CN111575190A

CN111575190A - Magnetic particle conjugate for enriching helicobacter pylori, preparation method, enrichment method and kit

Info

Publication number: CN111575190A
Application number: CN202010399965.8A
Authority: CN
Inventors: 冯宇阳; 何晓奕; 唐彬; 刘文正; 卜姣; 邓铃; 张雨; 解庆华
Original assignee: Chongqing Bolida Medical Technology Co ltd
Current assignee: Chongqing Bolida Medical Technology Co ltd
Priority date: 2020-05-12
Filing date: 2020-05-12
Publication date: 2020-08-25

Abstract

The invention provides a magnetic particle conjugate for enriching helicobacter pylori, a preparation method, an enrichment method and a kit, and relates to the technical field of biology. Meanwhile, the invention effectively improves the enrichment rate and the detection rate of the helicobacter pylori by optimizing the coupling amount of the magnetic beads and the anti-helicobacter pylori antibody, so that the reactivity of the positive sample and the reactivity of the negative sample have higher discrimination.

Description

Magnetic particle conjugate for enriching helicobacter pylori, preparation method, enrichment method and kit

Technical Field

The invention relates to the technical field of biology, in particular to a magnetic particle conjugate for enriching helicobacter pylori, a preparation method, an enrichment method and a kit.

Background

Helicobacter pylori is a gram-negative bacillus, is spiral and gull-shaped under the microscope, mainly inhabits the stomach of a human body, and can grow and reproduce under microaerophilic conditions. The composition is closely related to the occurrence and development of chronic atrophic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer and the like, and therefore, the composition attracts clinical wide attention. The traditional biopsy tissue isolation culture is the gold standard for diagnosing helicobacter pylori, but is low in detection rate due to harsh sampling part and laboratory culture conditions, long in time and the like, is not favorable for quick diagnosis and belongs to invasive examination on an examinee. Immunological detection of antibodies (IgG) in serum or saliva has the disadvantage that the presence of symptomatic infection is not immediately reflected. The positive rate and accuracy of directly detecting the antigen components in the excrement are not high. Metagenomic sequencing has high requirements on equipment and technology, high cost and long time consumption, is not suitable for detection of common laboratories and clinical specimens, and cannot carry out quantitative detection on helicobacter pylori by the methods. Digital PCR assays require specialized equipment and conditions, are expensive, and are not as suitable for detection in common laboratory and clinical specimens. The probe method real-time quantitative fluorescent PCR technology needs to synthesize a fluorescent probe, has higher cost and has certain requirements on the loading capacity of helicobacter pylori in a sample. The loop-mediated isothermal amplification (LAMP) method has high sensitivity, but is easily polluted by aerosol, and the false positive problem is serious because most laboratories in China cannot be strictly partitioned at present.

In conclusion, how to rapidly, accurately and non-invasively identify and quantitatively analyze helicobacter pylori is an urgent problem to be solved by the technical personnel in the field.

Disclosure of Invention

It is a first object of the present invention to provide a magnetic particle conjugate for helicobacter pylori enrichment, which alleviates at least one of the technical problems of the prior art.

The second purpose of the invention is to provide a preparation method of the magnetic particle conjugate for enriching helicobacter pylori.

The third purpose of the invention is to provide a method for enriching helicobacter pylori.

The fourth purpose of the invention is to provide a kit for enriching helicobacter pylori.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides a magnetic particle conjugate for enriching helicobacter pylori, which comprises magnetic beads and an anti-helicobacter pylori antibody coupled with the magnetic beads;

wherein, 90-110 μ g of anti-helicobacter pylori antibody is coupled to each 5mg of magnetic beads.

Further, 100. mu.g of anti-helicobacter pylori antibody was coupled to 5mg of magnetic beads;

preferably, the anti-helicobacter pylori antibody comprises an anti-helicobacter pylori polyclonal antibody.

The invention also provides a preparation method of the magnetic particle conjugate, which comprises the steps of coupling the anti-helicobacter pylori antibody with the activated magnetic beads to obtain the magnetic particle conjugate for enriching the helicobacter pylori;

preferably, the activated magnetic beads comprise activated carboxylated magnetic beads;

preferably, the activation conditions of the carboxylated magnetic beads include: performing magnetic separation and cleaning on every 10mg of carboxylated magnetic beads by using 1ml of MES (methyl ethyl ketone), repeating for 3 times, removing the supernatant, adding an EDC (ethylene diamine tetraacetic acid) solution and an NHS (polyethylene glycol) solution, keeping the carboxylated magnetic beads suspended in a state for activation, and removing the supernatant to obtain activated carboxylated magnetic beads;

preferably, the MES has a concentration of 100mM, pH 5.0;

preferably, the EDC solution has a concentration of 200mM and is added in an amount of 100. mu.l;

preferably, the concentration of the NHS solution is 200mM, and the addition amount is 100 μ l;

preferably, the activation time is 30min and the temperature is 25 ℃;

preferably, the method further comprises a step of washing with PBS after activation, and then activated carboxylated magnetic beads are obtained;

preferably, 1ml of magnetic beads with the concentration of 10mg/ml after ultrasonic dispersion is put into a 1.5ml centrifuge tube and placed on a magnetic separation frame, after solid-liquid separation, the supernatant is discarded, magnetic separation and washing are carried out for 3 times by using 1ml of MES, and the supernatant is removed; then adding 100 μ l EDC solution with concentration of 200mM and 100 μ l NHS solution with concentration of 200mM, vortex mixing to make the magnetic beads fully suspended and kept, activating for 30min at 25 ℃, using magnetic frame to magnetically separate and discard the supernatant, washing 3 times with ice PBS buffer solution, and re-suspending in 200 μ l PBS buffer solution;

preferably, the anti-helicobacter pylori antibody includes an anti-helicobacter pylori polyclonal antibody;

preferably, the preparation method of the anti-helicobacter pylori polyclonal antibody comprises the steps of preparing immunogen, preparing polyclonal antibody and purifying antibody;

preferably, inactivated H.pylori is selected, washed with PBS and adjusted to a final concentration of 10⁷CFU/ml to obtain immunogen;

preferably, the helicobacter pylori is one or more of helicobacter pylori cocci, helicobacter pylori, or helicobacter pylori sinuses;

preferably, helicobacter pylori is inactivated by formaldehyde fixation at 37 ℃;

preferably, the immunogen is used for immunizing rabbits to obtain polyclonal antibodies against helicobacter pylori;

preferably, 2ml of immunogen is injected per rabbit ear rim at a time;

preferably, the number of immunizations is four, preferably the first immunization is 10 days apart from the second immunization, the second immunization is 7 days apart from the third immunization, and the third immunization is 7 days apart from the fourth immunization;

preferably, the purification of the antibody comprises centrifuging the rabbit antiserum, taking the supernatant, and precipitating the supernatant by saturated ammonium sulfate;

further, coupling is carried out by mixing 100. mu.l of the activated magnetic bead solution with 100. mu.l of an anti-helicobacter pylori antibody solution at a concentration of 0.5 to 4mg/ml, wherein 4 to 6mg of the activated magnetic beads are contained in 100. mu.l of the activated magnetic bead solution;

preferably, the concentration of the anti-helicobacter pylori antibody solution is 1 mg/ml;

preferably, 5mg of activated beads are contained per 100. mu.l of activated bead solution.

Further, the coupling time is 1-3h, preferably 2 h;

preferably, the temperature of the coupling is between 25 and 45 ℃, preferably 25 ℃.

Further, the coupling step comprises the step of removing unconjugated anti-helicobacter pylori antibody, and then obtaining the magnetic particle conjugate for enriching helicobacter pylori;

preferably, after removing unconjugated anti-helicobacter pylori antibody, the method further comprises the step of blocking the remaining binding sites on the magnetic particle conjugate, and then obtaining the magnetic particle conjugate for enrichment of helicobacter pylori.

The invention also provides a helicobacter pylori enrichment method, which comprises the step of enriching the helicobacter pylori in a sample by using the magnetic particle conjugate.

Further, 0.4-0.6mg of magnetic particle conjugate is added into each 1ml of sample solution for enriching the helicobacter pylori in the sample;

preferably 0.5mg of magnetic microparticle conjugate per 1ml of sample solution;

preferably, the enrichment temperature is from 25 to 45 ℃, preferably from 25 to 37 ℃, more preferably 37 ℃;

preferably, the enrichment time is 15-60min, preferably 30-45min, more preferably 30 min.

Further, the sample is a stool sample.

In addition, the invention also provides a kit for enriching helicobacter pylori, which comprises the magnetic particle conjugate;

preferably, the kit further comprises one or more of a negative control, a positive control, a PBS buffer or a helicobacter pylori nucleic acid quantification standard.

Compared with the prior art, the invention has the following beneficial effects:

the magnetic particle conjugate for enriching the helicobacter pylori, provided by the invention, couples the magnetic beads with the anti-helicobacter pylori antibody, can effectively and specifically recognize and enrich the helicobacter pylori in a sample, especially in a fecal sample, has high enrichment efficiency, overcomes the influence of other matrix components on an experimental result in the process of directly extracting a fecal sample genome, and compared with the traditional invasive mode of gastroscope sampling, the method for directly extracting the sample from the feces of a detection object belongs to a non-invasive material taking mode, has a simple mode, is less influenced by other factors in the process, has small physical and psychological trauma to the detection object and has high acceptance. Meanwhile, the invention effectively improves the enrichment rate and the detection rate of the helicobacter pylori by optimizing the coupling amount of the magnetic beads and the anti-helicobacter pylori antibody, so that the reactivity of the positive sample and the reactivity of the negative sample have higher discrimination.

The preparation method of the magnetic particle conjugate provided by the invention can be obtained by coupling the anti-helicobacter pylori antibody and the activated magnetic beads, has the advantages of simple preparation method and low cost, and is suitable for large-scale production and application.

The enrichment method of helicobacter pylori provided by the invention comprises the step of enriching the helicobacter pylori in a sample by using the magnetic particle conjugate provided by the invention, the enrichment time is short, generally 15-30 minutes, the method is not influenced by the material-taking part and the culture condition, and the helicobacter pylori in a feces sample also has a high-efficiency enrichment effect, so that the traumatic sampling of an examinee is effectively avoided. In addition, the enrichment method has no special requirements on equipment and technology, can effectively save cost, and is suitable for detection of common laboratories and clinical specimens.

Based on the inventive concept of the magnetic particle conjugate for enriching helicobacter pylori provided by the invention, the invention also provides a kit for enriching helicobacter pylori, which comprises the magnetic particle conjugate provided by the invention. The kit also has the beneficial effects of high enrichment rate of positive samples and high detection precision, can quickly diagnose the infection dosage of an infected person in clinical application by combining the existing detection technology, and provides powerful reference for clinical diagnosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of magnetic beads dispersed by ultrasound under a 1000-fold mirror according to an embodiment of the present invention;

FIG. 2 is a graph showing the results of gram staining of a conjugate after capturing helicobacter pylori in a sample under a 1000-fold microscope, provided in an example of the present invention;

FIG. 3 is a graph showing the results of the 400-fold microscope after the conjugate of the polyclonal antibody and the magnetic bead is incubated with helicobacter pylori and then a fluorescent secondary antibody is added;

FIG. 4 is a graph showing the results of the addition of a fluorescent secondary antibody under a 400-fold microscope after the direct incubation of the polyclonal antibody with H.pylori provided in the example of the present invention;

FIG. 5 is a graph showing the results of the incubation of H.pylori with a fluorescent secondary antibody directly under a 400-fold microscope, according to the example of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to one aspect of the present invention, there is provided a magnetic particle conjugate for enrichment of helicobacter pylori, comprising a magnetic bead, and an anti-helicobacter pylori antibody coupled to the magnetic bead; 90-110. mu.g of anti-H.pylori antibody was conjugated to each 5mg of magnetic beads.

The magnetic particle conjugate for enriching the helicobacter pylori, provided by the invention, couples the magnetic beads with the anti-helicobacter pylori antibody, can effectively and specifically recognize and enrich the helicobacter pylori in a sample, especially a fecal sample, has high enrichment efficiency, and overcomes the influence of other matrix components on an experimental result in the process of directly extracting a fecal sample genome. Meanwhile, the invention effectively improves the enrichment rate and the detection rate of the helicobacter pylori by optimizing the coupling amount of the magnetic beads and the anti-helicobacter pylori antibody, so that the reactivity of the positive sample and the reactivity of the negative sample have higher discrimination.

Wherein, the amount of coupling anti-H.pylori antibody per 5mg magnetic bead can be, but is not limited to, 90. mu.g, 95. mu.g, 100. mu.g, 105. mu.g or 110. mu.g, and when 100. mu.g anti-H.pylori antibody is coupled per 5mg magnetic bead, the enrichment efficiency of the magnetic particle conjugate for H.pylori is higher.

In some preferred embodiments, the magnetic beads include carboxylated magnetic beads, the surfaces of the carboxylated magnetic beads are modified by carboxyl groups and have hydrophilicity, and the magnetic beads can be used for immunoassay, separation and purification, immunoprecipitation and the like, and have a wide application range. Preferably comprising carboxylated magnetic beads having a diameter of 3 μm. The method is not limited to the source of the carboxylated magnetic beads, and the conventional commercially available carboxylated magnetic beads can be used for preparing the magnetic particle conjugate provided by the invention, preferably JSR IVD&Life Science ResearchMagnosphere^TMMS300/Carboxyl magnetic beads produced.

In some preferred embodiments, the anti-H.pylori antibody comprises an anti-H.pylori polyclonal antibody. The source of the polyclonal antibody against helicobacter pylori in the present embodiment is not limited, and the polyclonal antibody may be a conventional commercial polyclonal antibody or a self-made polyclonal antibody in a laboratory. Optionally, the rabbit is immunized with the particle antigen after the helicobacter pylori inactivation to obtain the serum of the anti-helicobacter pylori polyclonal antibody, and the anti-helicobacter pylori polyclonal antibody is obtained after the purification by saturated ammonium sulfate. Wherein, the helicobacter pylori is optionally the deposit number: helicobacter pylori (Helicobacter pylori) of ATCC 700392. The specificity of the magnetic particle conjugate provided by the invention can be further increased by coupling an anti-helicobacter pylori polyclonal antibody.

preferably, the activation conditions of the carboxylated magnetic beads include: performing magnetic separation and cleaning on every 10mg of carboxylated magnetic beads by using 1ml of MES, repeating the steps for 3 times, removing the supernatant, adding 100 mu l of EDC solution and 100 mu l of NHS solution, keeping the carboxylated magnetic beads suspended in a state, activating, and removing the supernatant to obtain activated carboxylated magnetic beads;

preferably, the MES has a concentration of 100mM, pH 5.0;

preferably, the activation time is 30min and the temperature is 25 ℃;

preferably, 1ml of magnetic beads with the concentration of 10mg/ml after ultrasonic dispersion is put into a 1.5ml centrifuge tube and placed on a magnetic separation frame, after solid-liquid separation, the supernatant is discarded, magnetic separation and washing are carried out for 3 times by using 1ml of MES, and the supernatant is removed; then adding 100 μ l EDC solution with concentration of 200mM and 100 μ l NHS solution with concentration of 200mM, vortex mixing to make magnetic beads fully suspended and maintained, activating at 25 deg.C for 30min, using magnetic frame to magnetically separate and discard supernatant, washing 3 times with ice PBS buffer solution, and resuspending in 200ul PBS buffer solution;

preferably, 2ml of immunogen is injected per rabbit ear rim at a time;

preferably, the purification of the antibody comprises centrifugation of the rabbit antiserum followed by supernatant precipitation with saturated ammonium sulfate.

The preparation method of the magnetic particle conjugate provided by the invention is simple to operate, low in cost and suitable for large-scale production and application.

In some preferred embodiments, 100. mu.l of the activated magnetic bead solution containing 4 to 6mg of activated magnetic beads per 100. mu.l of the activated magnetic bead solution is mixed with 100. mu.l of an anti-H.pylori antibody solution at a concentration of 0.5 to 4mg/ml for coupling. By limiting the use amount of the coupled magnetic beads and the anti-helicobacter pylori antibody, on one hand, the utilization rate of raw materials can be higher, the cost is effectively saved, and on the other hand, the enrichment efficiency of the prepared magnetic particle conjugate on helicobacter pylori can also be higher.

Wherein, the concentration of the anti-helicobacter pylori antibody solution may be, for example, but not limited to, 0.5mg/ml, 0.8mg/ml, 1mg/ml, 2mg/ml, 3mg/ml or 4 mg/ml; the amount of activated beads per 100. mu.l of activated bead solution may be, for example, but not limited to, 4mg, 5mg, or 6 mg. When the concentration of the anti-helicobacter pylori antibody solution is 1mg/ml and 5mg of activated magnetic beads are contained in each 100. mu.l of the activated magnetic bead solution, the utilization rate of the raw material is higher.

In some preferred embodiments, the coupling time is 1 to 3 hours, for example, and can be, but is not limited to, 1 hour, 2 hours, or 3 hours; the temperature of the coupling is 25-45 deg.C, for example, but not limited to, 25 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 35 deg.C, 37 deg.C, 40 deg.C or 45 deg.C. The coupling reaction conditions are further adjusted and optimized, so that the coupling efficiency is higher. When the coupling time is 2h and the coupling temperature is 25 ℃, the efficiency of the coupling reaction is higher.

In some preferred embodiments, the coupling step further comprises removing unconjugated anti-H.pylori antibody, and then obtaining the magnetic particle conjugate for H.pylori enrichment. Optionally, the unconjugated anti-H.pylori antibody is removed by washing with PBS. The unconjugated anti-helicobacter pylori antibody is removed, so that the prepared magnetic particle conjugate has higher enrichment efficiency on helicobacter pylori in the subsequent use process, and ineffective combination is avoided.

Preferably, after removing unconjugated anti-helicobacter pylori antibody, the method further comprises the step of blocking the remaining binding sites on the magnetic particle conjugate, and then obtaining the magnetic particle conjugate for enrichment of helicobacter pylori. Alternatively, the remaining binding sites were blocked using PBST (pH7.3) buffer containing 1% BSA. So as to improve the accuracy in the subsequent use process.

The enrichment method of helicobacter pylori provided by the invention has the advantages that the enrichment time is short, generally 15-30 minutes, the enrichment method is not influenced by the material taking part and the culture condition, and the enrichment method also has a high-efficiency enrichment effect on the helicobacter pylori in the excrement sample, so that the wound sampling of an examinee is effectively avoided. In addition, the enrichment method has no special requirements on equipment and technology, can effectively save cost, and is suitable for detection of common laboratories and clinical specimens.

Wherein, the sample can be a biological sample for detecting helicobacter pylori clinically and conventionally, such as gastroscope drawing, feces or urine and the like.

In some preferred embodiments, 0.4-0.6mg of magnetic microparticle conjugate is added to 1ml of sample solution for enrichment of H.pylori in the sample; wherein the amount of the magnetic particle conjugate added per 1ml of the sample solution may be 0.4mg, 0.5mg or 0.6 mg. Preferably 0.5mg of magnetic microparticle conjugate is added to each 1ml of sample solution. By limiting the addition of the magnetic particle conjugate, the utilization rate of the magnetic particle conjugate is higher on the basis of ensuring the enrichment effect in the enrichment reaction, and the cost is effectively saved.

Preferably, the enrichment temperature is 25-45 ℃, for example, but not limited to, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃ or 45 ℃. The enrichment time is 15-60min, such as, but not limited to, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60 min. The enrichment efficiency can be higher by further adjusting and optimizing the conditions of the enrichment reaction. When the enrichment temperature is 37 ℃ and the enrichment time is 30min, the efficiency of the enrichment reaction is higher.

In some preferred embodiments, the sample is a stool sample.

The magnetic particle conjugate provided by the invention can effectively enrich helicobacter pylori in a stool sample, and compared with the separation culture after traditional gastroscope biopsy, the magnetic particle conjugate can be used for extracting the helicobacter pylori on the gastric mucosa, and directly taking the stool of a detected person belongs to non-invasive detection, so that the welfare of a patient can be effectively improved.

Optionally, after the magnetic particle conjugate provided by the invention is used for enriching helicobacter pylori in a sample, a real-time fluorescence quantitative PCR method can be used for detection, so that the infection dose of an infected person can be rapidly diagnosed, the result can be obtained only by taking 5 hours, and the magnetic particle conjugate has the advantages of rapidness and sensitivity.

In addition, the invention also provides a kit for enriching the helicobacter pylori, which comprises the magnetic particle conjugate.

The kit for enriching the helicobacter pylori also has the beneficial effects of high enrichment rate of positive samples and high detection precision based on the inventive concept of the magnetic particle conjugate for enriching the helicobacter pylori, and can quickly diagnose the infection dose of an infected person in clinical application by combining the existing detection technology, thereby providing powerful reference for clinical diagnosis.

Optionally, the magnetic particle conjugate is dissolved in PBS buffer solution, and the molar concentration of the PBS buffer solution is 0.05 mol/L; the concentration of the magnetic particle conjugate in the buffer solution is 50 mug/ul; the negative control product is PBS buffer solution, wherein the molar concentration of the PBS buffer solution is 0.05 mol/L; the positive control is helicobacter pylori positive control, and the bacterium is helicobacter pylori ATCC 700392; the helicobacter pylori nucleic acid quantitative Standard is a positive plasmid quantified by an international Standard Human DNA quantification Standard.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagent information used in the examples of the present invention is as follows:

helicobacter pylori thallus: purchased from ATCC with accession numbers: ATCC 700392.

Immunomagnetic bead microspheres: purchased from JSR IVD&Life Science Research Magnosphere^TMMS300/Carboxyl was produced with a diameter of 3.0. mu.m.

A dialysis bag: MWCO 14K W34 mm.

PBS buffer: 0.05mol/L, pH7.3, 0.22 μm for membrane preparation.

Saturated ammonium sulfate solution: 780g of ammonium sulfate is added into 1L of water at 25 ℃, the mixture is heated by a microwave oven until the mixture is dissolved, then the mixture is placed overnight, crystals are separated out after the overnight, and the supernatant is a saturated ammonium sulfate solution.

Magnetic beads: magnosphere MS300/CarboxyI magnetic microspheres with a diameter of 3.0 μm were selected.

MES buffer: 100mM MES, pH 5.0.

EDC solution: 200mM, using the ice bath MES buffer as dispersant.

Sulfo-NHS solution: 200mM, using the ice bath MES buffer as dispersant.

Blocking solution PBST: 0.05mol/L PBS buffer, pH7.3, 0.05% Tween20, 1% BSA.

EXAMPLE 1 preparation and purification of anti-helicobacter pylori antibody

1. Preparation of anti-helicobacter pylori polyclonal antibody

The deposited ATCC700392 helicobacter pylori was taken out at-80 ℃ and plated in Skirrow's solid medium containing 5% O₂Culturing at 37 deg.C for 2-3 days in microaerophilic culture box, transferring into Skirow liquid at a ratio of 5% after transparent coniform bacteria growIn the culture medium, the following components are respectively: 1) 5% O₂Culturing the strain in a microaerophilic incubator at 37 ℃ for 36 hours to grow a conventional colony morphology; 2) culturing at 37 deg.C for 36 hr under normal atmosphere to allow the pellet to deform under aerobic stress. Centrifuging at 3500rpm/min for 5min, discarding supernatant, washing the bacterial pellet with sterile PBS for 3 times, and resuspending the bacterial concentration to 10 with PBS⁷cfu/ml, adding formaldehyde with final concentration of 0.4%, inactivating at 37 deg.C for 24 hr, washing the bacterial pellet with sterile PBS for 3 times, and resuspending the bacterial concentration to 10 with PBS⁷The cfu/ml is stored at 4 ℃, 100 μ l of Skirrow's solid medium is taken for sterility test, and after the growth of the germ-free colony on the plate, the two inactivated bacteria solutions can be mixed together to be used as antigen. The particulate antigen prepared above was injected into the auricular vein of rabbit in 3 times, each time 2ml, with 2 weeks interval between the first and second immunizations and 1 week interval between the second and third immunizations. Collecting blood 2 days before each immunization, measuring serum titer by indirect ELISA method until the serum titer reaches 10⁶In the above steps, blood is collected from heart, and the obtained product is placed at 37 deg.C for 1 hr, then at 4 deg.C for 5-7 hr, the upper precipitated serum is sucked out, centrifuged at 1500rpm/min at 4 deg.C for 10min, and the supernatant is collected to obtain rabbit polyclonal antibody against helicobacter pylori, and stored at-80 deg.C.

2. Purification of antibodies

Centrifuging rabbit antiserum 20000g at 4 deg.C for 30min to obtain supernatant, removing cell debris, and collecting supernatant. The supernatant was mixed with PBS (volume ratio 2:1), added with SAS (saturated ammonium sulfate) of the same volume after mixing, and the protein was precipitated thoroughly overnight in a refrigerator at 4 ℃. Centrifuging the protein solution at 10000g for 30min at 4 ℃, discarding the supernatant, leaving the precipitate, dissolving the precipitate in a small amount of PBS (2-3ml), adding into a dialysis bag (MWCO 14K W34 mm), sealing with dialysis clamps, dialyzing in sufficient amount of PBS (making the dialysis bag completely immersed in the PBS), placing at 4 ℃, replacing PBS every 6-8h to completely remove ammonium sulfate for 24-48 h. Removing the solution from the bag after dialysis, centrifuging at 12000g and 4 ℃ for 30min, discarding the precipitate, subpackaging 1 ml/branch of supernatant, detecting the protein concentration by BCA, detecting the purity by SDS-PAGE electrophoresis, and storing at-80 ℃ for later use.

Example 2 activation of Immunocarboxylated magnetic beads

Taking 1ml of magnetic beads (10mg/ml), fully and uniformly mixing by ultrasonic dispersion, taking 1ml to a 1.5ml centrifuge tube, placing on a magnetic separation frame, discarding supernatant after solid-liquid separation, performing magnetic separation and washing for 3 times by using 1ml of MES (100mM, pH5.0, 0.05% Tween), and removing the supernatant. Freshly prepared 100. mu.l EDC solution (200mM) and 100. mu.l NHS solution (200mM) were added rapidly, vortexed to fully suspend the beads, activated at 25 ℃ for 30 minutes, during which the beads were kept in suspension, magnetically separated with a magnetic frame and discarded the supernatant, washed 3 times with ice PBS buffer, and resuspended in 200ul PBS buffer.

Example 3 conjugation of immunomagnetic beads to anti-helicobacter pylori antibodies

Taking 200 mu L PBS buffer solution to resuspend and preserve activated magnetic beads, adding 200 mu L (1mg/ml) of purified anti-helicobacter pylori polyclonal antibody, gently mixing uniformly, vertically mixing for 2 hours at 25 ℃ (the process leads the magnetic beads to be in a suspension state), using a magnetic frame for solid-liquid separation, then discarding supernatant, using 0.5ml ice bath PBS buffer solution for at least 3 times to remove unconjugated polyclonal antibody, using 1ml PBST buffer solution containing 1% BSA to seal the residual binding sites of the conjugate of the polyclonal antibody, whirling for 30s, placing the centrifuge tube in a mixer for sealing reaction for 2 hours at 25 ℃, placing the centrifuge tube on the magnetic frame after the reaction is finished, removing supernatant after solid-liquid separation, then adding 0.5ml PBS buffer solution, magnetically separating, repeating the step for 3 times, adding 200 mu L PBS buffer solution for resuspension to obtain the magnetic particle conjugate for enriching the helicobacter pylori, the mixture was stored at 4 ℃.

Example 4 Observation of magnetic beads

1) Observation under mirror of magnetic beads before capture

After 30 minutes of ultrasonic dispersion of the purchased magnetic beads, a small amount of the beads was applied to a clean slide glass, and the dispersion was observed under 1000-fold magnification, as shown in FIG. 1.

2) Magnetic particle conjugate enriched bacteria for enrichment of helicobacter pylori under microscope observation

The prepared magnetic particle conjugate was used to enrich H.pylori in the sample, after magnetic separation, washed 3 times with sterile PBS buffer, finally resuspended with 500. mu.l PBS, 50. mu.l was spread on a clean glass slide, gram-stained, and the capture was observed under a biological microscope, the results are shown in FIG. 2.

Example 5 specific binding of multiple antibodies to H.pylori bacteria

To a concentration of 10⁵26695 (of helicobacter pylori standard strain) bacteria solution (cfu/ml) 100 mul is dripped on a glass slide, natural air drying is carried out on a super clean bench, 100 mul of anti-Hp rabbit polyclonal antibody (PBS is diluted by 1: 500) is dripped, PBST is washed for 3 times at 37 ℃ for 1 hour (wet box and dark), 100 mul of FITC labeled goat anti-rabbit secondary antibody (diluted by 1: 1000) is dripped after natural drying, PBST is washed for 3 times at 37 ℃ for 1 hour (wet box and dark), a cover glass is covered, and observation is carried out under a fluorescence microscope, so that the results are shown in fig. 3, fig. 4 and fig. 5, and the result graph can show that the anti-helicobacter pylori polyclonal antibody prepared by the invention has better specific binding with helicobacter pylori thallus.

Example 6 optimization of conditions for coupling of polyclonal antibodies to carboxylated magnetic beads

1) Optimization of antibody concentration

5mg (100. mu.l) of activated carboxylated magnetic beads are respectively reacted with 100. mu.l of antibody series concentration gradient of 4mg/ml, 2mg/ml, 1mg/ml and 0.5mg/ml to form a reaction volume of 200. mu.l, the reaction volume is vertically and uniformly mixed for 2 hours at 25 ℃, and the amount of unconjugated antibody remaining in the supernatant is detected by using a BCA kit to determine the optimal antibody use concentration. As can be seen from the results in Table 1, when the amount of magnetic beads was 5mg and the antibody concentrations were 1mg/ml and 0.5mg/ml, the coupling rates reached 86% and 87%, respectively; when the antibody concentration is 4mg/ml and 2mg/ml, the coupling rate can reach 51% (102.45 μ g) and 26% (104.5 μ g), respectively, so that the maximum antibody loading of 5mg magnetic beads can be calculated to be about 100 μ g, and in summary analysis, when the volume of the activated 5mg magnetic beads and the coupled antibody is taken as 100 μ L, the optimal antibody concentration is 1 mg/ml.

TABLE 1

2) Optimization of coupling time

5mg (100. mu.l) of activated carboxylated magnetic beads and 100. mu.l of antibody (2mg/ml) are coupled for 0.5h, 1h, 2h and 3h respectively at 25 ℃, the magnetic beads are vertically and uniformly mixed during the coupling, the magnetic beads are kept in a suspension state, and the BCA kit is used for detecting the amount of the remaining unconjugated antibody in the supernatant so as to determine the optimal coupling time. As is clear from the results in Table 2, when the amount of magnetic beads was 5mg, the amount of antibody was 200. mu.g, and the temperature was 25 ℃, the coupling rate was the highest between 2h and 3h, and the analysis-by-synthesis antibody was a biologically active substance, and 2h was used as the optimum coupling time.

TABLE 2

3) Optimization of coupling temperature

5mg (100. mu.l) of activated carboxylated magnetic beads and 100. mu.l of antibody (2mg/ml) were coupled at 4 ℃, 25 ℃, 37 ℃ and 45 ℃ for 2 hours, respectively, and were mixed vertically and uniformly, so that the magnetic beads were kept in a suspended state, and the amount of the remaining unconjugated antibody in the supernatant was measured by using the BCA kit to determine the optimal coupling temperature. As is clear from the results in Table 3, when the amount of magnetic beads was 5mg, the amount of antibody was 200. mu.g, and the coupling time was 2 hours, the coupling rates were highest at 25 ℃ and 37 ℃, and the assay-integrated antibody was a biologically active substance, with 25 ℃ being the optimum coupling temperature.

TABLE 3

Example 7 Condition optimization of magnetic particle conjugate enriched bacteria for enrichment of helicobacter pylori

1) Confirmation of optimal amount of magnetic microparticle conjugate

Adding 25 μ l, 50 μ l, 100 μ l, 200 μ l, 400 μ l of magnetic particle conjugate (5 mg/ml) to 1ml of 10mg/ml⁶CFU/ml and 10³Reacting in bacterial solution of CFU/ml at 37 deg.C for 30min, placing on magnetic frame for 2min, discarding supernatantAnd extracting the bacterial genome enriched by the magnetic particle conjugate, and quantitatively detecting the bacterial genome by using a Real-time PCR method to determine the optimal working concentration of the magnetic particle conjugate. As is clear from the results in Table 4, when the number of the enriched target bacteria was 10, respectively⁶CFU and 10³In CFU, the change of the antibody concentration of 25 μ L from the CT value of more than 25 μ L is not obvious, so that the maximum number of bacteria captured by the 25 μ L magnetic particle conjugate is more than 10⁶CFU, but in order to ensure the best possible enrichment of the bacteria of interest in the sample, the optimal amount of magnetic particle conjugate to be used is 100. mu.L for the integrated assay.

TABLE 4

2) Optimal reaction temperature of magnetic particle conjugate

100. mu.l of magnetic microparticle conjugate (5 mg/ml) was added to 1ml of 10⁶CFU/ml and 10³And (3) respectively acting the bacterial strains in CFU/ml bacterial liquid at the temperature of 4 ℃, 25 ℃, 37 ℃ and 45 ℃ for 30min, placing the bacterial strains on a magnetic frame for 2min, removing supernatant, extracting bacterial genomes enriched on the magnetic particle conjugates, and quantitatively detecting the bacterial genomes by using a Real-time PCR method to determine the optimal reaction temperature of the magnetic particle conjugates. As can be seen from the results in Table 5, when the number of the enriched target bacteria was 10³In CFU, the CT value is minimum when the enrichment temperature is 25 ℃, and when the number of the enriched target bacteria is 10⁶CFU, enrichment temperature is 25 ℃ and 45 ℃, CT value is minimum, difference is not obvious, but considering that the magnetic particle conjugate is a bioactive substance, in order to ensure the purpose bacteria enriched in the sample as much as possible, comprehensive analysis, the optimal reaction temperature of the magnetic particle conjugate is 37 ℃.

TABLE 5

3) Optimal reaction time for magnetic microparticle conjugates

100. mu.l of magnetic microparticle conjugate (5 mg/ml) was added to 1ml of 10⁶CFU/ml and 10³And (3) reacting the bacterial strain solution of CFU/ml at 37 ℃ for 15min, 30min, 45min and 60min, placing the bacterial strain solution on a magnetic frame for 2min, removing the supernatant, extracting bacterial genomes enriched on the magnetic particle conjugate, and quantitatively detecting the bacterial genomes by using a Real-time PCR method to determine the optimal reaction temperature of the magnetic particle conjugate. As can be seen from the results in Table 6, when the number of the enriched target bacteria was 10³In CFU, the difference of CT values is not obvious when different enrichment times are used, but when the number of the enriched target bacteria is 10⁶In CFU, when the enrichment time is 30min and 45min, the CT value is minimum, and is respectively 25.65 and 24.73, but the difference is not obvious, but considering that the magnetic particle conjugate is a bioactive substance which cannot be placed at 37 ℃ for a long time, in order to ensure that the target bacteria enriched in the sample as far as possible can be obtained, the comprehensive analysis is carried out, and the optimal reaction time of the magnetic particle conjugate is 30 min.

TABLE 6

EXAMPLE 8 sensitivity of enrichment of magnetic particle conjugates for isolating bacteria in samples

1) Sensitivity in enrichment isolation of pure bacterial samples

Adding 100 μ l of magnetic microparticle conjugate (5 mg/ml) into 1ml of 10-concentration solution¹CFU/ml、10²CFU/ml、10³CFU/ml、10⁴CFU/ml、10⁵CFU/ml、 10⁶CFU/ml、10⁷CFU/ml、10⁸And (3) reacting the CFU/ml at 37 ℃ for 30min, placing the CFU/ml on a magnetic frame for 2min, discarding the supernatant, extracting the bacterial genome enriched on the magnetic particle conjugate, and quantitatively detecting the bacterial genome by using a Real-time PCR method to determine the sensitivity in the enriched and separated bacterial sample. As can be seen from the results in Table 7, when the CT value detected is less than 35.04, the sample is determined to be positive, and when the sample is pure bacteria, the minimum bacteria number that can be enriched by 100. mu.l of the magnetic particle conjugate is 10²CFU。

TABLE 7

Enrichment of pure bacterial sample number (sensitivity)	CT value
		10¹	35.33
10²	31.91
		10³	29.17
10⁴	25.26
		10⁵	22.25
10⁶	19.09
		10⁷	14.71
10⁸	12.15

2) Sensitivity for enriching and separating bacteria in fecal sample

100 μ l of magnetic microparticle conjugate (5 mg/ml) was added to a volume of 1ml of bacteria 10¹CFU/ml、10²CFU/ml、10³CFU/ml、10⁴CFU/ml、10⁵CFU/ml、10⁶CFU/ml、 10⁷CFU/ml、10⁸CFU/ml, adding 200mg of pretreated negative feces into each sample, mixing uniformly, acting at 37 ℃ for 30min, placing on a magnetic frame for 2min, removing supernatant, and extracting bacterial genome enriched on the magnetic particle conjugate; 200mg of pretreated negative feces are added to another group of samples, and then 1ml of bacteria with a concentration of 10 is added¹CFU/ml、10²CFU/ml、 10³CFU/ml、10⁴CFU/ml、10⁵CFU/ml、10⁶CFU/ml、10⁷CFU/ml、10⁸CFU/ml, mixing at 37 deg.C for 30min, directly extracting genome with fecal genome kit, and quantitatively detecting with Real-time PCR method to determine sensitivity of bacteria in enriched and separated fecal sample. As can be seen from the results in Table 8, when the CT value detected was less than 35.04, the sample was judged to be positive, and when the sample was a mixture of bacteria and feces, the minimum bacteria amount enriched by 100. mu.l of the conjugate was 10²CFU, whereas the minimum number of genomic bacteria extracted directly from the fecal sample was 10³CFU, it can be seen that the sensitivity of the method is higher than that of extracting bacterial genome directly from fecal sample. To further validate the lowest detection line of the method, 20 aliquots of 1ml bacterial concentration 10 were prepared²And adding 200mg of pretreated negative excrement into each CFU/ml, uniformly mixing, acting for 30min at 37 ℃, placing the mixture on a magnetic frame for 2min, removing supernatant, extracting bacterial genomes enriched on the magnetic particle conjugates, and quantitatively detecting the bacterial genomes by using a Real-time PCR method, wherein the result is shown in table 9, the repetition is carried out for 20 times, and the experimental detection rate is 100%.

TABLE 8

Enrichment of bacterial count in fecal samples	Direct CT value extraction	CT value extraction after enrichment
			10¹	38.39	36.42
10²	35.16	33.71
			10³	31.94	29.67
10⁴	28.15	25.86
			10⁵	25.81	22.47
10⁶	21.22	18.90
			10⁷	17.25	15.73
10⁸	14.94	13.68

TABLE 9

Number of repetitions	CT mean value	Number of repetitions	CT mean value
				1	34.05	11	34.11
2	33.19	12	33.92
				3	33.74	13	33.98
4	33.56	14	33.45
				5	33.91	15	33.71
6	34.01	16	34.06
				7	34.15	17	34.23
8	33.62	18	34.27
				9	33.43	19	33.95
10	33.75	20	33.72

Example 9 enrichment of magnetic particulate conjugate for specificity of bacteria in an isolated sample

Adding 100 μ l of magnetic microparticle conjugate (5 mg/ml) into 1ml of 10-concentration solution¹CFU/ml、10²CFU/ml、10³CFU/ml、10⁴CFU/ml、10⁵CFU/ml、10⁶CFU/ml、 10⁷CFU/ml、10⁸CFU/ml, adding 200mg pretreated negative feces and 500 μ l common gastrointestinal bacteria (100 μ l each of Escherichia coli, Pseudomonas aeruginosa, Bacillus dysenteriae, Salmonella typhi, and Bacillus proteus) into each sample, mixing, acting at 37 deg.C for 30min, placing on magnetic frame for 2min, discarding supernatant, and extracting bacteria gene enriched on magnetic particle conjugateAnd (3) performing quantitative detection on the obtained sample by using a Real-time PCR method to determine the specificity in the enrichment and separation of the pure fecal sample. As can be seen from the results in Table 10, the number of the objective bacteria in the mixed gastrointestinal tract bacteria was 10²In CFU, the CT value is 34.19 and is more than 35.04, the test result is positive, the test result accords with the lowest test line in the embodiment 8, the test result still can reach the lowest test line after the mixed sample is enriched, and the method has good specificity.

Watch 10

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A magnetic particle conjugate for enrichment of helicobacter pylori, which comprises magnetic beads and an anti-helicobacter pylori antibody coupled with the magnetic beads;

2. The magnetic particle conjugate of claim 1, wherein 100 μ g of anti-helicobacter pylori antibody is coupled per 5mg of magnetic beads; preferably, the magnetic beads comprise carboxylated magnetic beads;

3. The method for preparing a magnetic particle conjugate according to claim 1 or 2, comprising coupling an anti-helicobacter pylori antibody to activated magnetic beads to obtain the magnetic particle conjugate for helicobacter pylori enrichment;

preferably, the activation conditions of the carboxylated magnetic beads include: performing magnetic separation and cleaning on every 10mg of carboxylated magnetic beads by using 1ml of MES, repeating for 3 times, removing the supernatant, adding an EDC solution and an NHS solution, keeping the carboxylated magnetic beads suspended in a state for activation, and removing the supernatant to obtain activated carboxylated magnetic beads;

preferably, the MES has a concentration of 100mM, pH 5.0;

preferably, the activation time is 30min and the temperature is 25 ℃;

preferably, 2ml of immunogen is injected per rabbit ear rim at a time;

4. The method according to claim 3, wherein the coupling is carried out by mixing 100. mu.l of the activated magnetic bead solution containing 4 to 6mg of activated magnetic beads per 100. mu.l of the activated magnetic bead solution with 100. mu.l of an anti-helicobacter pylori antibody solution at a concentration of 0.5 to 4 mg/ml;

5. The method of claim 3, wherein the coupling time is 1 to 3 hours, preferably 2 hours;

6. The production method according to any one of claims 3 to 5, further comprising a step of removing unconjugated anti-helicobacter pylori antibody after conjugation, and then obtaining the magnetic microparticle conjugate for enrichment of helicobacter pylori;

7. A method for enrichment of helicobacter pylori, comprising enriching helicobacter pylori in a sample using the magnetic particle conjugate according to claim 1 or 2.

8. The enrichment method according to claim 7, wherein 0.4-0.6mg of the magnetic microparticle conjugate is added to every 1ml of the sample solution for enriching helicobacter pylori in the sample;

9. The enrichment method according to claim 7 or 8, wherein the sample is a stool sample.

10. A kit for the enrichment of helicobacter pylori, comprising the magnetic particle conjugate according to claim 1 or 2;