Neutrophil apolipoprotein homodimer-specific monoclonal antibodies and uses thereof

Abstract

The application discloses a monoclonal antibody which can specifically recognize only neutrophil apolipoprotein homodimer HNL / HNL and cannot recognize monomer form HNL. The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody can realize accurate, simple and rapid quantification of the homodimer form HNL / HNL, promote the wide application of the detection index in the clinic, accurately distinguish bacterial infection and viral infection, and reduce the unreasonable use of antibiotics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to a monoclonal antibody specifically recognizing neutrophil lipocalin homodimer and application thereof. BACKGROUND

[0002] Human neutrophil lipocalin (HNL), also known as neutrophil gelatinase-associated lipocalin (NGAL) and lipocalin-2 (Lcn2), is mainly secreted by immune cells such as neutrophils, macrophages and dendritic cells, in addition to which it can also be secreted by small tubular epithelial cells of various human organs, including kidney, heart, lung, liver, stomach, colon, etc. HNL is a secreted glycosylated protein composed of 198 amino acids, with a signal peptide sequence of 20 amino acid residues at the N-terminus, and a mature protein of 178 amino acids. HNL mainly exists in three molecular forms, namely monomer, homodimer and heterodimer, wherein the monomer HNL has a molecular weight of 25 kDa and is mainly derived from epithelial cells; the monomer HNL forms homodimer HNL / HNL by intermolecular disulfide bond, with a molecular weight of about 45 kDa and being mainly derived from neutrophils; the heterodimer is formed by intermolecular disulfide bond between HNL and matrix metalloproteinase-9 (MMP-9), with a molecular weight of about 135 kDa. Different molecular forms of HNL are related to different pathological processes, and in clinical diagnosis, monomer HNL is closely related to critical conditions such as acute kidney injury, cardiovascular disease and multiple organ failure; homodimer is closely related to bacterial infection, and when bacterial infection occurs, the level of HNL homodimer in body fluid rapidly increases, while when viral infection occurs, the level of HNL homodimer does not change significantly, so HNL homodimer has high specificity and sensitivity for differential diagnosis of acute bacterial infection and viral infection.

[0003] However, so far, clinical detection has been unable to accurately distinguish monomer HNL from homodimer HNL / HNL, resulting in inability to accurately diagnose and identify bacterial infection, and the key limiting factor is the lack of molecular form-specific monoclonal antibodies, and the existing monoclonal antibodies and polyclonal monomers can recognize both monomer HNL and homodimer HNL / HNL, and there is no monoclonal antibody that specifically recognizes only homodimer HNL / HNL. Although non-molecular form-specific monoclonal antibodies or polyclonal antibodies can be used to distinguish different molecular forms by WB technology according to different molecular weights, but due to the defects of this technology such as complex operation, difficulty in accurate quantification, long operation period, difficulty in standardization, etc., it cannot be widely used in clinical diagnosis.

[0004] Therefore, the purpose of the present application is to prepare and screen a monoclonal antibody capable of specifically recognizing only the homodimer of neutrophil lipocalin, but not the monomer form HNL, so as to realize accurate, simple and rapid quantification of the homodimer form HNL / HNL of neutrophil lipocalin, promote the wide application of the detection index in clinic, accurately distinguish bacterial infection from viral infection, and reduce the unreasonable use of antibiotics. SUMMARY

[0005] To this end, the purpose of the present application is to provide a monoclonal antibody prepared by using a fused hybridoma cell strain, which specifically recognizes only the homodimer of neutrophil lipocalin, and the monoclonal antibody obtained through experiments does not recognize the monomer form HNL and does not have cross-reaction with the monomer form HNL, and can be used for accurate quantification of the homodimer HNL / HNL of neutrophil lipocalin.

[0006] Therefore, one aspect of the present application relates to a monoclonal antibody or an antigen-binding fragment thereof specifically recognizing only the homodimer of neutrophil lipocalin, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein,

[0007] the amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO. 2 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 2;

[0008] the amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO. 3 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 3;

[0009] the amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO. 4 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 4;

[0010] the amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO. 6 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 6;

[0011] the amino acid sequence of the light chain CDR2 is LYP or an amino acid sequence having 1 conservative amino acid substitution compared with LYP;

[0012] the amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO. 7 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 7.

[0013] In a further aspect, the present application also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO. 1, and the light chain variable region amino acid sequence is as set forth in SEQ ID NO. 5.

[0014] The present application also relates to the above-mentioned monoclonal antibody or antigen-binding fragment thereof, which is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody. These antibodies or antigen-binding fragments can specifically recognize neutrophil lipocalin homodimer because they retain the variable region of the light chain and the heavy chain, or only the variable region of the heavy chain.

[0015] In addition, the present application also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or antigen-binding fragment thereof, and an expression vector comprising the above-mentioned nucleic acid molecule, which can express the above-mentioned antibody or antigen-binding fragment thereof. Meanwhile, the present application also relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned antibody or antigen-binding fragment thereof. On the other hand, the present application relates to a monoclonal antibody hybridoma cell strain that specifically recognizes neutrophil lipocalin homodimer, which secretes the above-mentioned monoclonal antibody. Further, the present application relates to a monoclonal antibody hybridoma cell strain that specifically recognizes only neutrophil lipocalin homodimer, which is a mouse hybridoma cell strain HNL11, and the preservation number is CGMCC No. 46548.

[0016] In a further aspect, the present application relates to the use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a product for detecting neutrophil lipocalin homodimer (HNL / HNL). Further, the present application relates to a kit for detecting neutrophil lipocalin homodimer (HNL / HNL), which comprises the above-mentioned monoclonal antibody or antigen-binding fragment thereof for specifically recognizing and binding neutrophil lipocalin homodimer (HNL / HNL). Preferably, the kit is a double antibody sandwich detection kit, and the monoclonal antibody or antigen-binding fragment thereof serves as a capture antibody.

[0017] Biological material preservation instructions

[0018] The monoclonal antibody hybridoma cell strain of the present application: mouse hybridoma cell strain HNL11, preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation center registration number is CGMCC No. 46548, and the preservation date is June 18, 2025. The address of the China General Microbiological Culture Collection Center is No. 3, Beichen West Road, Chaoyang District, Beijing, China, with a postcode of 100101. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the alignment result of the E. coli genetic code preference optimized nucleotide sequence of the HNL gene with the original nucleotide sequence, wherein the upper sequence is the optimized sequence, the lower sequence is the original sequence, "=" represents the same nucleotide, and "*" represents the different nucleotide.

[0020] Figure 2 is an SDS-PAGE electrophoresis map showing prokaryotic expression of neutrophil apolipoprotein, wherein each label is: "M" is Marker, "non-reduced" is that the sample loaded is in a non-reduced state, and "reduced" is that the sample loaded is in a reduced state.

[0021] Figure 3 is the identification result of the recognition specificity of the anti-neutrophil apolipoprotein monoclonal antibody, wherein "polyclonal antibody" is a rabbit anti-HNL polyclonal antibody, and "HNL1-12" is a mouse anti-HNL monoclonal antibody.

[0022] Figure 4 is a graph of the identification result of the anti-neutrophil apolipoprotein homodimer (HNL / HNL) monoclonal antibody HNL11 subtype. DETAILED DESCRIPTION

[0023] The purpose of the present application is to provide a monoclonal antibody that specifically recognizes only neutrophil apolipoprotein homodimer (HNL / HNL) prepared by using a fused hybridoma cell strain. The specific preparation process is as follows: first, the HNL is prokaryotic soluble expressed to obtain a soluble expressed HNL antigen that can form a homodimer; then the HNL antigen is used as an immunogen to immunize mice to prepare a monoclonal antibody, and after screening by a WB method, a high-specificity monoclonal antibody that specifically recognizes neutrophil apolipoprotein homodimer (HNL / HNL) is obtained, the mouse hybridoma cell strain secreting the monoclonal antibody is named HNL11, which specifically recognizes only neutrophil apolipoprotein homodimer (HNL / HNL) and has no cross reaction with monomer HNL, and has very high specificity. The present inventors preserved the cell strain in the China General Microbiological Culture Collection Center on June 18, 2025, and the preservation number is CGMCC No. 46548.

[0024] Subsequently, the present inventors sequenced and immunoglobulin domain sequence analyzed the monoclonal antibody secreted by the mouse hybridoma cell strain CGMCC No. 46548, and found that the heavy chain variable region amino acid sequence is: EVQLVESGGGLVKPGGSLKLSCAASGFTFNSYAMSWVRQTPEKRLEWVATINSGGGFTYYPDSLRGRFTISRANANSTLYLQMSSLRSEDTAMYYCARPTATDVMDYWGQGTTVTVSS (SEQ ID NO. 1), wherein the heavy chain CDR1 amino acid sequence is GFTFNSYA (SEQ ID NO. 2), the heavy chain CDR2 amino acid sequence is INSGGGFT (SEQ ID NO. 3), and the heavy chain CDR3 amino acid sequence is ARPTATDVMDY (SEQ ID NO. 4). The light chain variable region amino acid sequence is: DIVLTQSPAPLYLWGRGPPSHTGPAKVSVHLAIVICTGTNRNQDSHPDSSSILYPTNLGSLPGSVAVGLGQTSPSTSILWRRRMLQPITVSTLGSLHVRRGHQAGNQ (SEQ ID NO. 5), wherein the light chain CDR1 amino acid sequence is KVSVHLAIVI (SEQ ID NO. 6), the light chain CDR2 amino acid sequence is LYP, and the light chain CDR3 amino acid sequence is STLGSLH (SEQ ID NO. 7).

[0025] The present inventors evaluated the specificity of the monoclonal antibody to the detection of neutrophil lipocalin homodimer (HNL / HNL) by WB method, and found that the monoclonal antibody only specifically recognizes neutrophil lipocalin homodimer (HNL / HNL), and does not recognize monomeric form HNL, and has very high specificity. Meanwhile, detection of clinical samples by enzyme-linked immunoassay showed that the monoclonal antibody of the present application can be used to prepare products for the diagnosis of bacterial infection, and for the differential diagnosis of bacterial infection and viral infection.

[0026] It is well known in the art that the CDR regions of the heavy chain and the CDR regions of the light chain of an antibody are important amino acid sequence regions for recognizing and binding to the corresponding antigen, and a conservative substitution of one amino acid in the above CDR region amino acid sequence can not change the structure of the protein, so a single amino acid conservative substitution in the above region can still have the property of binding to the corresponding antigen. Therefore, the monoclonal antibody or antigen-binding fragment thereof obtained after a conservative substitution of one amino acid in the heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still specifically recognize neutrophil lipocalin homodimer (HNL / HNL). In the present patent application, the conservative substitution of amino acids refers to the substitution of one amino acid in a protein with another chemically similar amino acid, such as the mutual substitution between aromatic amino acids Phe, Trp, Tyr, the mutual substitution between aliphatic amino acids Ala, Gly, Leu, Ile, Val, the mutual substitution between polar amino acids Gln, Asn, the mutual substitution between basic amino acids Lys, Arg, His, the mutual substitution between acidic amino acids Asp, Glu, and the mutual substitution between hydroxyl amino acids Ser, Thr, etc.

[0027] The skilled person in the art can also prepare various antibody fragments, i.e. antigen-binding fragments, such as but not limited to Fab, Fab', F(ab')2, which can specifically recognize only neutrophil lipocalin homodimer (HNL / HNL) from the monoclonal antibody of the present application, according to the existing technology in the art. The Fab fragment is the region of the antibody structure that can bind to the antigen, which is composed of one complete light chain and the variable region VH and constant region CH1 domain (Fd fragment) of the heavy chain. There is one constant region and one variable region in the light chain and the heavy chain, and there is a disulfide bond between the light chain and the heavy chain. The antigen-binding fragment can be prepared, for example, by using papain to degrade the antibody IgG into two Fab fragments and one Fc fragment. Under the action of pepsin, the antibody IgG is degraded into one F(ab')2 fragment and one pFc' fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragment can still bind to the corresponding antigen, it can be used to prepare products for detecting neutrophil lipocalin homodimer (HNL / HNL).

[0028] The skilled person in the art can also prepare single chain antibodies (scFv) from the monoclonal antibodies of the present application by the techniques available in the art. Single chain antibodies are antibodies having only one chain, which are artificially synthesized antibodies, and are formed by connecting the variable region of the heavy chain and the variable region of the light chain of the antibody with a short peptide linker of several amino acids. The single chain antibodies can also be formed by only the variable region of the heavy chain. The length and the amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibodies of the present application can be determined by simple repetition experiments. The single chain antibodies of the present application thus prepared have the property of specifically recognizing the homodimer of neutrophil lipocalin (HNL / HNL), and can be used in the detection of the homodimer of neutrophil lipocalin (HNL / HNL).

[0029] The skilled person in the art can design and synthesize nucleic acid molecules encoding the variable region of the monoclonal antibodies of the present application based on the amino acid sequences of the monoclonal antibodies of the present application that specifically recognize the homodimer of neutrophil lipocalin (HNL / HNL), and can insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors that can express the monoclonal antibodies or antigen-binding fragments thereof that specifically recognize the homodimer of neutrophil lipocalin (HNL / HNL). The skilled person in the art can also introduce the synthesized nucleic acid molecules or the constructed expression vectors into organisms such as cells, bacteria, and yeasts to obtain recombinants, and can produce the antibodies or antigen-binding fragments thereof of the present application by expression from the recombinants, and the antibodies or antigen-binding fragments thereof thus expressed can specifically recognize the homodimer of neutrophil lipocalin (HNL / HNL), and thus the nucleic acid molecules, the expression vectors, and the recombinants are within the scope of the claims of the present application. Furthermore, the above-mentioned techniques are well known in the art, and the skilled person in the art can perform the above-mentioned techniques without inventive labor.

[0030] As described above, the antibodies or antigen-binding fragments thereof of the present application can specifically recognize the homodimer of neutrophil lipocalin (HNL / HNL), and thus can be used in the preparation of kits for detecting the homodimer of neutrophil lipocalin (HNL / HNL). The kits can be any kits using the binding reaction of the antibodies or antigen-binding fragments thereof of the present application with the homodimer of neutrophil lipocalin (HNL / HNL), such as, but not limited to, enzyme-linked immunosorbent assay, fluorescent immunochromatography, chemiluminescence, immunoblotting, colloidal gold immunochromatography, and immunohistochemical methods. Preferably, the kits are double antibody sandwich type detection kits in the above-mentioned methods, and the monoclonal antibodies or antigen-binding fragments thereof are used as capture antibodies.

[0031] In order to describe the technical content of the technical scheme, the purposes and effects achieved, the following will be described in combination with specific embodiments.

[0032] Example 1: Optimization of mature neutrophil lipocalin (HNL) gene nucleotide sequence

[0033] According to the nucleotide sequence and protein amino acid sequence of human neutrophil lipocalin (NGAL) gene published by NCBI GenBank database (GenBank: X99133.1), the gene sequence of HNL is 597 bp, i.e. ATGCCCCTAGGTCTCCTGTGGCTGGGCCTAGCCCTGTTGGGGGCTCTGCATGCCCAGGCCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATATCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCTGA (SEQ ID NO. 8). The protein sequence of HNL is 198 aa, i.e. MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKYLGLPENHIVFPVPIDQCIDG (SEQ ID NO. 9).

[0034] Firstly, the signal peptide sequence 1-20 aa was removed, and the mature HNL length was 178 amino acids (21-198 aa). Then, the BIOSUN biological software was used to deduce the mature HNL optimized nucleotide sequence according to the genetic code preference of E. coli, and the length was 534 bp, i.e. CAGGATAGCACCTCTGACCTGATTCCGGCGCCACCGTTGTCCAAAGTGCCACTGCAACAGAACTTTCAGGATAATCAATTCCAGGGCAAGTGGTATGTTGTGGGTTTAGCCGGCAACGCGATCCTGCGTGAAGATAAAGACCCGCAGAAAATGTACGCAACGATTTATGAGCTTAAGGAAGATAAATCGTACAATGTCACTAGCGTTCTGTTTCGCAAAAAGAAATGCGACTATTGGATTCGTACCTTCGTACCAGGTTGTCAACCGGGCGAATTTACTCTCGGTAACATTAAAAGTTACCCAGGCCTGACGTCTTATCTGGTGCGCGTTGTCTCGACCAATTACAACCAGCATGCTATGGTGTTCTTTAAGAAAGTTAGCCAGAATCGTGAGTATTTCAAAATCACTTTGTATGGTCGCACCAAGGAACTGACCAGCGAATTAAAAGAGAACTTTATTCGTTTTTCTAAATACCTGGGCCTTCCGGAAAACCACATCGTGTTCCCGGTCCCAATTGATCAATGCATTGATGGC (SEQ ID NO. 10). Compared with the original nucleotide sequence, the HNL optimized nucleotide sequence had a total of 126 base substitutions, as shown in Table 1, wherein the upper sequence is the optimized sequence, the lower sequence is the original sequence, "=" indicates the same nucleotide, and "*" indicates the different nucleotide. Figure 1

[0035] Example 2: Soluble expression of mature neutrophil lipocalin (HNL)

[0036] ​The HNL gene Escherichia coli genetic code preference optimized nucleotide sequence in Example 1 was synthesized by Beijing Qikexin Biotechnology Co., Ltd., and a recombinant pCold I plasmid was constructed by using BamH I and EcoR I enzyme cutting sites. The used upstream primer was 5'-GCGGATCCCAGGATAGCACCTCTG-3' (SEQ ID NO. 11), and the downstream primer was 5'-GCGAATTCTCAGCCATCAATGCAT-3' (SEQ ID NO. 12). The full-length gene was amplified by using the synthesized optimized nucleotide sequence as a template, and the amplification conditions were as follows: 95℃ for 2 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 45 s, for a total of 30 cycles; and then 72℃ for 5 min. 2% agarose electrophoresis identification showed that the relative molecular weight of the amplified fragment was about 550 bp. The purified PCR product was double-digested by using BamH I and EcoR I enzyme cutting sites, and then was connected into the pCold I plasmid to obtain the pCold-HNL recombinant plasmid. The recombinant expression plasmid with correct sequencing was transformed into E. coli Rosetta (DE3) competent cells, a single colony was picked and inoculated in 5 mL LB liquid medium containing ampicillin sodium, and was cultured at 37℃ for overnight. The next day, it was inoculated in 250 mL fresh LB liquid medium, and was cultured to the logarithmic growth phase. The temperature was adjusted to 16℃, 150 μl of 1 mol / L IPTG induction liquid was added after 30 min, and the induction was performed at 16℃ for 12-14 h. The induced bacterial bodies were collected by centrifugation, resuspended with 25 mmol / L Tris-HCl (pH 8.5), and were broken by ultrasonic wave. The supernatant was collected by high-speed centrifugation at 20000 g at 4℃ for 30 min, and was subjected to Ni column purification. First, the Ni column was equilibrated with the equilibration buffer (25 mmol / L TE, 6 mol / L urea, pH 8.5), and then the supernatant was added to the Ni column. After the sample was completely added, the target protein was eluted by using the washing liquid containing 25 mmol / L and 250 mmol / L imidazole, respectively. Meanwhile, SDS-PAGE gel electrophoresis was performed under non-reducing conditions and reducing conditions. The mature HNL was expressed in a soluble form, most of the proteins were eluted by using the 250 mmol / L imidazole washing liquid, the molecular weight of HNL was about 23 kDa under reducing conditions, and in addition to the 23 kDa HNL monomer, the HNL / HNL in a homodimer form was also observed under non-reducing conditions, and the molecular weight was about 45 kDa. The results are shown in Figure 2 .

[0037] Example 3: Preparation of rabbit anti-neutrophil apolipoprotein polyclonal antibody

[0038] Healthy male rabbits were selected, 1.0 mg of E. coli prokaryotic expressed neutrophil apolipoprotein in Example 2 was mixed with 1.0 ml of Freund's complete adjuvant, and after complete emulsification by a stirrer, subcutaneous injection was performed at both sides of the spine of the rabbit, with 0.2 ml per injection; 4 weeks later, 1.0 mg of RacR antigen was mixed with 1.0 ml of Freund's incomplete adjuvant, and after complete emulsification by a stirrer, the second immunization was performed at different points of the above-mentioned parts; 4 weeks later, the third boost immunization was performed for the preparation of polyclonal antibody serum. After 1 week, the blood was taken from the heart, and after the blood clot contracted, centrifugation was performed at 5000 rpm for 15 minutes, and the serum was stored in a refrigerator at -20°C for standby. The operation of the indirect ELISA method for determining the titer of the polyclonal antibody is as follows: the neutrophil apolipoprotein was used to coat the enzyme-linked plate, with a concentration of 2.0 μg / ml, 150 μl per well, and 4°C overnight; the plate was washed 2 times with washing solution; 200 μl / well of blocking solution was added and blocked at room temperature for 6 hours; the plate was washed 5 times with washing solution. The rabbit serum was diluted with PBS, with a dilution ratio of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000 and 1:4096000, with 100 μL per well; 37°C incubation for 45 min; 5 times of washing of the plate with 200 μL per well of washing solution; HRP-labeled goat anti-rabbit secondary antibody was incubated at 37°C for 45 min; the plate was washed 5 times with 200 μL per well of washing solution; 100 μL of freshly prepared substrate solution was added per well, and incubated at 37°C for 10 min; 50 μL of 2 M H2SO4 was added per well to terminate the reaction, and the absorbance value of each well was determined by an enzyme-labeled instrument at a wavelength of 450 nm, and the reading was performed within 10 min after termination, and the results are shown in Table 1. The rabbit serum before immunization diluted 1:2000 was used as a negative control, and the titer of the polyclonal antibody prepared by immunizing the rabbit with the neutrophil apolipoprotein could reach 1:2048000.

[0039] Table 1: Determination results of rabbit anti-neutrophil apolipoprotein polyclonal antibody titer (OD450nm)

[0040] Dilution ratio 1:2000 1:8000 1:32000 1:128000 1:512000 1:1024000 1:2048000 1:4096000 Negative control OD450nm 3.594 2.901 2. 377 1.492 0.735 0.406 0.212 0.096 0.084

[0041] Example 4: Preparation of anti-neutrophil apolipoprotein monoclonal antibody

[0042] The 6-8 week old BALB / c female mice were immunized with 100 μg of the neutrophil lipocalin antigen and an equal amount of Freund's complete adjuvant, which was thoroughly emulsified by a stirrer, and then the mice were injected subcutaneously on the back and intraperitoneally. Three mice were immunized. The second immunization was performed after 4 weeks, and the third immunization was performed after 8 weeks. 50 μg of the antigen and incomplete Freund's adjuvant were thoroughly emulsified by a stirrer, and then the mice were injected subcutaneously on the back and intraperitoneally. One week after the third immunization, the mice were bled from the tail vein to detect the titer of the immune serum, and the mouse with the highest titer was selected for intraperitoneal booster immunization (50 μg per mouse). Three days later, the spleen cells were collected for fusion. The SP20 myeloma cells were recovered and cultured until they were in the logarithmic growth phase. The spleen of the immunized BALB / c mouse was removed to prepare a spleen cell suspension. The spleen cells and the myeloma cells were mixed in a ratio of 9:1 in a serum-free DMEM medium, centrifuged at 1500 rpm for 5 minutes, and the supernatant was aspirated. The cells were dispersed by gentle shaking, fused in a 37°C water bath, and 1 mL of preheated 50% PEG was added to the cells within 1 minute. After the addition was completed, the cells were allowed to stand for 90 seconds, and then the fusion was terminated by adding a serum-free DMEM medium. The cells were allowed to stand at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, suspended in a HAT medium, and then dispensed into a 96-well cell plate containing feeder cells. After 5 days of culture in a 37°C, 5% CO2 cell incubator, the medium was replaced with a HAT medium once, and then the medium was replaced with a HAT medium on the 10th day. When the fusion cells covered about 60% of the bottom of the well, the cell culture supernatant was collected, and positive clones were screened by indirect ELISA. Specifically, the neutrophil lipocalin antigen was diluted in a carbonate coating buffer to a concentration of 2.0 μg / mL, and 150 μL was coated in each well at 4°C overnight. The plate was washed twice with a washing solution. 200 μL of blocking solution was added to each well, and the plate was blocked at room temperature for 6 hours. The plate was washed five times with the washing solution. 100 μL of a sample diluent was added to each well, and then 10 μL of the cell culture supernatant was added. The plate was incubated at room temperature for 30 minutes, and then the liquid was discarded. The plate was washed five times, and then the washed plate was inverted on an absorbent paper to dry. 100 μL of HRP-labeled goat anti-mouse IgG antibody was added to each well, and the plate was incubated at room temperature for 30 minutes. The plate was washed five times. 50 μL of TMB color developing solution A and 50 μL of TMB color developing solution B were added to each well, and the plate was developed at room temperature for 15 minutes in the dark. 50 μL of 2 M H2SO4 was added to each well to terminate the reaction. The detection wavelength of the microplate reader was set to 450 nm, and the OD value of each well was measured within 10 minutes after termination.

[0043] A total of 12 strong positive clones were obtained, and the hybridoma cell strains were cultured in a 1640 medium containing 10% fetal bovine serum. Each BALB / c male mouse was injected intraperitoneally with 0.5 mL of liquid paraffin. After 10 days, the cells were collected, resuspended in 10 mL of normal saline, and the cell density was adjusted to 1×10 7The mice were injected intraperitoneally with 0.5 mL of the antibody solution at a concentration of 10 μg / mL. Two weeks later, ascites were collected. The antibodies were purified using a Thermo Melon Gel Monoclonal IgG Purification Kit, and the purified antibodies were stored at -20 °C after being aliquoted.

[0044] Example 5: Screening of Anti-Neutrophil Lipocalin Homodimer (HNL / HNL) Monoclonal Antibodies

[0045] The mature neutrophil lipocalin expressed in Example 2 was subjected to non-reducing SDS-PAGE gel electrophoresis, with 20 μg loaded per well, and a pre-stained protein standard was added as a molecular weight control. After electrophoresis, the proteins were transferred to a PVDF membrane by electroblotting. The wells were cut open after being stained with ponceau red and destained by TBST washing. The wells on the PVDF membrane were blocked with 5% skim milk in TBST at room temperature for 1.5 hours to remove non-specific binding, and 12 strains of anti-neutrophil lipocalin monoclonal antibodies prepared in the present application and rabbit anti-neutrophil lipocalin polyclonal antibodies were added to each well, with a final antibody concentration of 10 μg / mL, and incubated at 4 °C overnight. The membrane was washed with TBST for 3 times, 5 min each time, and HRP-labeled goat anti-mouse secondary antibody (1:5000) or goat anti-rabbit secondary antibody (1:5000) was added, and incubated at room temperature for 2 hours. The membrane was washed with TBST for 3 times, 5 min each time. ECL color development was performed for 2 min, and the photograph was taken under a Bio-Rad developer.

[0046] Among the 12 strains of anti-neutrophil lipocalin monoclonal antibodies prepared in the present application, only one monoclonal antibody, HNL11, specifically recognized neutrophil lipocalin homodimers (HNL / HNL); the remaining 11 monoclonal antibodies, like the polyclonal antibody, could recognize both neutrophil lipocalin monomers (HNL) and neutrophil lipocalin homodimers (HNL / HNL). The results are shown in Figure 3 The monomer has a molecular weight of about 23 kDa, and the dimer has a molecular weight of about 45 kDa.

[0047] Example 6: Specific detection of neutrophil lipocalin homodimers (HNL / HNL) in blood samples

[0048] The monoclonal antibody HNL11 prepared by the application and only recognizing the neutrophil lipocalin homodimer (HNL / HNL) is used as a capture antibody to specifically capture the neutrophil lipocalin homodimer (HNL / HNL) in a sample, the rabbit anti-neutrophil lipocalin polyclonal antibody prepared in Example 3 is used as a detection antibody, and a neutrophil lipocalin homodimer (HNL / HNL) specific detection method based on a double antibody sandwich method is established. The specific steps are as follows: the enzyme-linked plate is coated with the monoclonal antibody HNL11 prepared by the application and only recognizing the neutrophil lipocalin homodimer (HNL / HNL), the concentration is 2.0 μg / mL, 100 μL per well, and the plate is coated at 4°C overnight; the plate is washed with the washing solution for 2 times; 120 μL / well of blocking solution is added and blocked at room temperature for 6 hours; the plate is washed with the washing solution for 5 times, 100 μL of sample diluent and 10 μL of serum sample are added to each well, incubated at 37°C for 45 min, and then the liquid is discarded. The plate is washed with the washing solution for 5 times, 100 μL of horseradish peroxidase-labeled rabbit anti-neutrophil lipocalin polyclonal antibody is added to each well, and incubated at 37°C for 30 min. The plate is washed for 5 times, dried, 50 μL of TMB color developing liquid A and B are added to each well, and the color is developed at room temperature in the dark for 15 min. 50 μL / well of 2 M H2SO4 termination liquid is added to terminate the reaction. The OD value of each well is determined by an enzyme-labeled instrument at a wavelength of 450 nm, and the value is read within 10 min after termination.

[0049] A total of 5 serum samples of patients with clinically diagnosed respiratory tract bacterial infection, 5 serum samples of patients with respiratory tract viral infection, and 5 serum samples of healthy persons are detected, and the detection results are shown in Table 2. The detection OD values of the 5 serum samples of patients with clinically diagnosed respiratory tract bacterial infection are all greater than 1.0, which are significantly higher than the detection OD values of the 5 serum samples of patients with respiratory tract viral infection and the 5 serum samples of healthy persons. There is no significant difference between the detection OD values of the 5 serum samples of patients with respiratory tract viral infection and the 5 serum samples of healthy persons. The above results show that the monoclonal antibody HNL11 prepared by the application and only recognizing the neutrophil lipocalin homodimer (HNL / HNL) can be used for specific detection of the neutrophil lipocalin homodimer (HNL / HNL) and differential diagnosis of bacterial infection.

[0050] Table 2 Specific detection of neutrophil lipocalin homodimer (HNL / HNL) in blood samples (OD 450nm )

[0051] Sample source Patients with bacterial infection Patients with viral infection Healthy subjects 1 1.245 0.206 0.089 2 2.376 0.099 0.032 3 1.900 0.143 0.157 4 3.021 0.192 0.066 5 1.137 0.058 0.183

[0052] Example 7: Specific detection of neutrophil lipocalin homodimer (HNL / HNL) in urine samples

[0053] 3 urine samples from patients diagnosed with urinary tract infection (UTI) and 3 urine samples from healthy persons were centrifuged at 3000 rpm for 15 minutes at 4°C immediately, 50 mL supernatant was taken, and the urine samples were subjected to gel filtration on a Superdex 75 HR 10 / 30 column using a FPLC system (Amersham Pharmacia Biotech AB, Sweden), and the collected components were subjected to ELISA detection. The detection procedure is described in Example 6. The detection results are shown in Table 3, the OD values of the 3 urine samples from UTI patients were significantly higher than those of the urine samples from healthy persons, and the main form of the urine samples from UTI patients was neutrophil lipocalin homodimer (HNL / HNL). The above results show that the monoclonal antibody HNL11 prepared in the application can be used for specific detection of neutrophil lipocalin homodimer (HNL / HNL) and diagnosis of bacterial urinary tract infection.

[0054] Table 3 Specific detection of neutrophil lipocalin homodimer (HNL / HNL) in urine samples (OD 450nm )

[0055] Sample source Patients with bacterial infection Healthy subjects 1 0.873 0.121 2 1.024 0.176 3 0.799 0.109

[0056] Example 8: Subtype analysis of anti-neutrophil lipocalin homodimer (HNL / HNL) monoclonal antibody HNL11

[0057] The heavy chain and light chain subtypes of the mouse antibody were identified using the mouse antibody subtype rapid detection card (product number: THJ-ISO-M8a-10 / 20) of Antigene (Beijing) Biotechnology Co., Ltd. First, the antibody was diluted to 1 μg / mL with PBS, then 100 μl of the diluted antibody was added to each well, and the results were observed and recorded after standing for 5-10 min. The results are shown in Table 4, the anti-neutrophil lipocalin homodimer (HNL / HNL) monoclonal antibody HNL11 is of mouse IgG2b subtype, and the antibody light chain is of Igκ subtype. Figure 4

[0058] Example 9: Determination of the variable region sequence of anti-neutrophil lipocalin homodimer (HNL / HNL) monoclonal antibody HNL11

[0059] ​The mouse hybridoma cell strain HNL11 was cultured, total RNA of the hybridoma cells was extracted by Trizol method, after reverse transcription of cDNA, PCR amplification was performed using the mouse monoclonal antibody Fab segment primer sequence synthesized by Beijing Genki Biological Technology Co., Ltd., 95°C preheating for 2 min, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds for 30 cycles, and finally 72°C for 5 min, ligated with pMD18-T vector, transformed into E. coli JM109, and positive clones were selected for sequencing. The determination sequence was compared and analyzed with the CDR region sequence of the mouse-derived monoclonal antibody in the IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) BLAST module in the NCBI website.

[0060] After sequence analysis, it was found that the heavy chain variable region amino acid sequence was 118 amino acids, and the sequence was as follows: EVQLVESGGGLVKPGGSLKLSCAASGFTFNSYAMSWVRQTPEKRLEWVATINSGGGFTYYPDSLRGRFTISRANANSTLYLQMSSLRSEDTAMYYCARPTATDVMDYWGQGTTVTVSS (SEQ ID NO. 1), wherein CDR1 was located at 26-33 aa, and the amino acid sequence was GFTFNSYA (SEQ ID NO. 2); CDR2 was located at 51-58 aa, and the amino acid sequence was INSGGGFT (SEQ ID NO. 3); and CDR3 was located at 97-107 aa, and the amino acid sequence was ARPTATDVMDY (SEQ ID NO. 4). The light chain variable region amino acid sequence was 107 amino acids, and the sequence was as follows: DIVLTQSPAPLYLWGRGPPSHTGPAKVSVHLAIVICTGTNRNQDSHPDSSSILYPTNLGSLPGSVAVGLGQTSPSTSILWRRRMLQPITVSTLGSLHVRRGHQAGNQ (SEQ ID NO. 5), wherein CDR1 was located at 26-35 aa, and the amino acid sequence was KVSVHLAIVI (SEQ ID NO. 6); CDR2 was located at 53-55 aa, and the amino acid sequence was LYP; and CDR3 was located at 91-97 aa, and the amino acid sequence was STLGSLH (SEQ ID NO. 7).

Claims

1. A monoclonal antibody or its antigen-binding fragment that specifically recognizes neutrophil apolipoprotein homodimers, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LYP; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by the mouse hybridoma cell line HNL11 with accession number CGMCC No.46548.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

5. A nucleic acid molecule, characterized in that, It comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A recombinant cell, bacterium, or yeast, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. A hybridoma cell line that secretes monoclonal antibodies that specifically recognize neutrophil apolipoprotein homodimers, characterized in that, It is the mouse hybridoma cell line HNL11 with accession number CGMCC No.46548.

9. The use of the monoclonal antibody or antigen-binding fragment thereof that specifically recognizes neutrophil apolipoprotein homodimer as described in any one of claims 1 to 4 in the preparation of products for detecting neutrophil apolipoprotein homodimer.

10. A kit for detecting apolipoprotein homodimers in neutrophils, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

11. The reagent kit according to claim 10, characterized in that, It is a detection kit using a double-antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment serves as the capture antibody.

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