uL / well on 96-well plates, for a total of 30 plates. 37°C, 5% CO 2 The cells were cultured in an incubator until the monoclones filled the wells. The monoclones with high expression levels were transferred to shake flasks for expansion culture. The final monoclones were used to construct CHO-K1 working cell banks and frozen in liquid nitrogen tanks for subsequent antibody expression.

[0092] (2) Expression of “anti-human PCT protein monoclonal antibody I / II” in CHO-K1 suspension expression system

[0093] Cell recovery: Take out the CHO-K1 working cell line constructed in step (1) from the liquid nitrogen tank, quickly thaw in a 37°C water bath, resuspend the cells in CHO cell culture medium, and place them in a cell culture shaker for 48 hours of recovery.

[0094] Cell transfer and fed-batch culture: After the cells recovered, they were transferred to the fermentation medium at a density of 0.5x10E6. Cultured until Day 5, fed-batch feeding was started, and cooled culture was performed until Day 7.

[0095] Collecting supernatant: When the culture is cultured to Day 15 or the cell viability is lower than 60%, centrifuge the culture supernatant, remove the cell precipitate, and retain the supernatant.

[0096] (3) Affinity purification of "anti-human PCT protein monoclonal antibody I / II"

[0097] Affinity column filling: calculate the required commercial Protein A filler, load the filler into the column, and wash the loaded filler with PBS equilibration buffer.

[0098] Loading and washing: The anti-human PCT protein monoclonal antibody I / II cell supernatant collected by centrifugation was loaded at a low flow rate. After loading, 10 column volumes were washed with equilibration buffer and 10 column volumes were washed with pre-elution buffer A at pH 5.0.

[0099] Elution and neutralization: The filler after washing with the pre-elution solution is eluted with a pH 3.2 citric acid eluent. The eluted protein is neutralized with a 2M Tris buffer. The protein concentration of the neutralized antibody solution is measured, and then the solution is dialyzed into PBS and stored in aliquots.

[0100] (4) ELISA affinity test of “anti-human PCT protein monoclonal antibody I / II”

[0101] Plating: Recombinant human PCT protein was diluted with carbonate buffer to a concentration of 0.5ug / mL, added to the Elisa plate at 100uL / well, and incubated at 4°C overnight.

[0102] Blocking: Wash the ELISA plate three times with PBS + 0.1% Tween 20 cleaning solution, add PBST + 2.5% skim milk powder blocking solution at a volume of 200uL / well, and block at 37°C for 1h.

[0103] Primary antibody binding: Wash the ELISA plate 3 times with PBS + 0.1% Tween 20 cleaning solution, dilute the antibody with PBST + 2.5% skim milk powder, the first well concentration is 1ug / mL, 3-fold gradient dilution, a total of 8 gradients. Add the diluted antibody to the Elisa plate, the addition amount is 100uL / well, and incubate at 37℃ for 2h.

[0104] Secondary antibody binding: Wash the ELISA plate 3 times with PBS + 0.1% Tween 20 cleaning solution, dilute the goat anti-human Fc-HRP secondary antibody with PBST + 2.5% skim milk powder. Add the diluted antibody to the Elisa plate at a volume of 100uL / well and incubate at 37°C for 1h.

[0105] Color development: Wash the ELISA plate 3 times with PBS + 0.1% Tween 20 cleaning solution, add OPD color development solution, the amount added is 100uL / well. After color development for 5-15 minutes, add 100uL 2M H 2 SO 4 The reaction was terminated and detected using an enzyme reader at a wavelength of OD450.

[0106] The test results were processed using Graphpad. The processing results are shown in Figure 1 .Depend on Figure 1 It can be seen that anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II have good binding activity with PCT protein, and the Ec50 range of the two antibodies is 1-10 ng / mL.

[0107] Example 4

[0108] A latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein in this embodiment includes reagent R1, reagent R2 and a calibrator.

[0109] Reagent R1 includes: 10 mg / mL trehalose, 0.5% PEG6000, 0.05% ProClin300, 50 mM Tris-HCl buffer (containing 2% NaCl, buffer pH 7.5), 100 ug / mL rheumatoid factor blocker (the blocker used in this kit is HIER-E-001 blocker from Feipeng Biotechnology Co., Ltd., product number: HIER-E-001).

[0110] Reagent R2 includes: 5 mg / mL bovine serum albumin, 15 mg / mL trehalose, 0.05% ProClin300, 0.01% Tween 20, 50 mM Hepes buffer (pH 7.5), and latex particles coated with PCT protein antibody at a final concentration of 0.15%.

[0111] The calibrators include: 0.05% sodium azide, 15 mg / mL bovine serum albumin, 100 mg / mL trehalose, 0-100 ng / mL PCT protein antigen, and 50 mM Tris-HCl buffer (pH 7.2).

[0112] Specifically, in this embodiment, the "latex particles coated with PCT protein antibodies" of the reagent R2 specifically use two antibodies, the anti-human PCT protein monoclonal antibody I of Example 1 and the anti-human PCT protein monoclonal antibody II of Example 2 (the mass ratio of antibody I to antibody II is 2:1), and the two antibodies are respectively connected to the latex particles through "streptavidin-Strep-II". Among them, the original amino acid sequence of the anti-human PCT protein monoclonal antibody I is shown in SEQ ID NO.1 and SEQ ID NO.2, and the Strep-II modification refers to the addition of a Strep-II tag to the C-terminus of the antibody (the sequence N-terminus-C-terminus after the modification is, the antibody's own sequence + Strep-II); the original amino acid sequence of the anti-human PCT protein monoclonal antibody II is shown in SEQ ID NO.3, and the Strep-II modification refers to the addition of a Strep-II tag to the C-terminus of the antibody (the sequence N-terminus-C-terminus after the modification is, the antibody's own sequence + Strep-II).

[0113] Specifically, in this embodiment, the "PCT protein antigen" of the calibrator adopts the "recombinant human PCT antigen" produced by Anhui Qiancheng Biotechnology Co., Ltd., which is derived from Ecoli, human, with a purity of ≥95%, and the product number is: AG00402 (recombinant PCT protein expressed and purified by other in vitro systems on the existing market can also be used).

[0114] Preparation method:

[0115] (1) Prepare reagent R1:

[0116] In 50 mM Tris-HCl buffer (containing 2% NaCl, buffer pH 7.5), add 10 mg / mL trehalose, 0.5% PEG6000, 0.05% ProClin300, and 100 ug / mL rheumatoid factor blocking agent and stir evenly to obtain the R1 reagent.

[0117] (2) Prepare dilution buffer of reagent R2:

[0118] 50 mM Hepes buffer (pH 7.5), 5 mg / mL bovine serum albumin, 15 mg / mL trehalose, 0.05% ProClin 300, and 0.01% Tween 20 were mixed to obtain a dilution buffer for reagent R2.

[0119] (3) Prepare reagent R2:

[0120] ① Washing and activation of latex particles

[0121] The latex particles were added to MES washing solution and centrifuged, the supernatant was discarded, and the washing was repeated 3 times;

[0122] The pellet was resuspended in MES coupling buffer and activated with EDC for 30 min;

[0123] The activated latex particles were washed three times with MES coupling buffer and then resuspended to make the final concentration of latex particles at a mass volume ratio of 1%;

[0124] ②Streptavidin coupling

[0125] Take 1 mL of activated and washed latex solution, add 0.5 mg / mL streptavidin, mix well, and couple at 37°C and 220 rpm for 3 h.

[0126] Wash the microspheres with Hepes buffer, repeat 3 times;

[0127] Add blocking agent and block for 2 hours; the blocking agent is Hepes buffer containing 5% BSA;

[0128] ③ Ligation of PCT protein antibody and streptavidin-coated particles

[0129] The latex particle solution prepared in step ② is mixed with two antibodies, "anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II", and the two antibodies are mixed in a ratio of 2:1 by mass, and incubated in a shaker at 37°C for 2 hours to form a "latex particle-streptavidin-anti-human PCT protein antibody complex"; wherein, 1L of 0.5% latex particle solution is used to add 100mg and 50mg of antibodies respectively for mixed incubation; and the mixture is diluted with the dilution buffer prepared in step (2) to a final mass-to-volume ratio of 0.15%.

[0130] (4) Preparation of calibration materials:

[0131] PCT protein antigens with concentrations of 0, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL were added to 50 mM Tris-HCl buffer at pH 7.2 containing 0.05% sodium azide, 15 mg / mL bovine serum albumin, and 100 mg / mL trehalose, and stirred evenly to serve as PCT protein calibrators.

[0132] Comparative Example 1

[0133] The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein in this comparative example has the same formulation and preparation method as those in Example 4, with the main difference being that the "latex particles coated with PCT protein antibodies" use the monoclonal antibodies I and II of the present invention without any modification.

[0134] That is, relative to Example 4, the original amino acid sequence of the Fab region of the antibody in this comparative example is the same, but its Fc sequence is a mouse IgG2a sequence, and the C-terminus is not fused with a Strep-II tag.

[0135] Comparative Example 2

[0136] The comparative example is a latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein, and its formulation and preparation method are basically the same as those of Example 4, with the main difference being that: the "latex particles coated with PCT protein antibody" use the monoclonal antibody I and antibody II of the present invention that have not been modified with Strep-II, but the antibody Fc sequence is a human IgG1 sequence.

[0137] That is, relative to Example 4, the amino acid sequence of the antibody in this comparative example is the same, but no Strep-II tag is added to its C-terminus.

[0138] Comparative Example 3

[0139] The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein in this comparative example has a formulation and preparation method that are basically the same as those in Example 4, with the main difference being that the "latex particles coated with PCT protein antibodies" use the inventive monoclonal antibodies I and II modified by Strep-II, but the antibody Fc sequence is a mouse IgG2a sequence.

[0140] That is, relative to Example 4, the amino acid sequence of the Fab region of the antibody in this comparative example is the same, a Strep-II tag is added to its C-terminus, but the Fc sequence of the antibody is a mouse IgG2a sequence.

[0141] Comparative Example 4

[0142] The present comparative example is a latex-enhanced immunoturbidimetric kit for quantitatively detecting PCT protein, and its formulation and preparation method are basically the same as those in Example 4, with the main difference being that: "latex particles coated with PCT protein antibodies" use commercially available common anti-human PCT protein monoclonal antibodies I and II (PCT antibody 12D12 and PCT antibody 7B7 from Wuhan Aokebot Biotechnology Co., Ltd., with article numbers A2801 and A2802, respectively).

[0143] Test Example 1

[0144] This test example is used to test the performance of the PCT protein latex enhanced immunoturbidimetric kit (kit prepared by the method of Example 4) of the present invention.

[0145] 1. Biochemical analyzer detection method:

[0146] Take Hitachi 7180 biochemical analyzer as an example: the measurement wavelength is 600nm, first add 180uL of R1 reagent, incubate at 37℃ for 30s; add 15uL of calibrator, react at 37℃ for 60s; add 60uL of R2 reagent, react at 37℃ for 60s; measure the absorbance value A1 of each tube, react for 5min, read the absorbance value A2 of each tube, calculate the absorbance difference ΔA=A2-A1; repeat the measurement for each tube at least twice, take the average value of the absorbance difference ΔA measured twice by the calibration tube as the ordinate, and the corresponding concentration as the abscissa, and draw the "concentration-absorbance difference" calibration curve.

[0147] Take the serum or plasma sample to be tested, measure the absorbance difference of the sample in the same way, substitute it into the calibration curve, and then calculate the concentration of PCT protein in the sample to be tested. If the concentration of PCT protein in the sample exceeds the range of the standard curve, the sample to be tested needs to be diluted before testing to ensure the accuracy of the test results.

[0148] 2. Sensitivity detection:

[0149] 5% bovine serum albumin solution was used as a blank sample. The test was repeated more than 20 times according to the above biochemical analyzer detection method. The blank mean plus two times the standard deviation was used as the minimum detection limit. The results showed that the minimum detection limit of the kit of the present invention was 0.08 ng / mL.

[0150] 3. Linear range detection:

[0151] A high concentration sample close to the PCT protein concentration of 100 ng / mL was diluted with physiological saline at 1 / 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256 to prepare a total of 9 gradient dilution concentrations of sample solutions, and the concentration of each diluted sample was determined using the biochemical analyzer detection method. Each concentration was measured 3 times, and the average value of the measurement results was calculated.

[0152] The linear regression equation was obtained with the dilution concentration as the independent variable and the average value of the test results as the dependent variable, and the linear regression correlation coefficient r was calculated according to the corresponding formula. The test results are shown in Table 1, which proves that the linear performance reaches 100 ng / mL, the correlation equation is y=0.962x+0.8935, and the correlation coefficient R 2 =0.9966, see Figure 2 .

[0153] Table 1. Linear range detection of the kit of the present invention

[0154] Dilution ratio Theoretical concentration (ng / mL) Detection concentration (ng / mL) Recovery rate (%) 1 / 256 0.39 0.42 107.7 1 / 128 0.78 0.82 105.1 1 / 64 1.56 1.60 102.6 1 / 32 3.125 3.21 102.7 1 / 16 6.25 6.53 104.5 1 / 8 12.5 13.19 105.5 1 / 4 25.0 26.48 105.9 1 / 2 50.0 53.15 106.3 1 / 1 100.0 94.67 94.7

[0155] 4. Repeatability and accuracy:

[0156] The PCT protein concentration was 6.25 ng / mL and 25 ng / mL of calibrators were used as samples and measured according to the biochemical analyzer detection method. Each concentration was repeated 10 times, and the mean and standard deviation were calculated. The results are shown in Table 2.

[0157] Table 2. Repeatability and accuracy test of the kit of the present invention

[0158]

[0159] It can be seen from Table 2 that the repeatability and accuracy of the PCT protein latex enhanced immunoturbidimetric kit of the present invention can meet the requirements of market application.

[0160] 5. Batch Difference:

[0161] Three batches of test kits were prepared according to the test kit preparation method described in Example 4 of the present invention. The same serum sample was repeatedly measured using the three batches of test kits according to the biochemical analyzer detection method. Each batch was measured three times, and the mean of the three measurements of each batch was calculated. The results are shown in Table 3.

[0162] Table 3. Batch difference detection of the kit of the present invention

[0163]

[0164] It can be seen from Table 3 that the batch differences of the three batches of PCT protein latex enhanced immunoturbidimetric kits are small, and the relative deviation is less than 5%.

[0165] VI. Comparison of the correlation between the kit of the present invention and the comparative kit in detecting clinical samples:

[0166] The comparison kit is a latex-enhanced turbidimetric detection kit for PCT protein produced by Anhui Daqian Biotechnology Co., Ltd., with a product number of 70-6080. 80 clinical samples were tested simultaneously using the present invention and the comparison kit.

[0167] The PCT protein concentration of the sample detected by the comparison kit was used as the horizontal axis, and the PCT protein concentration detected by the kit of the present invention was used as the vertical axis for regression analysis. The results are as follows: Figure 3 As shown, the correlation equation is: y = 0.9531x + 2.5495, the linear correlation coefficient R 2 =0.9981.

[0168] The above results show that the detection values ​​and trends of clinical samples by the kit of the present invention and the control kit have good clinical correlation.

[0169] Test Example 2

[0170] This test example is used to verify the performance difference between the kit of the present invention and the kits of comparative examples 1 to 4.

[0171] ELISA experiments were performed on the antibodies in the kits of Example 4 of the present invention and Comparative Examples 1, 2, 3, and 4 to detect their affinities. The results are shown in Table 4.

[0172] At the same time, the same standard PCT sample was tested using the kits of Example 3 of the present invention and Comparative Examples 1, 2, 3, and 4, respectively. The results are shown in Table 5.

[0173] At the same time, the kits of Example 3 of the present invention and Comparative Examples 1, 2, 3, and 4 were used to detect special samples (serum samples with strong non-specific binding), and the results are shown in Table 6.

[0174] Table 4. Comparison of ELISA affinity of the antibodies of the present invention and the comparative examples

[0175] sample Ec50(ng / mL) sample Ec50(ng / mL) Comparative Example 1-Antibody I 5.68 Comparative Example 1-Antibody II 8.92 Comparative Example 2-Antibody I 6.20 Comparative Example 2-Antibody II 9.31 Comparative Example 3-Antibody I 6.35 Comparative Example 3-Antibody II 9.53 Comparative Example 4-Antibody I 8.63 Comparative Example 4-Antibody II 16.5 The present invention - Antibody I 5.86 The present invention - Antibody II 9.62

[0176] Table 5. Comparison of test results of the reagents of the present invention and the comparative examples

[0177]

[0178] Table 6. Comparison of test results of the reagents of the present invention and the comparative examples

[0179]

[0180] From the results of Table 4, Table 5 and Table 6, we can see that:

[0181] (1) For antibodies of the present invention based on specific sequences, changing the basic sequence will affect the affinity and specificity of the antibodies (combined with the affinity comparison data of the present invention and comparative examples 1, 2, 3, and 4 in Table 4).

[0182] (2) For the antibodies of the present invention based on specific sequences, changing the Fc of different subtypes and adding Strep-II tags at the C-terminus will not have a significant impact on the affinity of the antibodies (combined with the affinity comparison data results of the present invention and comparative examples 1, 2, and 3 in Table 4).

[0183] (3) Adding a Strep-II tag to the C-terminus of the antibody can further improve the sensitivity of the final detection (combining the data results of the present invention and comparative example 2 in Table 5);

[0184] (4) The Fc end of the antibody uses a human Fc sequence to reduce the nonspecific interference caused by abnormal heterotrophic antibodies, thereby increasing the specificity and detection sensitivity of the kit (combining the data results of the present invention and comparative example 3 in Table 6).

[0185] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A PCT protein antibody, characterized in that: Including anti-human PCT protein monoclonal antibody I and / or anti-human PCT protein monoclonal antibody II; The anti-human PCT protein monoclonal antibody I comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2; The anti-human PCT protein monoclonal antibody II is formed by fusion of nano antibody VHH and Fc of human IgG1, and the sequence after fusion is shown in SEQ ID NO.

3.

2. A PCT protein antibody, characterized in that: Including anti-human PCT protein monoclonal antibody I and / or anti-human PCT protein monoclonal antibody II; The anti-human PCT protein monoclonal antibody I comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2; at the same time, the antibody C-terminus of the anti-human PCT protein monoclonal antibody I is also fused with a Strep II tag; the amino acid sequence of the Strep II tag is shown in SEQ ID NO.4; The anti-human PCT protein monoclonal antibody II is formed by the fusion of nano antibody VHH, Fc of human IgG1 and Strep II tag; wherein, the sequence after the fusion of nano antibody VHH and human IgG1 is shown in SEQ ID NO.3, and the C-terminus of the SEQ ID NO.3 sequence is fused with the Strep II tag; the amino acid sequence of the Strep II tag is shown in SEQ ID NO.

4.

3. A latex enhanced immunoturbidimetric kit for quantitative detection of PCT protein, characterized in that: Comprising the PCT protein antibody as described in claim 1 or 2.

4. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 3, characterized in that: Specifically including reagent R1, reagent R2 and calibrator; The reagent R1 includes: a stabilizer, a coagulant, a blocking agent, a preservative, and a buffer; The reagent R2 includes: a stabilizer, a preservative, a surfactant, a buffer, and latex particles coated with PCT protein antibodies; The calibrator comprises: a preservative, a stabilizer, a PCT antigen, and a buffer; Among them, the "latex particles coated with PCT protein antibodies" of the reagent R2 specifically use two antibodies, anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II, and the two antibodies are respectively connected to the latex particles through "streptavidin-Strep-II".

5. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 4, characterized in that: In the reagent R1: The stabilizer is 10 mg / mL trehalose; The coagulant was PEG6000 with a final concentration of 0.5%; The blocking agent was a rheumatoid factor blocking agent with a final concentration of 100ug / mL; The preservative is 0.05% ProClin300; The buffer consisted of 50 mM Tris-HCl buffer and 2% NaCl, buffer pH 7.

5.

6. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 4, characterized in that: In the reagent R2: Stabilizers included 5 mg / mL bovine serum albumin and 15 mg / mL trehalose; The preservative is 0.05% ProClin300; The surfactant was 0.01% Tween 20; The buffer was 50 mM Hepes buffer, pH 7.5; Latex particles coated with PCT protein antibody, final concentration 0.15%.

7. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 4, characterized in that: Among the calibrators: The preservative is 0.05% sodium azide; Stabilizers included 15 mg / mL bovine serum albumin and 100 mg / mL trehalose; PCT antigen is PCT protein antigen with a concentration of 0-100 ng / mL; The buffer was a Tris-HCl buffer with a concentration of 50 mM and a pH of 7.

2.

8. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 4, characterized in that: The "latex particles coated with PCT protein antibodies" are obtained by the following method: (1) Washing and activation of latex particles ① Add the latex particles to MES washing solution and centrifuge, discard the supernatant, and repeat the washing several times; ② Resuspend the precipitate in MES coupling buffer and add EDC for activation; ③ The activated latex particles were washed with MES coupling buffer and then resuspended; (2) Streptavidin coupling ① Take the activated washed latex solution and add streptavidin; mix well and couple on a shaker; ② Wash the microsphere particles with Hepes buffer and repeat several times; ③Add sealing agent to seal; (3) Ligation of PCT protein antibodies and streptavidin-coated particles The latex particle solution prepared in step (2) is mixed with the two pre-mixed PCT antibodies and incubated in a shaker to form a "latex particle-streptavidin-PCT protein antibody complex", i.e., the desired "latex particle coated with PCT protein antibody".

9. The latex-enhanced immunoturbidimetric kit for quantitative detection of PCT protein according to claim 4, characterized in that: In the "latex particles coated with PCT protein antibodies" of the reagent R2, the mixing mass ratio between the anti-human PCT protein monoclonal antibody I and the anti-human PCT protein monoclonal antibody II is 2:

1.

10. A method for preparing a latex enhanced immunoturbidimetric kit for quantitatively detecting PCT protein according to any one of claims 3 to 9, characterized in that: The steps include: (1) Prepare reagent R1: The stabilizer, coagulant, blocking agent, preservative and buffer of reagent R1 are mixed in the same container and mixed evenly to obtain reagent R1; (2) Prepare dilution buffer for reagent R2: Mixing the stabilizer, preservative, surfactant and buffer of reagent R2 to obtain a dilution buffer of reagent R2; (3) Prepare reagent R2: ① Washing and activation of latex particles Add MES washing solution to the latex particles and centrifuge, discard the supernatant, and repeat the washing several times; The pellet was resuspended in MES coupling buffer and activated by adding EDC; The activated latex particles were washed with MES coupling buffer and then resuspended; ②Streptavidin coupling Take the activated washed latex solution, add streptavidin, mix well and couple on a shaker; Wash the microsphere particles with Hepes buffer and repeat several times; Add sealing agent to seal; ③ Ligation of PCT protein antibody and streptavidin-coated particles The latex particle solution prepared in step ② is mixed with the two antibodies "anti-human PCT protein monoclonal antibody I and anti-human PCT protein monoclonal antibody II", and incubated in a shaker to form a "latex particle-streptavidin-PCT protein antibody complex", and then diluted with the dilution buffer prepared in step (2) to the target concentration; (4) Preparation of calibration materials: The preservative, stabilizer, PCT antigen and buffer of the calibrator are mixed to obtain the calibrator.

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