CN109456409B

CN109456409B - Specific antibody group for detecting klebsiella pneumoniae and application thereof

Info

Publication number: CN109456409B
Application number: CN201811342422.1A
Authority: CN
Inventors: 张金阳; 张立定; 石耀强; 宋玉竹; 夏雪山
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-11-13
Filing date: 2018-11-13
Publication date: 2021-10-29
Anticipated expiration: 2038-11-13
Also published as: CN109456409A

Abstract

The invention discloses a specific antibody group for detecting klebsiella pneumoniae, which comprises a monoclonal antibody 1E6 and a monoclonal antibody 2F1; according to the invention, a nano magnetic bead sensor reaction system is constructed by adopting a pair of monoclonal antibodies for identifying different membrane proteins on the surface of the Klebsiella pneumoniae, HRP, TMB, carboxyl nano magnetic beads and the like, so that a method for rapidly detecting the Klebsiella pneumoniae nano magnetic bead sensor is established, and the Klebsiella pneumoniae can be rapidly and accurately detected; the technology integrates the specificity of the antibody and the characteristic of high-efficiency enrichment of the antigen by the nano magnetic beads, has the characteristics of accuracy, rapidness, high efficiency, specificity and sensitivity, does not need long-time amplification culture of thalli, and extraction of genes and plasmids, is suitable for on-site rapid detection of import and export quarantine, food sanitation inspection, clinical samples of patients and the like, and has important significance for guaranteeing food safety and timely and accurately guiding the use of medicaments.

Description

Specific antibody group for detecting klebsiella pneumoniae and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nano magnetic bead sensor for detecting klebsiella pneumoniae.

Background

Klebsiella pneumoniae is a gram-negative bacillus, and exudate in lesions is sticky and heavy, causing the space between the leaves to descend. The bacteria have a capsule and when growing and multiplying in the alveoli, cause tissue necrosis, liquefaction, and the formation of single or multiple abscesses. When the pleural and pericardial diseases are involved, exudative or purulent fluid accumulation may occur. The fibrous tissue of the focus is proliferated actively and is easy to organize; cellulosic pleural effusions can develop adhesions early. In septicemia caused by nosocomial infection, klebsiella, pseudomonas aeruginosa, serratia and the like are important pathogenic bacteria, and the high fatality rate is one of the main pathogenic bacteria causing nosocomial infection. In China, the infection rate is second to that of Escherichia coli, and the diseases such as pneumonia, septicemia, urinary tract infection and the like are mainly caused.

The detection methods of the Klebsiella pneumoniae which are clinically applied at present comprise bacteria separation identification, immunological methods, molecular biology detection methods and the like, but the methods have some defects. The traditional detection method mainly adopts the steps of bacteria pre-enrichment culture, separation culture, colony morphology observation, a series of biochemical identification, serotype identification and the like, generally takes 4 to 7 days, has long culture time, large workload and complicated experimental steps, and can not reflect accurate data in time in many emergencies. The immunological method is easy to pollute, and has more serious cross reaction, more false positive and lower sensitivity. The conventional PCR method has low detection sensitivity, and enrichment of thalli and extraction of DNA are required before detection. The LAMP method has high detection sensitivity, but false positive easily occurs, a reaction system is unstable, the bacteria of a detection sample cannot be enriched, and the bacteria still need to be enriched manually. The immunomagnetic separation technology is a technology for coupling a specific antibody with magnetic beads with a certain size, combining magnetic spheres with cells by utilizing the principle that the specific antibody can be combined with cell surface antigens, separating a magnetic sphere-cell compound from the environment under the action of an external magnetic field to obtain target cells, but the technology can only realize separation when being used alone, and needs to be combined with other means for detection, but the sensitivity of the existing detection method for separating by using the immunomagnetic separation technology is still to be improved.

Disclosure of Invention

The invention aims to provide a specific antibody group for detecting klebsiella pneumoniae, which comprises a monoclonal antibody 1E6 (capture antibody), a monoclonal antibody 2F1; 1E6 recognizes Klebsiella pneumoniae antigen outer membrane protein C, and 2F1 recognizes Klebsiella pneumoniae antigen outer membrane protein A;

the amino acid sequence of the variable region of the light chain of the monoclonal antibody 1E6 is shown as SEQ ID NO. 1, and the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.

The amino acid sequence of the light chain variable region of the monoclonal antibody 2F1 is shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4.

The invention also aims to apply the specific antibody group for detecting klebsiella pneumoniae in the preparation of a nano magnetic bead sensor detection kit, wherein monoclonal antibody 1E6 is coated on nano magnetic beads to form nano immunomagnetic beads, monoclonal antibody 2F1 labeled with horseradish peroxidase is used as a second antibody, and an immunomagnetic separation technology is adopted to detect klebsiella pneumoniae;

the magnetic nanobead combined with the klebsiella pneumoniae specific antibody 1E6 is prepared by adopting the principle that carboxyl nanobead can be specifically combined with antibody amino, and the preparation method specifically refers to a 300 nm carboxyl magnetic bead [ Chinese engineering Biotechnology, cat #: (PC 31) ] product specification.

The kit of the invention also comprises a conventional developing solution (TMB) and a washing solution (PBS-T).

The invention combines a double-antibody sandwich detection reagent with immune nano magnetic beads, optimizes reaction conditions and establishes a klebsiella pneumoniae nano magnetic bead sensor detection technology.

The invention also aims to provide a method for detecting the Klebsiella pneumoniae, which comprises the steps of coupling carboxyl nano magnetic beads with the monoclonal antibody of the Klebsiella pneumoniae to form immune nano magnetic beads to capture the Klebsiella pneumoniae in a sample, adding the monoclonal antibody 2F1 which is used for identifying different membrane proteins and is marked with HRP, and carrying out TMB color development to confirm whether the Klebsiella pneumoniae exists or not.

The method can be used for detecting water, food, body fluid of human and animals, excrement samples and the like polluted by the klebsiella pneumoniae.

The method for detecting the Klebsiella pneumoniae comprises the following specific steps:

(1) adding nanometer immunomagnetic beads coupled with Klebsiella pneumoniae specific antibody 1E6 into a sample to be detected, uniformly mixing, reacting at 37 ℃ for 30min, performing magnetic separation, removing supernatant, adding PBS-T solution for resuspension, performing magnetic separation, and repeating for 2-3 times;

(2) adding 100 mu L of monoclonal antibody 2F1 marked with HRP, and reacting for 20min at 37 ℃;

(3) performing magnetic separation, discarding the supernatant, adding PBS-T solution for resuspension, performing magnetic separation, and repeating for 2-3 times; adding 100 mu L of color development liquid, and reacting for 10min at 37 ℃ in a dark place;

(4) directly observing whether the liquid turns blue by naked eyes, and judging whether the klebsiella pneumoniae exists or not; the detection method is a conventional detection method.

After the reaction is finished, directly observing the result by naked eyes; and (4) judging a result: if the blue color appears, the detection result is positive, and the sample contains the Klebsiella pneumoniae; and if the detection result is negative, the sample does not contain the Klebsiella pneumoniae.

Experimental results show that the nano magnetic bead sensor kit for Klebsiella pneumoniae disclosed by the invention has a good positive effect on the aspect of detecting Klebsiella pneumoniae.

Compared with the prior art, the invention has the positive effects that:

(1) the invention has the characteristics of simple and convenient operation, rapidness and the like, does not need large-scale detection equipment, has low requirements on other conditions except that the magnetic frame is a necessary accessory (for example, a 37-degree incubator or a water bath kettle is lacked, the detection work can be directly carried out at room temperature), and is suitable for basic popularization and application; in addition, the double antibody sandwich technology is combined with the immune nano magnetic bead technology, the result can be directly observed and judged by naked eyes, only 1h is needed, 6 days are saved compared with the traditional biochemical culture method, 9h is shortened compared with a PCR (agarose gel electrophoresis) method for extracting genome, and the enrichment of thalli and the extraction of genome and the like are not needed, so that the detection is more convenient and efficient;

(2) based on the advantages of sensitivity, specificity, low cost, convenient operation and the like of the nano magnetic bead sensor, the invention improves the detection capability of the Klebsiella pneumoniae strain in China to a certain extent, provides a new technical source for the clinical real-time rapid diagnosis of the Klebsiella pneumoniae disease and the rapid and accurate detection of the Klebsiella pneumoniae strain in the entry-exit inspection and quarantine of animals and foods, and has important significance for improving the epidemic situation monitoring and rapid detection level of the Klebsiella pneumoniae strain in the inspection and quarantine institution in China and popularizing the popularization and application of advanced technology in the basic inspection and quarantine institution; particularly for the detection of special samples such as blood and the like, the method solves the difficult problems of obtaining and enriching thalli from the special samples such as blood and the like, and has higher sensitivity than the conventional detection methods such as PCR, multiple fluorescence quantitative PCR, LAMP and the like.

Drawings

FIG. 1A is a flow chart of the preparation of immunomagnetic beads of Klebsiella pneumoniae; FIG. 1B is a flow chart of mab-HRP probe preparation; FIG. 1C is a flow chart of the detection of the magnetic nanobead sensor of Klebsiella pneumoniae;

FIG. 2 shows the prepared 3 titers against ascites in Klebsiella pneumoniae mice;

FIG. 3 shows the results of detection of the specificity of the 3-strain antibody;

FIG. 4A shows the SDS-PAGE of the purified 3-strain antibody; FIG. 4B shows the result of SDS-PAGE verification after successful coupling of monoclonal antibody 1E6 to the nanobead;

FIG. 5 shows Western Blot results of monoclonal antibodies 1E6, 2F1, and 4E 6;

FIG. 6 shows the mass spectrum of the target protein (Klebsiella pneumoniae outer membrane protein C) of mAb 1E 6;

FIG. 7 shows the mass spectrum of the target protein (Klebsiella pneumoniae outer membrane protein A) of mAb 2F1;

FIG. 8 shows the mass spectrum of the target protein of mAb 4E6 (Klebsiella pneumoniae catalase HPII protein);

FIG. 9A, B, C shows the titer of HRP on the prepared 3-strain antibody label;

FIG. 10 shows the results of the pairing of 3 antibodies;

FIG. 11 shows the affinity curves of mAbs 1E6 (A) and 2F1 (B);

FIG. 12 shows the specificity results of the combination of mAb 1E6+2F 1;

FIG. 13 shows the optimal capture antigen times for the combination of mAb 1E6+2F 1;

FIG. 14A shows the sensitivity of the nano-magnetic bead sensor, with the concentration of bacteria in tubes 1-9 being 10⁸、10⁷、10⁶、10⁵、10⁴、10³、10²、10¹、10⁰CFU/mL, tube 10 negative control; FIG. 14B is a graph showing 10 of the optical density sensitivity test results of the nano-magnetic bead test sensor, wherein the curves 1 to 9⁸、10⁷、10⁶、10⁵、10⁴、10³、10²、10¹、10⁰CFU/mL, curve 10 negative control;

FIG. 15 shows the sensitivity of a sensor for detecting nano-magnetic beads by a PCR method;

FIG. 16 is a standard curve of a nano magnetic bead sensor;

FIG. 17A shows the specific detection result of the nano-magnetic bead sensor, wherein tube 1 is Klebsiella pneumoniae, tubes 2-7 are Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Pseudomonas aeruginosa, and Shigella in sequence, and tube 8 is blank control; FIG. 17B shows the specific detection result of the optical density of the nano magnetic bead sensor, wherein a curve 1 shows Klebsiella pneumoniae, curves 2 to 7 show Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Pseudomonas aeruginosa, and Shigella in sequence, and a curve 8 shows a blank control;

FIG. 18 shows the specific results of the nano magnetic bead sensor detected by the PCR method;

FIG. 19 shows the results of 40 clinical samples detected by the nano-magnetic bead sensor, and 30 positive clinical samples detected by the tubes 1-30; tubes 31-40 are 10 negative clinical sample test results, respectively;

fig. 20 shows the detection result of the clinical sample of the nanobead sensor by the PCR method. Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and conventional methods.

Example 1: preparation of anti-Klebsiella pneumoniae specific monoclonal antibody

1. Preparation of antigens

Taking out the standard strain of Klebsiella pneumoniae from a refrigerator at-80 deg.C, streaking on LB solid culture medium, culturing overnight at 37 deg.C, selecting single colony in 250mL LB liquid culture medium, performing amplification culture, centrifuging, collecting thallus, washing with PBS for 3 times, dissolving in PBS to final concentration of 10¹⁰CFU/mL, then inactivating at 80 ℃;

2. immunization of mice

Mixing 100 mu L of prepared antigen with equivalent volume of Freund's complete adjuvant, and performing primary immunization; carrying out second immunization 14 days after the first immunization, and mixing 100 mu L of prepared antigen with equivalent volume of Freund incomplete adjuvant for immunization; the third immunization was identical to the second, with an interval of 14 days; after the third immunization, 100 mu L of prepared antigen is taken for tail vein boosting immunization;

3. cell fusion screening of Positive clones

3 days after the boosting, splenocytes of the mice are collected and fused with SP2/0 cells; culturing the fused cells in HAT medium containing 20% serum, and replacing HT medium after one week; performing first ELISA detection 12 days after cell fusion, and performing first subcloning on a positive hole; the first subcloning was performed for 14 days, the second ELISA assay was performed, and positive wells were used for the second subcloning. Subcloning for 14 days for the second time, and performing ELISA detection for the third time; if the ELISA detection results are positive after 3 times of subcloning, the monoclonal antibody becomes a monoclonal antibody, and the monoclonal antibody is frozen and stored in liquid nitrogen after the amplification culture.

4. Preparation and purification of ascites

3 female BALB/c mice of 6-7 weeks old are injected with 0.5mL sterile liquid paraffin into the abdominal cavity, and each mouse is injected with 10 percent of sterile liquid paraffin into the abdominal cavity after one week⁸(ii) individual hybridoma cells; after one week, collecting ascites; ascites prepared by purifying Protein A magnetic beads;

5. mass spectrometric identification of antigens recognized by sandwich antibodies

Coupling the purified ascites with the protein A/G immunoprecipitation magnetic beads respectively for co-immunoprecipitation with reference to the specification of the protein A/G immunoprecipitation magnetic beads; centrifuging the expanded Klebsiella pneumoniae 13,400 Xg for 10min, discarding the supernatant, and washing the precipitate with PBS 3 times; then 20mL of PBS was added and ultrasonication was performed on ice; adding the crushed bacterial liquid into the prepared immunoprecipitation magnetic beads, incubating for 30min at 37 ℃, washing for 3 times by PBS-T, and performing SDS-PAGE and Western Blot; cutting off a band corresponding to the Western Blot on the SDS-PAGE gel for mass spectrum identification;

as a result: 3 rounds of subcloning are carried out, 3 monoclonal antibody cell strains which are used for successfully screening the anti-Klebsiella pneumoniae are named as 1E6, 4E6 and 2F1, the prepared ascites titer is shown in figure 2, the 3 antibody strains only react with the Klebsiella pneumoniae in a specific way, but do not react with other 6 similar strain strains, and the specificity is shown in figure 3. 3 monoclonal antibodies were successfully purified using Protein A magnetic beads, and the SDS-PAGE results are shown in FIG. 4A. The mass spectrum results of the corresponding target protein of the 3-strain antibody are as follows: the mass spectrum result of the target protein (Klebsiella pneumoniae outer membrane protein C) of the monoclonal antibody 1E6 is shown in FIG. 6; the mass spectrum result of the target protein (Klebsiella pneumoniae outer membrane protein A) of the monoclonal antibody 2F1 is shown in FIG. 7; the mass spectrum results of the target protein (Klebsiella pneumoniae catalase HPII protein) of mAb 4E6 are shown in FIG. 8.

Example 2: the establishment of the detection method of the nano magnetic bead sensor is shown in figure 1

1. Preparation of HRP-2F1 Probe

1) 5mg of HRP was dissolved in 0.5mL0.1mol/L NaHCO₃To the solution, 0.5mL of 10mmol/L NaIO was added₄Mixing the solution, tightly covering the bottle stopper, and keeping away from light at room temperature for 2 hours;

2) 0.75mL of 0.1mol/L Na is added₂CO₃Mixing uniformly;

3) adding 0.75mL of the mouse-treated ascites or the purified monoclonal antibody (15mg/mL), and mixing uniformly;

4) weighing 0.3G of Sephadex G25 dry powder, and adding into a 5mL syringe outer cylinder with a lower mouth pad of glass wool; then transferring the cross-linked substance into the outer sleeve of the syringe; covering tightly, acting at room temperature (keeping out of the sun) for 3 hours or staying overnight at 4 ℃;

5) the cross-links were washed out completely with a little PBS and the eluate was collected and added 1/20 volumes of freshly prepared 5mg/mL NaBH₄Mixing the solution, and acting at room temperature for 30 min; additional 3/20 volumes of NaBH were added₄Mixing the solution, and acting at room temperature for 1 hr (or overnight at 4 deg.C);

6) purifying the cross-linked substance by Sephadex g200 or Sepharose 6B (2.6X 50cm), and collecting peaks by tubes;

7) preservation of HRP-2F1 antibody conjugate: adding equivalent glycerol, packaging at-20 deg.C;

as a result: HRP-2F1 probe was successfully prepared, and the results of its activity by ELISA are shown in FIGS. 9A, B and C.

2. Search for paired antibodies

Diluting 20 μ L of purified anti-Klebsiella pneumoniae antibodies 1E6, 4E6 and 2F1, mixing with 2mL of CBS, coating 100 μ L of each mixture in a 96-well plate at 37 deg.C for 2 h; after coating, adding 200 mu L of 5% skim milk into each hole and sealing for 2 h; then adding 100 mu L of HRP-labeled 1E6, 4E6 and 2F1 into each hole, reacting for 1h at 37 ℃, removing the supernatant, washing each hole for 5 times by PBS-T, and adding 100 mu L of color development liquid respectively to react for 15min in a dark place; finally 50. mu.L of 2M H was added to each well₂SO₄Terminating the reaction and measuring the absorbance at 450 nm;

as a result: successfully find 4 groups of paired antibodies, wherein the combination of 1E6+2F1 has the best effect, and the pairing result is shown in figure 10. The affinity curves of mAbs 1E6 and 2F1 are shown in FIG. 11; western Blot results further demonstrate that mAbs 1E6 and 2F1 recognize different membrane proteins of Klebsiella pneumoniae, and the results are shown in FIG. 5. The specificity results for the 1E6+2F1 combination are shown in figure 12.

3. Preparation of immune nano magnetic bead

A group of paired antibodies (1E 6, 2F 1) obtained in the step 1 are used for preparing immune nano magnetic beads; based on the affinity test, the antibody 1E6 was found to have the best affinity, and thus 1E6 was used to prepare immunomagnetic beads, which were performed as follows:

1) uniformly mixing the magnetic beads, adding 10mg of the magnetic beads into a 2mL centrifuge tube, and carrying out magnetic separation to remove supernatant;

2) adding 1mL of deionized water, mixing uniformly, and performing magnetic separation to remove supernatant (repeating for 2 times);

3) adding 500-1000 muL of reaction buffer (0.02M MES, pH 5.0), mixing and resuspending magnetic beads, and magnetically separating to remove supernatant (repeating for 2 times);

4) adding 200 mu L of reaction buffer solution, and mixing and resuspending magnetic beads;

5) adding 200-300 mug of antibody or protein solution (dissolved by 200 muL of reaction buffer solution in advance), and uniformly mixing for 30min at room temperature in a vortex manner;

6) adding 100 mu L of on-site coupling reagent (EDC-HCl solution), carrying out magnetic separation for 12-16 h, and removing supernatant;

7) adding 1mL of washing buffer solution (PBS, pH 7.4), mixing the heavy suspension magnetic beads, and magnetically separating to remove supernatant (repeating for 3-5 times);

8) adding 1mL of blocking buffer (1% BSA, 0.02M MES, pH 5.0), mixing and resuspending magnetic beads, carrying out vortex mixing at room temperature for 2-6 h, and carrying out magnetic separation to remove supernatant;

9) dispersing the above magnetic beads in 0.5mL PBS, storing at pH7.4 for a short period, or dispersing in PBS, pH7.4, 0.1% BSA, 0.02% NaN₃Long-term preservation;

as a result: the monoclonal antibody 1E6 was successfully coupled to the nanobead, and was verified by SDS-PAGE, the result is shown in FIG. 4B. The optimal capture time for capturing antigen by immunomagnetic beads is shown in FIG. 13.

Example 3: the specificity and sensitivity of the method of the invention are determined as shown in FIG. 1

1. Culture of Klebsiella pneumoniae

Taking out the frozen bacterium liquid, performing LB plate streaking, and culturing overnight in a constant-temperature incubator at 37 ℃; selecting a single clone to be cultured in an LB liquid culture medium for 4-5 hours at 37 ℃ at a speed of 180 r/h on the next day;

2. antigen capture using immunomagnetic beads

(a) Preparation of antigen: the bacterial solutions were diluted with mouse blood to a final concentration of 4X 10⁸、4 × 10⁷、4 × 10⁶、4 × 10⁵、4 × 10⁴、4× 10³、4× 10²、4×10¹And 4X 10⁰ CFU/mL；

(b) Pretreating immune nano magnetic beads: carrying out vortex oscillation on the prepared immune nano magnetic beads for 1min to fully resuspend the magnetic beads; placing 30 mu L of magnetic bead suspension in a 1.5mLEP tube; adding 200 mu L of LPBS-T for washing, performing magnetic separation (placing an EP tube on a magnetic frame to adsorb magnetic beads on the tube wall until the solution is clarified; the operation is omitted below), discarding supernatant, taking down the EP tube from the magnetic separator, repeating the washing once, and finally adding 200 mu L of PBS-T for re-suspension of the magnetic beads for later use;

(c) antibody binding reaction: adding the antigen obtained in the step (a) into an immune nano magnetic bead solution, and reacting at 37 ℃ for 0.5 hour at 100 rpm; then, magnetic separation was performed, and after resuspension by adding 1mL of PBS-T, magnetic separation was performed (this step was repeated 3 times).

3. Construction of double antibody Sandwich ELISA

(a) Adding an HRP-2F1 probe: adding 100 mu LHRP-2F1 probe (1: 1000) into the nano magnetic bead-antibody-antigen complex in the step (c) and reacting for 20 minutes at 37 ℃;

(b) color development: washing with PBS-T for 5 times, removing supernatant, adding 100 μ L TMB, and developing in dark for 10 min;

(c) and (4) terminating: finally 50. mu.L of 2M H was added₂SO₄Measuring the absorbance at 450 nm;

4. specificity detection

Respectively capturing a mixed bacterial liquid containing klebsiella pneumoniae, escherichia coli, staphylococcus aureus, salmonella, pseudomonas aeruginosa, shigella and acinetobacter baumannii and a mixed bacterial liquid containing no klebsiella pneumoniae by using immune nano magnetic beads (nano magnetic beads combined with klebsiella pneumoniae specific antibodies), and performing the steps according to the step 3 in the embodiment 3 after the capturing is finished;

as a result: the mixed bacteria (containing Klebsiella pneumoniae) can be detected by the nano magnetic bead detection sensor, and the positive tube of the mixed bacteria is blue; as shown in tube 1 of fig. 17A; however, neither of the mixed bacteria (containing Klebsiella pneumoniae) could be detected, and the corresponding tube was colorless, and the results are shown in

tubes

2, 3,4, 5, 6, and 7 in FIG. 17A; the detection light density curve of the immunomagnetic beads is shown in fig. 17B; the specific results of the nano magnetic bead sensor detected by the PCR method are shown in fig. 18.

4. Sensitivity detection

The bacterial solutions were diluted with mouse blood to a final concentration of 4X 10⁸、4 × 10⁷、4 × 10⁶、4 × 10⁵、4 × 10⁴、4× 10³、4× 10²、4×10¹And 4X 10⁰CFU/mL; sequentially adding 30 mu L of nano magnetic beads into the bacterial liquid, and reacting at 37 ℃ for 0.5 hour at 100 rpm; after magnetic separation, carrying out sensitivity test on the nano magnetic bead detection sensor according to the step 3 in the embodiment 3;

as a result: the detection method of the nanometer magnetic beads can quickly and accurately detect the Klebsiella pneumoniae in the simulated blood sample, and the lower limit of the detection reaches 4 multiplied by 10⁰CFU/mL, visualization results as in FIG. 14A; wherein the

tubes

1, 2, 3,4, 5, 6, 7, 8 and 9 respectively represent samples containing Klebsiella pneumoniae detected by the nano-magnetic bead sensor (the sample bacteria concentration is 4 × 10 in sequence)⁸，4×10⁷，4×10⁶，4×10⁵，4×10⁴，4×10³，4×10²，4×10¹，4 ×10⁰CFU/mL). The sensitivity optical density curve of the nano magnetic bead sensor is shown in fig. 14B. The sensitivity results of the nano magnetic bead sensor detected by the PCR method are shown in FIG. 15; FIG. 16 is a standard curve of the nano magnetic bead sensor for detecting Klebsiella pneumoniae.

Example 4: detection of clinical samples

(1) Immune nano magnetic bead pretreatment

Carrying out vortex oscillation on the immune nano magnetic beads for 1min to fully resuspend the magnetic beads; placing 30 mu L of magnetic bead suspension in a 1.5mLEP tube; 200 μ L of PBS-T was added for washing, magnetic separation was performed (the EP tube was placed on a magnetic frame to adsorb the magnetic beads to the tube wall until the solution was clarified; the operation is described below), the supernatant was discarded, the EP tube was removed from the magnetic separator, washing was repeated once, and finally 200 μ L of PBS-T was added to resuspend the magnetic beads for use.

(2) Antigen capture reaction

Adding the immune nano magnetic beads pretreated in the step (1) into a clinical detection sample containing Klebsiella pneumoniae, gently mixing uniformly, reacting for 0.5h at 37 ℃ at 100 rpm, then performing magnetic separation, adding 1mL of PBS-T for resuspension, performing magnetic separation (repeating the step for 3 times),

(3) adding HRP-2F1 probe

Adding 100 mu L of HRP-2F1 probe (1: 1000) into the immune nano magnetic bead-antigen complex in the step (2) and reacting for 20 minutes at 37 ℃;

(5) color reaction

After magnetic separation, the mixture was washed 5 times with PBS-T, and then 100. mu.L of TMB developing solution was added thereto to conduct reaction at 37 ℃ for 10 minutes.

(6) Termination of the reaction

Add 50. mu.L of stop solution (2M H) to each well₂SO₄) Measuring the absorbance at 450 nm;

(6) nano magnetic bead sensor result analysis

30 positive samples are detected from 40 clinical samples through the detection of the nano magnetic bead sensor, and 10 negative samples are consistent with the traditional physiological and biochemical detection result. The positive detection results are shown in the tubes 1-30 of FIG. 19, and the negative results are shown in the tubes 31-40 of FIG. 19; the results of the detection of the clinical samples by the PCR method are shown in FIG. 20; the traditional bacteria culture identification detection method is utilized to verify that the nano magnetic bead sensor can quickly and accurately detect the Klebsiella pneumoniae, has the characteristics of quickness, sensitivity, high specificity and the like, and is suitable for detecting clinical samples;

the method and the traditional bacterial culture identification of the invention are used for detecting results of clinical samples

。

Sequence listing

<110> university of Kunming science

<120> specific antibody group for detecting klebsiella pneumoniae and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 110

<212> PRT

<213> mouse (Mus musculus)

<400> 1

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Lys Ala Ser Val Ser Trp Thr Ser Ala

20 25 30

Ser Gly Thr Met Leu His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Val Ser Ala Leu Glu Leu Asn Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Arg Ile His Ser

85 90 95

Thr Leu Ile Arg Ser Glu Gly Gly Pro Ser Trp Lys Tyr Leu

100 105 110

<210> 2

<211> 115

<212> PRT

<213> mouse (Mus musculus)

<400> 2

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Tyr Ile His Thr Phe Thr Gly

20 25 30

Tyr Ala Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Pro Gly Ser Ser Ile Asn Tyr Lys Asp Ala Tyr Gln Asn Tyr Gln Ser

50 55 60

Phe Lys Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asp Gly Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 3

<211> 110

<212> PRT

<213> mouse (Mus musculus)

<400> 3

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Gln Ala Asp Thr Ser Ser His Gly Met

20 25 30

Arg Thr Gly Gly Trp Lys Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Ala Lys Gly Leu Glu Ala Val Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His Thr Gln Arg

85 90 95

Ile Leu Ser Arg Ser Glu Gly Gly Pro Ser Trp Lys Tyr Leu

100 105 110

<210> 4

<211> 115

<212> PRT

<213> mouse (Mus musculus)

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ile Ser Asn Ala Tyr

20 25 30

His Phe Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Ala Thr Val Ser Gln Arg Gly Gly Leu Thr Ala Ala Val Leu Thr Thr

50 55 60

Val Leu Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asp Gly Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

Claims

1. A specific antibody panel for detecting klebsiella pneumoniae, characterized in that: comprises monoclonal antibody 1E6, monoclonal antibody 2F1;

the amino acid sequence of the light chain variable region of the monoclonal antibody 1E6 is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2;

2. The use of the specific antibody set for detecting klebsiella pneumoniae of claim 1 in the preparation of a nano magnetic bead sensor detection kit, wherein: and (3) coating the monoclonal antibody 1E6 on the nano magnetic beads to form nano immunomagnetic beads, and detecting the Klebsiella pneumoniae by adopting an immunomagnetic separation technology by taking the monoclonal antibody 2F1 marked with horseradish peroxidase as a second antibody.