WO2023151240A1 - Carbapenemase conserved antigen, antibody and use thereof - Google Patents

Abstract

Provided are a carbapenemase conserved antigen, an antibody, and the use thereof. The conserved antigen comprises any one or several amino acid sequences as shown in SEQ ID NO: 1-15. The antigen used is the carbapenemase conserved antigen that comprises amino acid fragments existing in all common subtypes, so that a monoclonal antibody or a polyclonal antibody obtained by immunizing an animal with the antigen can identify all the common subtypes, and the probability of false negative is extremely low.

Description

A kind of carbapenemase conserved antigen, antibody and application thereof technical field

The invention belongs to the field of kit preparation, and in particular relates to a carbapenemase conserved antigen, antibody and application thereof.

Background technique

At present, bacterial drug resistance has become a major challenge in the field of global public health, especially the infection situation caused by carbapenem-resistant Enterobacterales (CRE) is the most severe. Carbapenem antibiotics, including imipenem, meropenem, and ertapenem, are one of the most effective antibacterial drugs for the treatment of infections caused by multidrug-resistant gram-negative bacilli. With the wide application of such drugs in clinical practice, the resistance rate of Enterobacteriaceae bacteria to carbapenem antibiotics is rapidly increasing year by year. According to the monitoring results of CHINET China Bacterial Resistance Surveillance Network over the years, the resistance rate of Klebsiella pneumoniae to carbapenems in clinical isolates in my country has risen rapidly from 3% in 2005 to more than 25% in 2019. up to 8 times. According to data from the National Antimicrobial Resistance Surveillance Network (CARSS) in 2018, the average resistance rate of Klebsiella pneumoniae clinically isolated from 1,429 hospitals across the country to carbapenem antibiotics was 10.1%, and even exceeded 20% in some provinces and cities. . Because CRE strains usually also carry genes resistant to other antibacterial drugs, they are characterized by extensive or even full resistance to antibacterial drugs, which makes clinical anti-infection treatment face the dilemma of no available drugs.

The production of carbapenemase is the most important mechanism of resistance to carbapenems in Enterobacteriaceae bacteria.

Carbapenemase refers to a type of β-2 lactamase that can obviously hydrolyze imipenem or meropenem, including Ambler molecular classification A, B, and D enzymes.

The types of carbapenemase produced by different countries, different regions, different hospitals, different populations and different bacteria are different. The carbapenemases produced by clinically isolated CRE strains in my country are mainly KPC and NDM types, and a few strains produce OXA-48, IMP and VIM carbapenemases. KPC-2 type enzymes are the most important subtype of KPC type carbapenemases, NDM-1 and NDM-5 are the most important subtypes of NDM type metalloenzymes, and OXA-181 and OXA-232 type enzymes are OXA The most dominant isoform of the -48 carbapenemase. According to the research results of CHINET China Bacterial Resistance Surveillance Network on 935 CRE strains collected from 39 hospitals across the country in 2018, the proportions of KPC, NDM and OXA-48 carbapenemase-producing strains were 51.6% (482/ 935), 35.7% (334/935) and 7.3% (68/935), of which a few strains produced carbapenemase complex enzymes.

Since different types of antibacterial drugs have different in vitro antibacterial activities against different carbapenemase-producing strains, accurate and rapid detection and typing of carbapenemase produced by CRE is helpful for precise drug use in clinical anti-infection treatment and hospitals. Infection prevention and control is of great value.

The current methods for laboratory detection of carbapenems are divided into phenotype detection and genotype detection. Phenotypic detection methods include Carba NP test, modified carbapenem inactivation test (including mCIM and eCIM), carbapenemase inhibitor enhanced test and time-of-flight mass spectrometry, etc. There are high requirements for personnel, most of the methods need to cultivate the strains to be tested overnight, and the Carba NP test, mCIM and eCIM tests still have the risk of false negative; The requirements are high, and the detection time is average. In comparison, the detection time of the colloidal gold technology detection method is short (about 20 minutes), the requirements for personnel training are low, and there is no need for equipment.

For the colloidal gold platform, the screening and matching of broad-spectrum antibodies is a major technical problem. Because if a single subtype of recombinant complete antigen is used to immunize animals, it is possible to obtain antibodies only against the subtype epitope, and when using these antibodies to detect, there will be false negatives that some subtypes are missed; in addition, using a single subtype Immunization of animals with recombinant complete antigens of the same type will also produce a large number of antibodies against a single epitope or adjacent epitopes. Such antibodies cannot be paired due to steric hindrance, which brings great difficulties to the screening of paired antibodies. In order to facilitate the detection of 5 kinds of carbapenemases (KPC, NDM, OXA-48, VIM and IMP) at the same time for customers, it is best to put these 5 kinds of carbapenemases on the same test card. The colloidal gold detection card is prepared by permuting and combining the carbapenemase antibodies with the characteristics of crossover or interference. In addition, the development of high-efficiency lysate is also an important factor affecting the sensitivity of detection reagents. The influence of lysate components on chromatographic reagents needs to be verified, and it is necessary to screen components that do not affect chromatographic reagents and have good lysing effects to prepare and optimize composition.

Contents of the invention

In view of this, the invention aims to overcome the defects in the prior art, and proposes a carbapenemase conserved antigen, antibody and application thereof.

In order to achieve the above object, the present invention provides a carbapenemase conserved antigen, said conserved antigen has an amino acid sequence as shown in any one or several of SEQ ID NO: 1-15, or has one or more An amino acid sequence formed by substitution, deletion or addition of amino acid residues.

The present invention also provides a broad-spectrum specific antibody to carbapenemase, and the broad-spectrum specific antibody is a monoclonal antibody or a polyclonal antibody prepared from the above-mentioned conserved antigen.

The present invention also provides a carbapenemase detection reagent, which includes lysate of Gram-negative bacteria and the above-mentioned conserved antigen or the above-mentioned broad-spectrum specific antibody.

Preferably, the active ingredient of the Gram-negative bacteria lysate is: one or a mixture of sodium lauroyl sarcosine, CHAPS, and SDS.

Preferably, the mass of sodium lauroyl sarcosine in the Gram-negative bacteria lysate is 0.1%-1%.

Preferably, the mass percentage of CHAPS in the Gram-negative bacteria lysate is 0.01%-0.1%.

Preferably, the mass percentage of SDS in the Gram-negative bacteria lysate is 0.1%-1%.

Preferably, the Gram-negative bacteria lysate comprises 0.2% sodium lauroyl acid, 0.02% CHAPS and 0.1% SDS in mass percentage.

The present invention also provides a carbapenemase detection kit, which includes the above-mentioned carbapenemase detection reagent.

Preferably, the carbapenemase detection kit is a colloidal gold detection kit, and the antibody marker in the kit is colloidal gold.

Compared with the prior art, the present invention has the following advantages:

(1) The antigen used in the present invention is a carbapenemase conserved antigen, an amino acid fragment that exists in common subtypes, and the monoclonal antibody or polyclonal antibody obtained by immunizing animals with it can recognize the common subtypes, and appears The probability of a false negative is extremely low.

(2) The Gram-negative bacteria lysate used in the present invention has little influence on the detection antibody and chromatographic effect, simple operation and short time, no equipment is needed, and the lysate effect can reach more than 90% of the physical fragmentation method.

Description of drawings

Fig. 1 is the SDS-PAGE electrophoresis figure of the carbapenemase that the embodiment of the present invention 1 makes;

Fig. 2 is the SDS-PAGE electrophoresis figure of the carbapenemase monoclonal antibody prepared in Example 2 of the present invention;

Fig. 3 is the microscopic examination picture of test group 1-6 and control group in test example 2 of the present invention

Fig. 4 is a microscopic examination picture of the control group and the experimental group in Test Example 2 of the present invention.

Detailed ways

Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

The present invention is described in detail below in conjunction with embodiment.

Embodiment 1 prepares carbapenemase antigen

1. Preparation of carbapenemase KPC

The present invention obtains the conserved sequence of carbapenemase KPC (KPC-2～KPC-82) through NCBI (National Center for Biotechnology Information) sequence alignment, including: KPC-N-terminal conserved region 19G～102V, KPC-C terminal conserved region 180S～238G, KPC-N+C conserved region), its amino acid sequence is shown in SEQ ID NO.1-3. High-purity protein is obtained through prokaryotic gene expression, and the gene expression process is as follows:

(1) Primer design

According to the base composition of the target sequence, primers were designed to amplify the target gene, and the upstream and downstream contained EcoR I and Xho I restriction sites respectively. The primer sequences are as follows:

KPC-N-terminal conserved region upstream primer: 5'ttgaattcGGCTTTTCTGCCA3'(EcoR I)

KPC-N-terminal conserved region downstream primer: 5'tctcgagGAACCAGCGCATTT3'(Xho I)

KPC-C terminal conserved region upstream primer: 5'ttgaattcCATCGCCGCGCGCC3'(EcoR I)

KPC-C terminal conserved region downstream primer: 5'tctcgagTCCGCAGGTTCCG3'(Xho I)

KPC-conserved region upstream primer: 5'ttgaattcGGCTTTTCTGCCA 3'(EcoR I)

KPC-conserved region downstream primer: 5'ttgaattcTCCGCAGGTTCCG3'(EcoR I)

(2) Gene amplification

The conventional SDS alkaline lysis method was used to crush the cell wall of the Gram-negative bacteria to be tested, and a commercially available DNA extraction kit was used to obtain the total DNA, which was used as a template to amplify the target gene. PCR reaction system:

PCR operating conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 56°C for 30 s, extension at 72°C for 20 s, a total of 30 cycles; extension at 72°C for 5 min.

(3) The expression plasmid pET-28a(+)-PM was constructed by conventional enzyme digestion and ligation techniques in the field of molecular biology, and the recombinant vector was transformed into Escherichia coli DH5a competent by CaCl ₂ heat shock method. Positive clones were screened using LB medium containing 100 μg/mL ampicillin. Escherichia coli was routinely cultured, and the plasmid was extracted for PCR identification to confirm the presence of the target gene.

(4) Expression and purification

Transform the extracted expression plasmid pET-28a(+)-PMAA into Escherichia coli BL21(DE3) competent cells, spread and culture on the selection medium, screen the single colony resistant to 100 μg/mL ampicillin, and then culture in liquid overnight . Take 1 mL of the overnight culture and inoculate it into 200 mL of LB medium containing 100 μg/mL ampicillin, culture with shaking until the logarithmic phase (OD600 is 0.5-0.6), add IPTG (1 mmol/L), induce culture at 16 ° C for 3 h, and ferment the Purified by nickel column, the purified product was detected by SDS-PAGE, and the fragment size was appropriate, which was the target protein. The SDS-PAGE electrophoresis diagram is shown in Figure 1 (M1 in the figure is Marker, and lane 1 is carbapenemase KPC expression protein) .

2. Preparation of carbapenemase NDM

The present invention obtains the conserved sequence of carbapenemase NDM (NDM1-15, NDM17-31) through NCBI sequence comparison, and its amino acid sequence is shown in SEQ ID NO.4-6. High-purity protein is obtained through prokaryotic gene expression, and the gene expression process is as follows:

(1) Primer design

According to the base composition of the target sequence, primers were designed to amplify the target gene, and the upstream and downstream contained EcoR I and Xho I restriction sites respectively. The primer sequences are as follows:

NDM-N-terminal conserved region upstream primer: 5'ttgaattcGAGCACCGCATTAG 3'(EcoR I)

NDM-N-terminal conserved region downstream primer: 5'ttgaattcGTTGGAAGCGACTG 3'(EcoR I)

NDM-C-terminal conserved region upstream primer: 5'ttgaattc ATGGCTGGGTCGAA 3'(EcoR I)

Downstream primer of NDM-C-terminal conserved region: 5'tctcgagGCGGGCCGTATGAG 3'(Xho I)

NDM-conserved region upstream primer: 5'ttgaattcGGTCGCGAAGCTGA 3'(EcoR I)

NDM-conserved region downstream primer: 5'ttgaattc ATGCGGGCCGTATG 3'(EcoR I)

(2) Gene amplification

The conventional SDS alkaline lysis method was used to crush the cell wall of the Gram-negative bacteria to be tested, and a commercially available DNA extraction kit was used to obtain the total DNA, which was used as a template to amplify the target gene. PCR reaction system:

PCR operating conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 1min, annealing at 54°C for 30s, extension at 72°C for 18s, a total of 30 cycles; extension at 72°C for 5min.

(3) The expression plasmid pET-28a(+)-PM was constructed by conventional enzyme digestion and ligation techniques in the field of molecular biology, and the recombinant vector was transformed into Escherichia coli DH5a competent by CaCl ₂ heat shock method. Positive clones were screened using LB medium containing 100 μg/mL ampicillin. Escherichia coli was routinely cultured, and the plasmid was extracted for PCR identification to confirm the presence of the target gene.

(4) Expression and purification

Transform the extracted expression plasmid pET-28a(+)-PMAA into Escherichia coli BL21(DE3) competent cells, spread and culture on the selection medium, screen the single colony resistant to 100 μg/mL ampicillin, and then culture in liquid overnight . Take 1 mL of the overnight culture and inoculate it into 200 mL of LB medium containing 100 μg/mL ampicillin, culture with shaking until the logarithmic phase (OD600 is 0.5-0.6), add IPTG (1 mmol/L), induce culture at 16 ° C for 3 h, and ferment the Purified by nickel column, the purified product was detected by SDS-PAGE, and the fragment size was appropriate, which was the target protein. The SDS-PAGE electrophoresis diagram is shown in Figure 1 (M1 in the figure is Marker, and lane 2 is carbapenemase NDM expressed protein) .

3. Preparation of carbapenemase OXA-48

The present invention obtains carbapenemase OXA-48 (OXA-48-162～163, OXA-48-181, OXA-48-204, OXA-48-232, OXA-48-247, The conserved sequence of OXA-48-244～245), its amino acid sequence is as shown in SEQ ID NO.7-9. High-purity protein is obtained through prokaryotic gene expression, and the gene expression process is as follows:

(1) Primer design

According to the base composition of the target sequence, primers were designed to amplify the target gene, and the upstream and downstream contained EcoR I and Xho I restriction sites respectively. The primer sequences are as follows:

OXA-48-N-terminal conserved region upstream primer: 5'ttgaattc CCAGCGGTAGCAAA 3'(EcoR I)

OXA-48-N-terminal conserved region downstream primer: 5'tctcgagAACCACGCCCAAAT 3'(Xho I)

OXA-48-C-terminal conserved region upstream primer: 5'ttgaattc ATTGGCTGGTGGGT 3'(EcoR I)

OXA-48-C-terminal conserved region downstream primer: 5'tctcgag TTTGTGATGGCTTG 3'(Xho I)

OXA-48-conserved region upstream primer: 5'ttgaattcAGCGGTAGCAAAGGAA 3'(EcoR I)

OXA-48-conserved region downstream primer: 5'ttgaattc CGCAAAAAACCACACA 3'(EcoR I)

(2) Gene amplification

The conventional SDS alkaline lysis method was used to crush the cell wall of the Gram-negative bacteria to be tested, and a commercially available DNA extraction kit was used to obtain the total DNA, which was used as a template to amplify the target gene. PCR reaction system:

PCR operating conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 1min, annealing at 54.5°C for 30s, extension at 72°C for 30s, a total of 30 cycles; extension at 72°C for 5min.

(3) The expression plasmid pET-28a(+)-PM was constructed by conventional enzyme digestion and ligation techniques in the field of molecular biology, and the recombinant vector was transformed into Escherichia coli DH5a competent by CaCl ₂ heat shock method. Positive clones were screened using LB medium containing 100 μg/mL ampicillin. Escherichia coli was routinely cultured, and the plasmid was extracted for PCR identification to confirm the presence of the target gene.

(4) Expression and purification

Transform the extracted expression plasmid pET-28a(+)-PMAA into Escherichia coli BL21(DE3) competent cells, spread and culture on the selection medium, screen the single colony resistant to 100 μg/mL ampicillin, and then culture in liquid overnight . Take 1 mL of the overnight culture and inoculate it into 200 mL of LB medium containing 100 μg/mL ampicillin, culture with shaking until the logarithmic phase (OD600 is 0.5-0.6), add IPTG (1 mmol/L), induce culture at 16 ° C for 3 h, and ferment the Purified by nickel column, the purified product was detected by SDS-PAGE, and the fragment size was appropriate, which was the target protein. The SDS-PAGE electrophoresis figure is shown in Figure 1 (M1 in the figure is Marker, and lane 3 is the expression of carbapenemase OXA-48 protein).

4. Preparation of carbapenemase IMP

The present invention obtains carbapenemase IMP (IMP-1～35, IMP-37～46, IMP-48～49, IMP-51～56, IMP-58～85, IMP-88～ 89), its amino acid sequence is shown in SEQ ID NO.10-12. High-purity protein is obtained through prokaryotic gene expression, and the gene expression process is as follows:

(1) Primer design

According to the base composition of the target sequence, primers were designed to amplify the target gene, and the upstream and downstream contained EcoR I and Xho I restriction sites respectively. The primer sequences are as follows:

IMP-N-terminal conserved region upstream primer: 5'ttgaattc AGTTAGAAAAGGGAAG 3'(EcoR I)

Downstream primer of IMP-N-terminal conserved region: 5'tctcgagATGAAAATGAGAGGAA 3'(Xho I)

IMP-C-terminal conserved region upstream primer: 5'ttgaattcCCGGGACACACTCCAG 3'(EcoR I)

Downstream primer of IMP-C-terminal conserved region: 5'tctcgagACCAGTTTTGCCTTAC 3'(Xho I)

IMP-conserved region upstream primer: 5'ttgaattcGGTCGATGTTTGATGT 3'(EcoR I)

IMP-conserved region downstream primer: 5'ttgaattcGGTTTTGATGGTTTTTT 3'(EcoR I)

(2) Gene amplification

The conventional SDS alkaline lysis method was used to crush the cell wall of the Gram-negative bacteria to be tested, and a commercially available DNA extraction kit was used to obtain the total DNA, which was used as a template to amplify the target gene. PCR reaction system:

PCR operating conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 25 s, a total of 30 cycles; extension at 72°C for 5 min.

(3) The expression plasmid pET-28a(+)-PM was constructed by conventional enzyme digestion and ligation techniques in the field of molecular biology, and the recombinant vector was transformed into Escherichia coli DH5a competent by CaCl ₂ heat shock method. Positive clones were screened using LB medium containing 100 μg/mL ampicillin. Escherichia coli was routinely cultured, and the plasmid was extracted for PCR identification to confirm the presence of the target gene.

(4) Expression and purification

Transform the extracted expression plasmid pET-28a(+)-PMAA into Escherichia coli BL21(DE3) competent cells, spread and culture on the selection medium, screen the single colony resistant to 100 μg/mL ampicillin, and then culture in liquid overnight . Take 1 mL of the overnight culture and inoculate it into 200 mL of LB medium containing 100 μg/mL ampicillin, culture with shaking until the logarithmic phase (OD600 is 0.5-0.6), add IPTG (1 mmol/L), induce culture at 16 ° C for 3 h, and ferment the Purified by nickel column, the purified product was detected by SDS-PAGE, and the fragment size was appropriate, which was the target protein. The SDS-PAGE electrophoresis diagram is shown in Figure 1 (M1 in the figure is Marker, and lane 4 is carbapenemase IMP expression protein) .

5. Preparation of carbapenemase VIM

The present invention obtains the conserved sequence of carbapenemase VIM (VIM-1-20, VIM-23-73) through NCBI sequence alignment, and its amino acid sequence is shown in SEQ ID NO.13-15. High-purity protein is obtained through prokaryotic gene expression, and the gene expression process is as follows:

(1) Primer design

According to the base composition of the target sequence, primers were designed to amplify the target gene, and the upstream and downstream contained EcoR I and Xho I restriction sites respectively. The primer sequences are as follows:

VIM-N-terminal conserved region upstream primer: 5'ttgaattc GCCGATGGTGTTTGGT3'(EcoR I)

Downstream primer of VIM-N-terminal conserved region: 5'tctcgagGAGAATGCGTGGGAAT 3'(Xho I)

VIM-C-terminal conserved region upstream primer: 5'ttgaattcAGGACTCTCATCGAGC 3'(EcoR I)

Downstream primer of VIM-C-terminal conserved region: 5'tctcgag GACGGGACGTATACAA 3'(Xho I)

VIM-conserved region upstream primer: 5'ttgaattcAGCCGAGTGGTGAGTA 3'(EcoR I)

VIM-conserved region downstream primer: 5'ttgaattcGAGCGATTTTTGTGTG 3'(EcoR I)

(2) Gene amplification

The conventional SDS alkaline lysis method was used to crush the cell wall of the Gram-negative bacteria to be tested, and a commercially available DNA extraction kit was used to obtain the total DNA, which was used as a template to amplify the target gene. PCR reaction system:

PCR operating conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 1min, annealing at 56°C for 30s, extension at 72°C for 22s, a total of 30 cycles; extension at 72°C for 5min.

(3) The expression plasmid pET-28a(+)-PM was constructed by conventional enzyme digestion and ligation techniques in the field of molecular biology, and the recombinant vector was transformed into Escherichia coli DH5a competent by CaCl ₂ heat shock method. Positive clones were screened using LB medium containing 100 μg/mL ampicillin. Escherichia coli was routinely cultured, and the plasmid was extracted for PCR identification to confirm the presence of the target gene.

(4) Expression and purification

Transform the extracted expression plasmid pET-28a(+)-PMAA into Escherichia coli BL21(DE3) competent cells, spread and culture on the selection medium, screen the single colony resistant to 100 μg/mL ampicillin, and then culture in liquid overnight . Take 1 mL of the overnight culture and inoculate it into 200 mL of LB medium containing 100 μg/mL ampicillin, culture with shaking until the logarithmic phase (OD600 is 0.5-0.6), add IPTG (1 mmol/L), induce culture at 16 ° C for 3 h, and ferment the Purified by nickel column, the purified product was detected by SDS-PAGE, and the fragment size was appropriate, which was the target protein. The SDS-PAGE electrophoresis diagram is shown in Figure 1 (M1 in the figure is Marker, and lane 5 is the carbapenemase VIM expressed protein) .

Example 2 Preparation of carbapenemase monoclonal antibody

1. Preparation of carbapenemase KPC monoclonal antibody

In this example, the product of the conserved region of carbapenemase KPC was used to immunize mice, the immunization dose was 50ug/mouse, immunized once every 2 weeks, and immunized 5 times in total. The ratio of complete adjuvant was 1:1 for the first immunization, and used for subsequent immunizations. The ratio of incomplete adjuvant is 1:1 to obtain pairable monoclonal antibodies. The specific operation process is: immunize mice with antigens, fuse lymphocytes and myeloma cells of selected mice to form hybridoma cells, and use ELISA In this method, the blank microtiter plate was coated with antigen 100ng/well, and 100uL hybridoma cell supernatant was added to the microtiter plate, incubated at 37°C for 1h, washed once with 1x washing solution and patted dry, then added 100uL goat anti-mouse IgG-HRP, 37 Incubate at ℃ for 30min, wash with 1x washing solution for 3 times and pat dry, add 100uL TMB chromogenic substrate, incubate at 37℃ for 25min, add 50uL stop solution to each well, read OD value at 450nm, screen for OD>0.5 to produce the desired monoclonal The positive hybridoma cells of the antibody were cloned and amplified, and the antibody pairing verification was carried out to the antibody produced by the clone to obtain the required capture antibody and detection antibody. Lane 1 is carbapenemase KPC monoclonal antibody).

2. Preparation of carbapenemase NDM monoclonal antibody

In this example, the product of the conserved region of carbapenemase NDM was used to immunize mice, the immunization dose was 50ug/mouse, immunized once every 2 weeks, and immunized 5 times in total. The ratio of complete adjuvant was 1:1 for the first immunization, and used for subsequent immunizations. The ratio of incomplete adjuvant is 1:1 to obtain pairable monoclonal antibodies. The specific operation process is: immunize mice with antigens, fuse lymphocytes and myeloma cells of selected mice to form hybridoma cells, and use ELISA In this method, the blank microtiter plate was coated with antigen 100ng/well, and 100uL hybridoma cell supernatant was added to the microtiter plate, incubated at 37°C for 1h, washed once with 1x washing solution and patted dry, then added 100uL goat anti-mouse IgG-HRP, 37 Incubate at ℃ for 30min, wash with 1x washing solution for 3 times and pat dry, add 100uL TMB chromogenic substrate, incubate at 37℃ for 25min, add 50uL stop solution to each well, read OD value at 450nm, screen for OD>0.5 to produce the desired monoclonal The positive hybridoma cells of the antibody were cloned and amplified, and the antibody pairing verification was carried out to the antibody produced by the clone to obtain the required capture antibody and detection antibody. Lane 2 is carbapenemase NDM monoclonal antibody).

3. Preparation of carbapenemase OXA-48 monoclonal antibody

In this example, mice were immunized with the conserved region product of carbapenemase OXA-48, the immunization dose was 50ug/mouse, once every 2 weeks, a total of 5 immunizations, the ratio of complete adjuvant used for the first immunization was 1:1, and the follow-up The incomplete adjuvant ratio of 1:1 is used for immunization to obtain pairable monoclonal antibodies. The specific operation process is: immunize mice with antigens, fuse lymphocytes and myeloma cells of selected mice to form hybridoma cells, Using the ELISA method, the blank plate was coated with antigen 100ng/well, and 100uL hybridoma cell supernatant was added to the plate, incubated at 37°C for 1h, washed once with 1x washing solution and patted dry, then added 100uL goat anti-mouse IgG-HRP , incubate at 37°C for 30min, wash with 1x washing solution 3 times and pat dry, add 100uL TMB chromogenic substrate, incubate at 37°C for 25min, add 50uL stop solution to each well, read the OD value at 450nm, and screen for OD>0.5 to produce the desired The positive hybridoma cells of the monoclonal antibody were cloned and amplified, and the antibody pairing verification was carried out to the antibody produced by the clone to obtain the required capture antibody and detection antibody. The SDS-PAGE electrophoresis figure is shown in Figure 2 (M in the figure is Marker, lane 3 is carbapenemase OXA-48 monoclonal antibody).

4. Preparation of carbapenemase IMP monoclonal antibody

In this example, the product of the conserved region of carbapenemase IMP was used to immunize mice, the immunization dose was 50ug/mouse, immunized once every 2 weeks, a total of 5 immunizations, the complete adjuvant ratio of 1:1 was used for the first immunization, and the subsequent immunization used The ratio of incomplete adjuvant is 1:1 to obtain pairable monoclonal antibodies. The specific operation process is: immunize mice with antigens, fuse lymphocytes and myeloma cells of selected mice to form hybridoma cells, and use ELISA In this method, the blank microtiter plate was coated with antigen 100ng/well, and 100uL hybridoma cell supernatant was added to the microtiter plate, incubated at 37°C for 1h, washed once with 1x washing solution and patted dry, then added 100uL goat anti-mouse IgG-HRP, 37 Incubate at ℃ for 30min, wash with 1x washing solution for 3 times and pat dry, add 100uL TMB chromogenic substrate, incubate at 37℃ for 25min, add 50uL stop solution to each well, read OD value at 450nm, screen for OD>0.5 to produce the desired monoclonal The positive hybridoma cells of the antibody were cloned and amplified, and the antibody pairing verification was carried out to the antibody produced by the clone to obtain the required capture antibody and detection antibody. Lane 4 is carbapenemase IMP monoclonal antibody).

5. Preparation of carbapenemase VIM monoclonal antibody

In this example, the product of the conserved region of carbapenemase VIM was used to immunize mice with an immunization dose of 50ug/mouse, once every 2 weeks, and a total of 5 immunizations. The ratio of complete adjuvant was 1:1 for the first immunization, and 1:1 for subsequent immunizations. The ratio of incomplete adjuvant is 1:1 to obtain pairable monoclonal antibodies. The specific operation process is: immunize mice with antigens, fuse lymphocytes and myeloma cells of selected mice to form hybridoma cells, and use ELISA The blank microtiter plate was coated with antigen 100ng/well, and 100uL of hybridoma cell supernatant was added to the microtiter plate, incubated at 37°C for 1h, washed once with 1x washing solution and patted dry, then added 100uL goat anti-mouse IgG-HRP, 37 Incubate at ℃ for 30min, wash with 1x washing solution for 3 times and pat dry, add 100uL TMB chromogenic substrate, incubate at 37℃ for 25min, add 50uL stop solution to each well, read OD value at 450nm, screen for OD>0.5 to produce the desired monoclonal The positive hybridoma cells of the antibody were cloned and amplified, and the antibody pairing verification was carried out to the antibody produced by the clone to obtain the required capture antibody and detection antibody. Lane 5 is carbapenemase VIM monoclonal antibody).

Embodiment 3 establishes carbapenemase KPC detection reagent

The carbapenemase detection reagent includes a colloidal gold detection card and a Gram-negative bacterial lysate;

Among them, the preparation process of the colloidal gold detection card is: coated with T1 line OXA-48 coated antibody, prepared according to the protein concentration of the coated T1 line OXA-48 antibody (protein concentration is formulated to be 1.0 mg/ml), and 6 mg/mL OXA-48 antibody as an example, measure 5.00mL of coating solution, add 1.00mL of OXA-48 antibody to it, mix well, and make a mark and record accordingly. The same method was used to establish the coating antibodies of carbapenemase NDM, KPC, IMP and VIM. Pipette the colloidal gold complexes of OXA-48 and KPC antibodies to the same centrifuge tube, 2 mL each, 4 mL in total. Then use a pipette to absorb 3mL of reconstituted solution to rinse the original container of each antibody colloidal gold complex, rinse twice, and mix 6mL of reconstituted solution with 4mL of antibody colloidal gold complex, totaling 10mL. Use a pipette to draw the colloidal gold complexes of IMP, NDM and VIM antibodies into the same centrifuge tube, 2 mL each, 6 mL in total. Then use a pipette to absorb 2mL of reconstituted solution to rinse the original container of each antibody colloidal gold complex, rinse twice, and mix 4mL of reconstituted solution with 6mL of antibody colloidal gold complex, totaling 10mL. Coating C-line goat anti-mouse IgG antibody was prepared according to the protein concentration of C-line goat anti-mouse IgG antibody (the protein concentration was prepared to be 1mg/ml). Taking 10mg/mL as an example, measure 4.50mL of coating solution, Add 0.50 mL of goat anti-mouse IgG antibody to it and mix thoroughly to obtain a coated nitrocellulose membrane, soak it in blocking solution, and dry it with air at 37°C for 2 hours. Measure 100mL of colloidal gold, add it to a beaker, and place it on a magnetic stirrer. Add 200 μL of 0.2M K ₂ CO ₃ solution under stirring and mix well. After that, add OXA-48 antibody to a final concentration of 12 μg/mL under stirring conditions, mix well, and place at 20-25°C for labeling for 2 hours; add 2.0 mL of blocking solution to the colloidal gold, stir and mix well, and then stand at room temperature for 40 minutes to block. Take out the colloidal gold solution and put it into a centrifuge tube, centrifuge at 10,000g at 4°C for 15 minutes, collect the precipitate, and reconstitute it together for subsequent use. Next, the supernatant was placed at 10,000 g and centrifuged at 4° C. for 15 minutes until the supernatant became clear. After aspirating the supernatant as much as possible, leave the precipitate. Add 2.00mL of reconstitution solution to the precipitate to fully dissolve it to obtain colloidal gold-labeled antibody. Spray the colloidal gold-labeled antibody on the conjugation pad, and dry it with air at 37° C. for 5 hours to obtain the conjugation pad after spraying gold. Assemble the polyvinyl chloride bottom plate, sample pad, gold-sprayed bonding pad, treated nitrocellulose membrane, and absorbent paper, cut into strips, and obtain a colloidal gold immunochromatography rapid detection test card.

The specific detection process of Gram-negative bacteria lysate is as follows: Take a small amount of bacteria (1-10 μL) and add 200 μL Gram-negative bacteria lysate (0.2% sodium lauroyl sarcosine + 0.01% CHAPS), mix well, and let stand 5~10min. Take 80 μL of the lysed product dropwise into the sample hole of the colloidal gold test card, wait for 15 minutes, and observe whether the T line and the C line come out.

The same method was used to establish detection reagents for carbapenemase NDM, OXA-48, IMP and VIM.

Test example 1

1. Minimum detection limit and HOOK effect detection

Use the finished reagent card to detect KPC, NDM, OXA-48, VIM and IMP samples with gradient concentrations, and repeat three times to test the minimum detection limit and HOOK effect. The test results are shown in Table 1. The results show: KPC, NDM, OXA-48 48. When the added concentration of VIM and IMP was 100ng/mL, there was no HOOK effect, and the lowest detection limit was in the range of 0.1-1ng/mL.

Table 1 The minimum detection limit of the finished reagent card and the detection results of HOOK effect

2. Repeatability detection

Use the finished reagent card to detect KPC, NDM, OXA-48, VIM and IMP negative samples (HRP conjugate stabilizer/diluent I), weakly positive samples (use the same HRP conjugate stabilizer/diluent I Dissolve KPC antigen, NDM antigen, OXA-48 antigen, IMP antigen, and VIM antigen to a final concentration of 5 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, and 20 ng/mL, vortex and mix to make a low value positive sample), medium positive sample (dissolve KPC antigen, NDM antigen, OXA-48 antigen, IMP antigen, VIM antigen to the final concentration of 100ng/mL, 200ng /mL, 100ng/mL, 50ng/mL, 400ng/mL, vortexed to make a median positive sample) to test the repeatability. The test results are shown in Table 2-1 to Table 2-5, and the results show that no abnormal results were found after repeated 10 times.

Table 2-1 Repeatability test results of KPC finished reagent cards

sample	negative sample	Weak positive samples	positive samples
repeat 1	-	+	++
repeat 2	-	+	++
repeat 3	-	+	++
Repeat 4	-	+	++
Repeat 5	-	+	++
Repeat 6	-	+	++
Repeat 7	-	+	++
Repeat 8	-	+	++
Repeat 9	-	+	++
Repeat 10	-	+	++

Table 2-2 Repeatability test results of OXA-48 finished reagent card

sample	negative sample	Weak positive samples	positive samples
repeat 1	-	+	++
repeat 2	-	+	++
repeat 3	-	+	++
Repeat 4	-	+	++
Repeat 5	-	+	++
Repeat 6	-	+	++
Repeat 7	-	+	++
Repeat 8	-	+	++
Repeat 9	-	+	++
Repeat 10	-	+	++

Table 2 Repeatability test results of finished NDM reagent cards

sample	negative sample	Weak positive samples	positive samples
repeat 1	-	+	++
repeat 2	-	+	++
Repeat 3	-	+	++
Repeat 4	-	+	++
Repeat 5	-	+	++
Repeat 6	-	+	++
Repeat 7	-	+	++
Repeat 8	-	+	++
Repeat 9	-	+	++
Repeat 10	-	+	++

Table 2-4 Repeatability test results of finished VIM reagent cards

sample	negative sample	Weak positive samples	positive samples
repeat 1	-	+	++
repeat 2	-	+	++
repeat 3	-	+	++
Repeat 4	-	+	++
Repeat 5	-	+	++

Repeat 6	-	+	++
Repeat 7	-	+	++
Repeat 8	-	+	++
Repeat 9	-	+	++
Repeat 10	-	+	++

Table 2-5 Repeatability test results of IMP finished reagent cards

sample	negative sample	Weak positive samples	positive samples
repeat 1	-	+	++
repeat 2	-	+	++
repeat 3	-	+	++
Repeat 4	-	+	++
Repeat 5	-	+	++
Repeat 6	-	+	++
Repeat 7	-	+	++
Repeat 8	-	+	++
Repeat 9	-	+	++
Repeat 10	-	+	++

3. Real-time stability detection

Use the finished reagent card to detect negative samples, low-value positive samples, Median positive samples to assess real-time stability. The test results are shown in Table 3-1 to Table 3-5, and the results show that the test results are consistent within 12 months.

Table 3-1 Real-time stability test results of KPC finished reagent cards

sample	negative sample	Weak positive samples	positive samples
0th month after production	-	+	++
1 month after production	-	+	++
2 months after production	-	+	++
3 months after production	-	+	++
4th month after production	-	+	++
6 months after production	-	+	++
8th month after production	-	+	++
10th month after production	-	+	++
12 months after production	-	+	++
14 months after production	-	+	++

Table 3-2 Real-time stability test results of OXA-48 finished reagent card

sample	negative sample	Weak positive samples	positive samples
0th month after production	-	+	++
1 month after production	-	+	++
2 months after production	-	+	++
3 months after production	-	+	++
4th month after production	-	+	++
6 months after production	-	+	++
8th month after production	-	+	++
10th month after production	-	+	++
12 months after production	-	+	++

14 months after production

-

+

++

Table 3-3 Real-time stability test results of finished NDM reagent cards

sample	negative sample	Weak positive samples	positive samples
0th month after production	-	+	++
1 month after production	-	+	++
2 months after production	-	+	++
3 months after production	-	+	++
4th month after production	-	+	++
6 months after production	-	+	++
8th month after production	-	+	++
10th month after production	-	+	++
12 months after production	-	+	++
14 months after production	-	+/-	++

Table 3-4 Real-time stability test results of finished VIM reagent cards

sample	negative sample	Weak positive samples	positive samples
0th month after production	-	+	++
1 month after production	-	+	++
2 months after production	-	+	++
3 months after production	-	+	++
4th month after production	-	+	++
6 months after production	-	+	++
8th month after production	-	+	++
10th month after production	-	+	++
12 months after production	-	+	++
14 months after production	-	+/-	++

Table 3-5 Real-time stability test results of IMP finished reagent cards

sample	negative sample	Weak positive samples	positive samples
0th month after production	-	+	++
1 month after production	-	+	++
2 months after production	-	+	++
3 months after production	-	+	++
4th month after production	-	+	++
6 months after production	-	+	++
8th month after production	-	+	++
10th month after production	-	+	++
12 months after production	-	+	++
14 months after production	-	+	++

Test example 2

Configure the lysate according to the table below to test the lysing effect of single-component and mixed-component lysates on Gram-negative bacteria (e.g. Escherichia coli producing KPC-type carbapenemase).

Table 4 Component and concentration grouping of lysate

serial number	components	concentration
Test group 1	SDS	0.1%
Test group 2	Triton X-100	1%
Test group 3	NaOH	10mM

Test group 4	CHAPS	0.02%
Test group 5	Sodium Lauroyl Sarcosine	0.2%
Test group 6	SDS, CHAPS, Sodium Lauroyl Sarcosinate	0.1%, 0.02%, 0.2%
control group	water	/

The test method includes the following steps:

1. Bacteria washing: centrifuge 1ml of bacterial solution at 8000g for 5min, wash with 1ml of pure water for 2 times, and finally redissolve with 1ml of pure water;

2. Bacteria separation: number 1-7 EP tubes, add 100ul of the above bacteria solution to each tube, centrifuge, and remove the supernatant;

3. Lysis: Add 200ul of the corresponding lysate according to the configuration in Table 4 to each tube, mix well, and let it stand for 1 hour;

4. Staining: Take 90ul of the above-mentioned lysate product, add 10ul trypan blue staining solution, mix well, and observe under a microscope. The results are shown in Figure 3.

It can be seen from Figure 3 that the number of bacteria observed in the 40X field of view of the test group 1 was significantly lower than that of the control group, and the multi-field observation was uniform; ; The number of bacteria observed in the 40X field of view of the test group 3 cells was significantly reduced compared with that of the control group, and the multi-field observation was uniform; The number of bacteria observed in the 40X field of view and the control group was significantly reduced, and the number of bacteria in the multi-field observation was uniform; the number of bacteria observed in the 6 single grids of the 40X field of view in the test group was significantly reduced compared with the control group, and the number of bacteria in the multi-field observation was uniform; the number of bacteria in the 40X field of view of the control group was more , Multi-field observation is uniform. It shows that each group has a lytic effect on Gram-negative bacterial strains.

Using 0.1% SDS, 0.02% CHAPS and 0.2% sodium lauroyl acid as the experimental group and pure water as the control group, the Gram-positive bacteria (taking Bifidobacterium as an example) were respectively lysed according to the above method. As can be seen from Figure 4, compared with the control group, the number of bacteria in the experimental group did not change significantly, indicating that the lysate had no significant effect on Gram-positive bacteria.

Use the colloidal gold reagent card to detect the detection effect of the single component and the mixed group after decomposition. The test results are shown in the table below. The results show the detection effect of the mixed use of 0.1% SDS, 0.02% CHAPS and 0.2% sodium lauroyl amate It is best to illustrate that the mixed use of 0.1% SDS, 0.02% CHAPS and 0.2% sodium lauroyl acid can not only effectively lyse Gram-negative bacterial strains to release the test substance (KPC type carbapenemase), but also does not affect colloidal gold Reagent card detection (although 1% TritonX-100 and 10mM NaOH can lyse Gram-negative bacteria, they will affect the colloidal gold detection system).

Table 5 Test results

To sum up, the conclusions of the above-mentioned test groups 1-6 and the control group on Gram-negative bacteria (taking KPC-type carbapenemase-producing Escherichia coli as an example) are shown in Table 5.

Table 5 Test conclusion

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the Within the protection scope of the present invention.

Claims (10)

1. A carbapenemase conserved antigen, characterized in that: the conserved antigen comprises any one or more of the amino acid sequences shown in SEQ ID NO: 1-15.
2. A broad-spectrum specific antibody to carbapenemase, characterized in that: the broad-spectrum specific antibody is a monoclonal antibody or a polyclonal antibody prepared from the conserved antigen described in claim 1.
3. A carbapenemase detection reagent, characterized in that: the detection reagent includes Gram-negative bacteria lysate and the conserved antigen according to claim 1 or the broad-spectrum specific antibody according to claim 2.
4. Carbapenemase detection reagent according to claim 3, is characterized in that: the active ingredient of described Gram-negative bacterial lysate is: one or more in sodium lauroyl sarcosine, CHA PS, SDS mixture of species.
5. The carbapenemase detection reagent according to claim 4, characterized in that: the mass percentage of sodium lauroyl sarcosine in the Gram-negative bacteria lysate is 0.1%-1%.
6. The carbapenemase detection reagent according to claim 4, characterized in that: the mass percentage of CHAPS in the Gram-negative bacteria lysate is 0.01%-0.1%.
7. The carbapenemase detection reagent according to claim 4, characterized in that: the mass percentage of SDS in the Gram-negative bacteria lysate is 0.1%-1%.
8. The carbapenemase detection reagent according to claim 4, characterized in that: the Gram-negative bacteria lysate comprises 0.2% sodium lauroyl amate, 0.02% CHAPS and 0.1% SDS in mass percentage .
9. A carbapenemase detection kit, characterized in that: the kit contains the carbapenemase detection reagent according to any one of claims 3-8.
10. The carbapenemase detection kit according to claim 9, characterized in that: the carbapenemase detection kit is a colloidal gold detection kit, and the antibody marker in the kit is colloidal gold.

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