CN105242040B - Human Haemophilus influenza quantum dot immunochromatography detection card, preparation method and application thereof - Google Patents

Abstract

The invention provides a human Haemophilus influenzae quantum dot immunochromatographic detection card and its preparation method and application. The detection card includes a bottom plate, a sample pad, a water-absorbing pad, a binding pad and a detection layer; the binding pad is coated with quantum dots Labeled anti-human Haemophilus influenzae nanoprobe; the detection layer is composed of a solid-phase nitrocellulose membrane with a detection line and a quality control line; the detection line is coated with mouse anti-human Haemophilus influenzae P6 protein poly Cloned antibody; the quality control line is coated with anti-rabbit IgG; the detection layer is pasted on the bottom plate; the binding pad and the water-absorbing pad are respectively arranged above the two ends of the detection layer and partially overlapped with the detection layer and pasted on the detection layer and the bottom plate respectively; The pad is arranged above the bonding pad and partially overlapped with the bonding pad and pasted with the bonding pad and the bottom plate respectively. The invention has the advantages of simple and convenient operation, rapid detection, quantification and high sensitivity.

Description

Human haemophilus influenzae quantum dot immunochromatographic detection card and its preparation method and application

technical field

The invention relates to the technical field of medical detection, in particular to a human Haemophilus influenzae quantum dot immunochromatographic detection card and a preparation method and application thereof.

Background technique

Haemophilus influenzae (Haemophilus influenzae, Hi) is an important respiratory pathogenic microorganism that infects humans. The bacterium was discovered by Polish bacteriologist Dr. Feffer in an influenza plague in 1892. been widely studied by researchers. Only humans are known to be the host of the pathogen. The elderly and children with poor immunity are susceptible groups, especially infants and young children under 5 years old. Hi can cause pneumonia, conjunctivitis, otitis media, meningitis and bacteremia, etc., and at least 3 million severe cases occur every year in the world, which can cause disability or even death in children. Hi is divided into six serotypes: a, b, c, d, e, and f with capsules, and nontypeable Haemophilus influenzae (NTHi) without capsules. At one time, Hi with capsule b was the most popular . At present, most of the Hi serological tests carried out in my country only focus on the more aggressive capsular type b. Due to the successful development and application of the capsular polysaccharide vaccine of this type of strain, its prevalence has been effectively controlled. Recent studies have shown that the most common strain in patients infected with Hi is NTHi, and its isolation rate has reached more than 50%.

Clinically, because the infection symptoms caused by Hi are similar to those caused by other respiratory pathogens, it is often difficult to draw conclusions based on clinical manifestations, X-ray examination, etc., and the diagnosis often relies on laboratory diagnosis. Infant diseases are characterized by rapid onset and rapid outcome, so sensitive, rapid and practical Hi detection is of great significance for early and effective clinical intervention.

Despite the global spread of Haemophilus influenzae, there are very few standardized commercial reagents available for laboratory diagnosis. Currently, the diagnostic methods for Hi infection include serological detection, nucleic acid detection and direct detection of pathogens. The commonly used methods for detecting Hi IgG, IgA, and IgM antibodies in serum include: microimmunofluorescence antibody detection (MIF), complement fixation test (CF), recombinant enzyme immunoassay (rEIA), serum complement fixation-enzyme immunoassay test (SeroCF- EIA) etc. However, the detection of Hi antibodies can only indicate that the individual has been infected with Hi, but it cannot reflect whether there are still live Hi bacteria in the body, and the detection of serological specific antibodies often needs to be judged based on the dynamic results of IgM antibodies, which takes a long time . More importantly, the main detection object of serological detection is anti-capsular antibody, and NTHi will cause missed detection because it does not have a capsule. At the same time, these techniques have disadvantages such as low sensitivity, complicated operation steps, professional operation, poor repeatability, long detection time, poor detection specificity, and high cost, so it is difficult to meet the actual clinical needs.

Nucleic acid detection includes nucleic acid hybridization and polymerase chain reaction (PCR). Nucleic acid hybridization detection of Hi has strong specificity but low sensitivity. It is mainly used for the detection and judgment of PCR results, and has not been directly used for the detection of clinical specimens; PCR It has high sensitivity, but the PCR test has special requirements for the laboratory, strict requirements for specimen processing, amplification and detection, and is prone to false positives, so it cannot be used as a commonly used clinical diagnostic method in my country.

The pathogen detection method is mainly the pathogen isolation and cultivation method. The specimens are inoculated on the chocolate blood agar medium added with V and X factors. After 24 hours of cultivation, suspicious colonies are picked and identified morphologically. Hi identification card, Vilek A 60 bacteria automatic analysis system identifies the species, and the system automatically performs biotyping. However, this method has obvious defects such as complex operation steps and long cell culture time, so it is not suitable for clinical application. More importantly, the capsular swelling test and the like are limited to the identification of capsulated strains, while the non-capsulated type (NTHi) is completely missed.

Therefore, it is very necessary to establish a rapid detection method for human Haemophilus influenzae antigen with high sensitivity and high specificity to meet the clinical detection needs.

Contents of the invention

Aiming at the technical bottleneck encountered in the detection methods of several existing Haemophilus influenzae in the background technology, the present invention proposes a human Haemophilus influenzae quantum Spot immunochromatography detection card and its preparation method and application.

The purpose of the present invention is achieved by the following technical means:

A human Haemophilus influenzae quantum dot immunochromatography detection card, characterized in that: the human Haemophilus influenzae quantum dot immunochromatography detection card includes a bottom plate, a sample pad, a binding pad, a detection layer and a water-absorbing pad; The binding pad is coated with anti-human Haemophilus influenzae nanoprobes labeled with quantum dots; the detection layer is composed of a solid-phase nitrocellulose membrane with a detection line and a quality control line; the detection line is coated with There is a mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody; the quality control line is coated with anti-rabbit IgG; the detection layer is pasted on the bottom plate; the binding pad and water-absorbing pad are respectively arranged at both ends of the detection layer above and partly overlapped with the detection layer, and then pasted together with the detection layer and the base plate; the sample pad is arranged above the combination pad and partially overlapped with the combination pad, and then pasted together with the combination pad and the bottom plate respectively.

Preferably, the quantum dots used in the present invention are water-soluble CdSe/ZnS quantum dots modified by carboxylated amphiphilic polymers.

Preferably, the anti-rabbit IgG used in the present invention includes but not limited to goat anti-rabbit IgG.

As preferably, the detection layer used in the present invention is 2cm long, and the detection layer is pasted on the middle section of the bottom surface whose length is 6.6-7.7cm; The water-absorbing pad overlaps 0.2-0.4cm; The detection layer overlaps the binding pad with a length of 0.5-0.8cm pasted on the detection layer and the base plate by 0.2-0.4cm; The length of the 2.5cm sample pad overlaps 0.2-0.4cm; the distance between the detection line and the quality control line is 0.5-0.8cm; the width of the bottom plate is 0.3-0.5cm.

Preferably, the water-absorbing pad used in the present invention is water-absorbing filter paper; the bottom plate is a PVC board.

A preparation method based on the above-mentioned human Haemophilus influenzae quantum dot immunochromatography detection card, characterized in that: the preparation method comprises the following steps:

1) Preparation of binding pad:

1.1) Preparation and purification of recombinant P6-His fusion protein:

1.1.1) Bioinformatics analysis was performed on the surface protein P6 of Haemophilus influenzae to obtain the peptide with the most abundant antigenic epitope in its extracellular domain;

1.1.2) Find the gene coding sequence corresponding to the peptide obtained in step 1.1.1), and introduce restriction sites at the 5' and 3' ends of the sequence and chemically synthesize the entire gene sequence, and mark it as P6; See the sequence listing for the sequence;

1.1.3) The P6 obtained in step 1.1.2) is cloned into the expression vector pET-28a(+) according to molecular biology methods and then transferred into Escherichia coli to express the recombinant P6-His fusion protein; the recombinant P6-His The fusion protein exists in the bacteria in the form of soluble expression;

1.1.4) Purify the recombinant protein obtained in step 1.1.3) with a nickel column. After the purity is detected by SDS-PAGE, the protein concentration is determined by the Bradford method, and the protein concentration is adjusted to 0.2 mg/mL for later use;

1.2) Preparation of rabbit and mouse anti-Haemophilus influenzae P6 protein polyclonal antibody IgG:

1.2.1) Using the recombinant P6-His fusion protein obtained in step 1.1.4) as the complete antigen, immunize New Zealand white rabbits and guinea pigs respectively; prepare rabbit anti-Hemophilus influenzae P6 protein antiserum and mouse anti-human influenza respectively Haemophilus P6 protein antiserum; the indirect ELISA titers of the rabbit anti-human Haemophilus influenzae P6 protein antiserum and the mouse anti-human Haemophilus influenzae P6 protein antiserum are both greater than 1×10 ⁵ ;

1.2.2) Purify the polyclonal antibody IgG in the rabbit anti-Hemophilus influenzae P6 protein antiserum and the mouse anti-Hemophilus influenzae P6 protein antiserum respectively by using Protein G affinity chromatography column;

1.2.3) Measure the concentration of the two polyclonal antibody IgG obtained in step 1.2.2) with the KGI Braford protein content detection kit, and adjust the protein concentration to 3 mg/mL for later use;

1.3) Preparation and purification of quantum dot-labeled anti-Haemophilus influenzae nanoprobes:

1.3.1) Add 2nmol of quantum dots, 300nmol of N-hydroxysuccinimide and 300nmol of carbodiimide to the microcentrifuge tube successively, and make the volume to 2ml with phosphate buffer solution, and set the temperature in a rotary mixer at 15rpm, 37 After reacting at ℃ for 30 minutes, dialyze to remove excess N-hydroxysuccinimide and carbodiimide; the contents of each component in the phosphate buffer are: 2.9g/L disodium hydrogen phosphate, 0.295g/L phosphoric acid Sodium dihydrogen and 2g/L sodium chloride, the pH=7.3 of the phosphate buffer;

1.3.2) Add 4-12nmol of the rabbit anti-Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step 1.2) to the activated quantum dots, react in the dark for 2 hours, and add terminal aminated polyethylene glycol to The final concentration was 1.5%, and the unreacted activated carboxyl sites were blocked, and the reaction was continued in the dark for 1 hour; the antibody aggregates were removed by filtration with a 0.2 μm PES filter, and then the filtrate was transferred to a 50,000 MW ultrafiltration centrifuge tube, at 4°C with a centrifugal force of 8,000g Centrifuge for 15 minutes to remove the antibody that has not undergone coupling reaction and the by-products in the reaction; collect the quantum dot-antibody conjugate solution on the upper layer of the ultrafiltration tube filter, dissolve it in 2ml of phosphate washing solution, and then transfer the solution to In a 50,000MW ultrafiltration centrifuge tube, centrifuge at 4°C for 15 minutes with a centrifugal force of 8,000g, collect the quantum dot-antibody conjugate solution on the upper layer of the ultrafiltration tube membrane, dissolve it in 1ml of phosphate preservation solution, and thus prepare the quantum dot-labeled antibody Human Haemophilus influenzae nanoprobe;

The contents of each component in the phosphate washing liquid are respectively: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml/L Tween-20 and 1g/L Sodium azide; the pH=7.3 of the phosphate washing solution; the contents of each component in the phosphate preservation solution are: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L Sodium chloride, 10g/LBSA and 1g/L sodium azide; pH=7.3 of the phosphate preservation solution;

1.4) Loading of quantum dot-labeled antibody:

Immerse the polyester fiber membrane in the quantum dot-labeled anti-Haemophilus influenzae nanoprobe solution obtained in step 1.3.2) for 1 hour, take it out, dry it at 25°C, and seal it at 4°C for later use, so as to prepare the conjugate pad;

2) Preparation of sample pad:

Take a piece of glass cellulose membrane, soak the glass cellulose membrane in the sample pad treatment solution for at least 3 hours, and then place it in a biological safety cabinet at 37°C to ventilate and dry, then store it in a sealed and dry place at 25°C; thus the sample pad is prepared;

The content of each component in the sample pad treatment liquid is respectively: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 20g/L bovine serum albumin (BSA), 10ml/L Tween-20, 20g/L sucrose and 5g/L polyvinylpyrrolidone, the pH of the sample pad treatment solution=7.3;

3) Preparation of detection layer:

3.1) Adjust the mouse anti-Haemophilus influenzae P6 protein polyclonal antibody prepared in step 1.2) and the anti-rabbit IgG with phosphate buffer to a final concentration of 0.5-2.5 mg/mL for later use; the phosphate buffer The contents of each component are: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, and the pH of the phosphate buffer is 7.3;

3.2) Put the diluted mouse anti-Haemophilus influenzae P6 protein polyclonal antibody into the nozzle of the BIODOT film-drawing instrument, set the amount of 0.8-2.5 μl/cm and spray it on the nitrocellulose membrane to form a detection line; Put the good anti-rabbit IgG into the nozzle of the BIODOT film-drawing instrument, set the amount of 0.8-2.5μl/cm and spray it on the nitrocellulose membrane at an interval of 0.5-0.8cm from the detection line as the quality control line;

3.3) Dry the nitrocellulose membrane sprayed with the test line and the quality control line at 37°C for 2 hours, and store in a sealed and dry place at 4°C; so far the detection layer is prepared;

4) Preparation of the bottom plate

Cut the bottom plate of PVC material according to the actual requirements and reserve it for later use;

5) Preparation of absorbent pad

Cut the water-absorbing filter paper according to the actual requirements for later use;

6) Assembly of human Haemophilus influenzae quantum dot immunochromatography detection card:

6.1) peel off the adhesive protective film on the bottom plate prepared in step 4);

6.2) Paste the detection layer prepared in step 3) to the middle area of the bottom plate, and smooth the film surface;

6.3) Assemble the water-absorbing pad prepared in step 5) on the bottom plate, so that the left side of the water-absorbing pad partially overlaps with the detection layer, and at the same time, align and glue the right edge of the water-absorbing pad to the right edge of the bottom plate and smooth it;

6.4) Overlay the binding pads prepared in step 1) on the left edge of the nitrocellulose membrane in a partially overlapping manner, and stick the binding pads on the bottom plate at the same time;

6.5) The sample pad prepared in step 2) is partially overlapped on the left edge of the bonding pad, and the other side is aligned with the left edge of the base plate, glued to the base plate and smoothed;

6.6) Cut the assembled Haemophilus influenzae quantum dot immunochromatography detection card, and store it in a sealed and dry place at 4°C;

The steps 6.1) to 6.6) are all performed in a biological safety cabinet.

As a preference, 6nmol of the rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step 1.3.2) used in the present invention is added;

As a preference, in step 3.1) used in the present invention, the mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody and anti-rabbit IgG prepared in step 1.2) are adjusted to a final concentration of 1.5-2.0 mg/ mL and 0.5-1.5mg/mL;

As a preference, in the step 3.2) used in the present invention, the diluted mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody is loaded into the nozzle of the BIODOT film-drawing instrument, and the amount of setting 1.0-2.0 μl/cm is sprayed on nitric acid On the cellulose membrane, a detection line is formed; put the diluted anti-rabbit IgG into the nozzle of the BIODOT film scratcher, and set the amount of 1.0-2.0 μl/cm to spray on the nitrocellulose membrane at an interval of 0.5-0.8cm from the detection line as a quality control line.

An application of the above-mentioned human Haemophilus influenzae quantum dot immunochromatographic detection card as a non-diagnostic detection of human Haemophilus influenzae.

A non-diagnostic detection method based on the above-mentioned human Haemophilus influenzae quantum dot immunochromatography detection card, characterized in that: the detection method comprises the following steps:

1) After fully dissolving the sample to be tested with 500 μl of sample treatment solution, take out 120 μL and drop it on the sample pad of the test card, and observe the test result under ultraviolet light after 15 minutes; the contents of each component in the sample treatment solution are respectively phosphoric acid Sodium hydrogen disodium 2.9g/L, sodium dihydrogen phosphate 0.295g/L, Triton x-100 10mL/L and sodium chloride 2g/L, the pH of the sample treatment solution=7.3;

2) If the sample to be tested contains human Haemophilus influenzae antigen, it will combine with the quantum dot-labeled anti-human Haemophilus influenzae nanoprobe in the binding pad, and first bind with the mouse antibody on the nitrocellulose membrane through chromatography. After the human Haemophilus influenzae P6 protein polyclonal antibody is combined, a fluorescent detection line visible to the naked eye will be formed at the detection line under ultraviolet excitation. Form the second fluorescent quality control line visible to the naked eye;

If there is no Haemophilus influenzae antigen in the sample to be tested, only one fluorescent quality control line will appear; if the fluorescent quality control line does not appear, the test card will be invalid.

Preferably, the samples to be tested used in the present invention include but not limited to throat swabs.

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

1. The method for detecting human Haemophilus influenzae antigen of the present invention is to combine immunochromatography and quantum dot labeling technology, and use the high titer and high specificity polyclonal antibody prepared by the present invention to detect the sample by exciting fluorescence , has the characteristics of high sensitivity (the bottom line of its detection of Haemophilus influenzae is 2×10 ⁴ CFU/ml), and its detection results of clinical samples are compared with the current "gold standard" for the detection of this pathogen - the culture method Compared with no statistical difference.

2. The antibodies used in the present invention are all polyclonal antibodies that recognize the extracellular conserved region of the human Haemophilus influenzae specific P6 antigen, which has high specificity and is cheaper to prepare than the most widely used monoclonal antibodies at present. Therefore, the detection cost of the present invention is relatively low.

3. Since the present invention uses the unique outer membrane protein P6 of human Haemophilus influenzae as an antigen target for the first time, this antigen not only exists on the surface of all types of Haemophilus influenzae cells, including capsulated and non-capsulated types , and highly conserved (protein conservation among bacterial strains is 100%), which belongs to the specific antigen of Haemophilus influenzae, so the test card can detect infection of all types of human Haemophilus influenzae with high specificity. However, there is currently no tool on the market that can rapidly detect all types of human Haemophilus influenzae as this test card.

4. The detection method of the present invention is simple, fast and easy to judge, and the result judgment is completed within 20 minutes. It can be detected qualitatively with an ultraviolet detector, or quantitatively detected in combination with technologies such as CCD scanning. The detection cost is low and overcomes the existing Existing technologies (such as ELISA) have the disadvantages of high detection cost, complex and cumbersome operation, long time-consuming, and the need for professionals to operate.

5. Since the test card detects human Haemophilus influenzae antigens rather than antibodies (the emergence of antibodies requires several days to weeks after infection), so this method is useful in the early clinical diagnosis and prevention of human Haemophilus influenzae, pathogen subtypes It has high practical value in identification and epidemiological investigation.

6. The clinical samples used in the detection method of the present invention are respiratory secretions such as sputum, rather than blood, which can avoid the pain of blood testing for infants and the psychological burden of parents, so it is easier to popularize.

Description of drawings

Fig. 1 is the longitudinal sectional structure schematic diagram of detection card provided by the present invention;

Fig. 2 is a schematic structural view of the detection card provided by the present invention after completion of assembly;

in:

1-sample pad; 2-binding pad; 3-detection layer; 4-detection line; 5-quality control line; 6-absorbent pad; 7-bottom plate.

detailed description

The working principle of the present invention is: under the premise of immunochromatographic assay (double antibody sandwich), the present invention is based on polyclonal antibody, adopts quantum dot labeling probe technology, and develops and detects human influenza tropism through quantum dot labeling technology. Quantum dot immunochromatographic detection card for Haemobacter antigen. The first is the preparation, purification and quantum dot labeling of rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody and mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody, followed by spraying the film, and then assembling the components of the detection card , and finally prepare the human Haemophilus influenzae quantum dot immunochromatographic detection card. The detection card provided by the invention has the characteristics of sensitivity, rapidity, good specificity, etc., can be quantitatively detected, can perform high-throughput screening of samples, and has good market application prospects.

As shown in FIG. 1 , the present invention provides a human Haemophilus influenzae quantum dot immunochromatographic detection card, which consists of a sample pad 1 , a binding pad 2 , a detection layer 3 , a water-absorbing pad 6 and a bottom plate 7 . The binding pad 2 is coated with quantum dot-labeled anti-Haemophilus influenzae nanoprobes; the detection layer 3 is a solid-phase nitrocellulose membrane sprayed with a detection line 4 and a quality control line 5, referred to as NC membrane; 4 packs of detection lines Mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody; quality control line 5 coated with anti-rabbit IgG; quantum dots are water-soluble CdSe/ZnS quantum dots modified by carboxylated amphiphilic polymers; water-absorbing pad 6 is made of Absorbent filter paper; base plate 7 is made of PVC.

Its specific structure is: the detection layer is 2cm long, and the detection layer is pasted on the middle section of the bottom surface with a length of 6.6-7.7cm; cm; the detection layer overlaps the 0.5-0.8cm-long binding pad pasted on the detection layer and the bottom plate by 0.2-0.4cm; 0.4cm; the distance between the detection line and the quality control line is 0.5-0.8cm; the width of the bottom plate is 0.3-0.5cm.

Wherein, the preferred scheme of the above-mentioned parameters is: the detection layer 3 is 2 cm long, and is pasted on the middle section of the surface of the base plate 7 with a length of 7.3 cm. The bonding pad 2 is 0.6 cm long and overlaps 0.3 cm; the bonding pad 2 overlaps the 2.5 cm long sample pad (1) pasted on the left end of the bottom plate 7 by 0.3 cm; the distance between the detection line 4 and the quality control line 5 on the detection layer 3 is 0.7 cm. The width of the whole test card is 0.4cm.

The method for preparing the above-mentioned human Haemophilus influenzae quantum dot immunochromatography detection card, its main steps include:

1. Preparation of conjugated pads

(1) Preparation and purification of recombinant P6-His fusion protein:

Bioinformatics analysis was carried out on the human Haemophilus influenzae P6 protein to obtain the peptide with the most abundant antigenic epitope in the extracellular domain of the human Haemophilus influenzae P6 protein, and to find its corresponding gene sequence; 5 of the two gene sequences Restriction sites were introduced into the 'end and 3' end respectively, and the whole gene sequence was chemically synthesized respectively, and marked as P6 at the same time. See the sequence listing for its gene sequence. The gene sequence was cloned into the expression vector pET-28a(+) according to the conventional method, and then the recombinant P6-His fusion protein was expressed. The fusion protein exists in the genetically engineered bacterium in the form of soluble expression. Purify the recombinant P6-His fusion protein in the genetically engineered bacteria with a nickel column, check its purity by SDS-PAGE, then measure the protein concentration by the Bradford method, and adjust the protein concentration to 0.2 mg/mL for later use;

(2) Preparation of rabbit and mouse polyclonal antibody IgG against Haemophilus influenzae P6 protein:

Using the purified recombinant P6-His fusion protein as the complete antigen, New Zealand white rabbits and guinea pigs were immunized according to conventional methods to prepare rabbit anti-H. influenzae P6 protein antiserum and mouse anti-H. influenzae P6 protein antiserum. The indirect ELISA titers of the two antisera were both greater than 1×10 ⁵ , and the polyclonal antibody IgG in the two antisera were purified by Protein G affinity chromatography column, and the antibody concentration was determined by KGI Braford protein content detection kit And adjusted to 3mg/mL for later use. Among them, the rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG is used as the quantum dot labeling test; the mouse anti-human Haemophilus influenzae influenzae P6 protein polyclonal antibody IgG is used as the coating of the detection line.

(3) Preparation and purification of quantum dot-labeled anti-Haemophilus influenzae nanoprobes

The operation steps are as follows: 2nmol quantum dots, 300nmol N-hydroxysuccinimide (sulfo-NHS) and 300nmol carbodiimide (EDC) were sequentially added to the microcentrifuge tube, and the phosphate buffer solution (2.9g/L phosphoric acid Disodium hydrogen disodium, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, pH 7.3) was fixed to a volume of 2ml, reacted at 15rpm at 37°C for 30min in a rotary mixer, and dialyzed to remove excess activator ( sulfo-NHS and EDC). In the activated quantum dots, add 4-12nmol (preferably 6nmol) of the rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step (2), react in the dark for 2 hours, and add terminal aminated polyethylene glycol Alcohol (PEG2000-NH ₂ ) was added to a final concentration of 1.5%, to block unreacted activated carboxyl sites, and to continue the reaction in the dark for 1 h. Use a 0.2 μm PES filter to remove antibody aggregates, then transfer the filtrate to a 50,000 MW ultrafiltration centrifuge tube, and centrifuge at 4°C for 15 min at 8,000 g to remove antibodies that have not undergone coupling reactions and by-products in the reaction. Collect the quantum dot-antibody conjugate solution on the upper layer of the filter membrane of the ultrafiltration tube, dissolve it in 2ml phosphate washing solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml /L Tween-20, 1g/L sodium azide, pH 7.3), then transfer this solution to a 50000MW ultrafiltration centrifuge tube, centrifuge at 4°C for 15min with a centrifugal force of 8000g, and collect the supernatant of the ultrafiltration tube membrane Dot-antibody conjugate solution, dissolved in 1ml phosphate preservation solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L BSA, 1g/L dihydrogen phosphate Nitrogen sodium, pH 7.3), prepared quantum dot-labeled anti-human Haemophilus influenzae nanoprobes.

(4) Loading of quantum dot-labeled antibody

Immerse the polyester fiber membrane in the quantum dot-labeled anti-Haemophilus influenzae nanoprobe solution obtained in step (3) for 1 hour, take it out, dry it at 25°C, cut it into a size of 4cm*0.6cm, and store it sealed at 4°C for later use , so far the bonded pad is prepared.

2. Preparation of sample pad

Take a piece of glass cellulose membrane, put it in the sample pad treatment solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 20g/L bovine serum albumin (BSA ), 10ml/L Tween-20, 20g/L sucrose, 5g/L polyvinylpyrrolidone (PVP-10), pH 7.3) soaked for at least 3 hours, then placed in a biological safety cabinet at 37 ℃ ventilated and dried, cut into 4cm*2.5cm, sealed and dry at 25°C. At this point, the sample pad is prepared. It has been proved by experiments that the release rate of the quantum dot-labeled antibody is significantly improved after the glass cellulose membrane is treated by this method.

3. Preparation of detection layer

The preparation of the detection layer is to form the detection line and the control line on the nitrocellulose membrane by using the mouse anti-Haemophilus influenzae P6 protein polyclonal antibody IgG and the anti-rabbit IgG prepared in step 1 respectively with a special spray film machine. ; Its concrete preparation method comprises the steps:

The mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step 1 and the anti-rabbit IgG were washed with phosphate buffer saline (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L Sodium chloride, pH 7.3) were adjusted to a final concentration of 0.5-2.5mg/mL, wherein the preferred dilution final concentration of mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG was 1.5-2.0mg/mL, anti-rabbit The preferred final dilution concentration of IgG is 0.5-1.5 mg/mL. Put the diluted mouse anti-Haemophilus influenzae P6 protein polyclonal antibody IgG into the nozzle of the BIODOT filming instrument, set the amount of 0.8-2.5μl/cm, preferably 1.0-2.0μl/cm, and spray it on the nitrocellulose membrane above, to form a detection line; put the diluted anti-rabbit IgG into the nozzle of the BIODOT filming instrument, set the amount of 0.8-2.5μl/cm, preferably 1.0-2.0μl/cm, and spray it on the nitrocellulose membrane as a quality control line, the distance between it and the detection line is 0.7cm. Dry the sprayed nitrocellulose membrane at 37°C for 2 hours, cut it into a size of 4cm*4cm, and store it sealed and dry at 4°C. So far the detection layer is prepared.

Fourth, the processing of the bottom plate

Cut the PVC base plate into a size of 4cm*7.3cm for later use.

5. Preparation of absorbent pad

Cut the water-absorbing filter paper into a size of 4cm*3cm to make a water-absorbing pad for later use.

6. Assembly of test card

The assembly work is performed in a biological safety cabinet. First, the adhesive protective film on the bottom plate described in step 4 is removed, and the detection layer described in step 3 above (that is, the one with 1 quality control line and 1 detection line) is removed. nitrocellulose membrane) to the central area of the base plate, and carefully smooth the membrane surface. Next, assemble the water-absorbing pad described in Step 5 above on the base plate, so that the left side overlaps the detection layer by 0.2 cm, and at the same time, align its right edge with the right edge of the base plate and carefully smooth it. Then overlap the binding pad described in the above step 1 on the left edge of the nitrocellulose membrane by 0.3 cm, and stick it on the bottom plate by 0.3 cm. Finally, overlap the sample pad described in step 2 above on the left edge of the bonding pad by 0.3 cm on one side, and align the other side with the left edge of the base plate, stick it on the base plate and smooth it carefully. The assembled test board was cut into 4.0mm wide test cards under a strip cutter, and stored in a sealed and dry place at 4°C. So far, the human Haemophilus influenzae quantum dot immunochromatographic detection card has been prepared.

The method for using the above-mentioned human Haemophilus influenzae quantum dot immunochromatography detection card, the steps are as follows:

Use 500μl of sample treatment solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 10mL/L Triton x-100, 2g/L sodium chloride , pH 7.3) after being fully dissolved, take out 120 μL and drop it on the sample pad of the test card, and observe the test result under ultraviolet light after 15 minutes. If the sample to be tested contains human Haemophilus influenzae antigen, it will combine with the quantum dot-labeled anti-human Haemophilus influenzae nanoprobe in the binding pad, and first combine with the mouse anti-human influenza on the nitrocellulose membrane through chromatography. After combining with the Haemophilus P6 protein polyclonal antibody, a fluorescent detection line visible to the naked eye will be formed at the detection line under ultraviolet excitation, and the unbound quantum dot-labeled antibody continues to be chromatographically combined with anti-rabbit IgG to form under ultraviolet excitation The second fluorescent quality control line visible to the naked eye; if there is no relevant antigen in the sample to be tested, only one fluorescent quality control line will appear. If the fluorescent quality control line does not appear, the test card is invalid.

The water-soluble CdSe/ZnS quantum dots modified by carboxylated amphiphilic polymers required by the present invention can be purchased from professional companies such as Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.; required PVC material bottom plate, water-absorbing filter paper, nitrocellulose Plain film, polyester fiber film, glass cellulose film, etc. can be purchased from professional companies such as Millipore and Shanghai Jinbiao Biotechnology Co., Ltd. Other conventional instruments, equipment, and biochemical drugs required are commercially available.

The present invention is further specifically described by the following examples.

The source of various materials used or adopted in the present invention and the preparation of related reagents

1. Sample pad treatment solution: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 2g bovine serum albumin (BSA), 1ml Tween-20, 2g sucrose, 0.5g poly Vinylpyrrolidone (PVP-10), dissolved in 90ml of deionized water, adjust the pH to 7.3 with 1mol/L NaOH, and then dilute to 100ml with deionized water.

2. Phosphate washing solution: Weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 0.5ml Tween-20, 0.1g sodium azide, dissolve in 90ml deionized water , adjust the pH to 7.3 with 1mol/L NaOH, and then dilute to 100ml with deionized water.

3. Phosphate preservation solution: Weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin (BSA), 0.1g NaN ₃ , dissolve in 90ml deionized water , adjust the pH to 7.3 with 1mol/L NaOH and then dilute to 100ml with deionized water.

4. Phosphate buffer saline (PBS): Weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, dissolve in 90ml deionized water, adjust the pH to 7.3 with 1mol/L NaOH Make up to 100ml with deionized water.

5. Rabbit anti-Haemophilus influenzae P6 protein polyclonal antibody IgG: self-made for the present invention, diluted with PBS, shaken well, so that the concentration of the polyclonal antibody in the solution is 3 mg/ml.

6. Mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG: self-made for the present invention, diluted with PBS, shaken, so that the concentration of the polyclonal antibody in the solution is 3 mg/ml.

7. Goat anti-rabbit IgG: It is a product of Boster Company, diluted with PBS, shaken well, so that the concentration of polyclonal antibody in the solution is 1mg/ml.

8. Quantum dots: The quantum dots used in the present invention are water-soluble CdSe/ZnS quantum dots modified by carboxylated amphiphilic polymers. The emission wavelength is 565nm. They are purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd. Water soluble quantum dot-565.

9. Glass cellulose membrane: thickness is 0.4 mm, water absorption is 42 mg/cm ² , glass fiber diameter is 0.6-3 μm, has good hydrophilicity, purchased from Shanghai Gold Standard Biotechnology Co., Ltd. (model BT40).

10. Polyester fiber membrane: the thickness is 0.48mm, the water absorption speed is 18s/4cm, it has excellent hydrophilicity, it is used for the preparation of the binding pad, purchased from Shanghai Jinbiao Biotechnology Co., Ltd. (model is DL42).

11. Nitrocellulose membrane: the model is Millipore Corp SHF135, with liner, purchased from Millipore Corp.

12. Water-absorbing filter paper: the thickness is 0.95mm, the water-absorbing speed is 60s/4cm, and the water-absorbing capacity is 700mg/cm ² . It has good water-absorbing properties and is used as a material for making water-absorbing pads. Purchased from Shanghai Gold Standard Biotechnology Co., Ltd. (model CH37K).

13. Bottom plate: It is made of high-whiteness PVC material, and the surface is coated with a single layer of high-polymer pressure-sensitive adhesive SM31. It was purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

14. Human Haemophilus influenzae strain: purchased from American Type Culture Collection (ATCC), No. ATCC53781.

15. The microbial samples used in the present invention were all purchased from the American Type Culture Collection (ATCC).

Below in conjunction with embodiment the technical scheme provided by the present invention is described in detail:

Embodiment 1 (preparation embodiment)

Conjugate pad preparation

(1) Preparation and purification of recombinant P6-His fusion protein

1. Cloning of related genes

Bioinformatics analysis was performed on the surface protein P6 of human Haemophilus influenzae (its accession number in the NCBI protein database is AAA24994), to obtain the most abundant epitope in its extracellular conserved domain, and to find its corresponding DNA code At the same time, the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (the whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd., and the delivery When the artificially synthesized gene fragment was connected to the vector pUC57), it was denoted as P6. The full sequence of its gene is shown in the sequence listing. Specifically, the protein sequence encoded by the P6 gene is 48-153aa of the natural human Haemophilus influenzae surface protein P6 (accession number: AAA24994). The vector pUC57 containing the artificially synthesized DNA fragment was double-digested with NdeI and XhoI, and the target fragment was recovered according to the conventional method for future use. At the same time, the vector pET-28a(+) was double-digested with NdeI and XhoI, and the P6 gene obtained after double-digestion was connected into the pET-28a(+) vector according to the conventional method, and transformed into Escherichia coli TOP10 to construct pET-P6 expression vector. It was confirmed by enzyme digestion and sequence determination that the expression vector was constructed correctly. The vector expresses recombinant P6-His fusion protein.

2. Expression and purification of recombinant P6-His fusion protein

After the identified positive clones were cultured, the plasmids were extracted, and transferred into competent E.coliBL21(DE3)plysS according to conventional techniques. The expression strains were screened by conventional methods. A single colony transformed with pET-P6 capable of expressing foreign proteins was picked and inoculated into 100 mL LB medium, and cultured overnight at 30°C. After taking out the bacterial liquid, inoculate in 100mL LB medium containing 50μg/mL kanamycin at a ratio of 1:100, cultivate at 30°C until OD ₆₀₀ =0.6, add 1mol/L IPTG to a final concentration of 1mmol/L, Shake the bacteria at 30°C to induce the expression of the fusion protein. After 4 hours of induction, the cells were collected by centrifugation at 8000r/min for 10 minutes. The cells were washed 3 times with 20mL phosphate buffer (8g/L NaCL, 0.2g/L KCl, 0.24g/L KH ₂ PO ₄ , 1.44g/L Na ₂ HPO ₄ pH=7.4) and loaded with 10mL Buffer solution (20mM Na ₃ PO ₄ , 0.5M NaCl; 30mM imidazole, pH 7.4) was resuspended and subjected to sonication. The operating conditions were: 50HZ, 200W, ultrasonication for 3S, intermittent for 5S, and 100 operations. After the ultrasonication was completed, the precipitate and supernatant were collected by centrifugation at 12000 g for 15 min, and then detected by electrophoresis. It was found that the recombinant P6-His fusion protein existed in the bacteria in a soluble expression form.

The sonicated supernatant obtained above was filtered with a 0.45 μm filter membrane, and then purified with His Trapaffinity columns (product of GE Healthcare) according to the instructions. The specific method is as follows:

1) Use a 5mL syringe to fill up with distilled water, unscrew the plug of the column, connect the column to the syringe with the provided connector, and wash the column at a flow rate of 1mL/min.

2) Equilibrate with 10mL loading buffer, flow rate 1mL/min.

3) Load the fusion protein with a flow rate of 1 mL/min.

4) Wash the column with 10 mL of loading buffer at a flow rate of 1 mL/min.

5) Use 10mL elution buffer (20mM Na ₃ PO ₄ , 0.5M NaCl, 300mM imidazole, pH7.4), elute at a flow rate of 1mL/min, collect in separate tubes, 1ml in each tube, detect by 12% SDS-PAGE, combine Samples containing the protein of interest in the elution fraction. After the protein concentration was determined by the Bradford kit, the concentration was adjusted to 0.2 mg/mL.

(2) Preparation of rabbit and mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG

1. Preparation of rabbit anti-Haemophilus influenzae P6 protein polyclonal antibody IgG

Use 200 μg (1 mL) of the recombinant P6-His fusion protein purified in step (1) to mix and emulsify with 1 mL of Freund’s complete adjuvant, and then immunize male New Zealand white rabbits (provided by the Hubei Provincial Center for Disease Control and Prevention), and subcutaneously in multiple spots on the back Inject and immunize again after an interval of 7 days. After another 14 days, use 200 μg (1 mL) of the above-mentioned purified recombinant P6-His fusion protein to mix and emulsify with 1 mL of Freund's incomplete adjuvant for booster immunization, and then booster immunization 7 days later. The same method as above was used to boost the immunization again. After 7 days, blood was taken to analyze the antibody titer. If unsatisfactory, one or two booster immunizations can be repeated until the antibody titer is satisfactory (the antibody titer measured by ELISA method is greater than 1×10 ⁵ ). If satisfied, the blood was collected from the heart, the serum was separated, and the polyclonal antibody IgG was purified with Protein G affinity chromatography column (GE healthcare company product) in strict accordance with the operation instructions, and the antibody concentration was determined with the KGI Braford protein content detection kit and buffered with phosphate buffer. solution (8g/L NaCL, 0.2g/L KCl, 0.24g/LKH ₂ PO ₄ , 1.44g/L Na ₂ HPO ₄ pH7.4) was adjusted to 2mg/mL, stored at -20°C for later use, and the rabbit antibody Human Haemophilus influenzae P6 protein polyclonal IgG. Western blot test showed that the polyclonal antibody IgG can specifically recognize the full-length P6 protein of human Haemophilus influenzae.

2. Preparation of mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG

The recombinant P6-His fusion protein purified in step (1) was used as the complete antigen to immunize guinea pigs (provided by the Hubei Provincial Center for Disease Control and Prevention), and 200 μg of antigen per guinea pig was injected subscapularly. For basic immunization, an equal volume of antigen was emulsified with Freund's complete adjuvant, and a booster immunization was carried out every two weeks. For the booster immunization, an equal volume of antigen was emulsified with an equal volume of Freund's incomplete adjuvant, and a total of 4 immunizations were performed. Blood was collected 10 days after the last immunization to analyze the antibody titer. If unsatisfactory, one or two booster immunizations can be repeated until the antibody titer is satisfactory (antibody titer greater than 1×10 ⁵ as determined by ELISA method). If satisfied, kill the guinea pigs and take serum, use Protein G affinity chromatography column (GE healthcare company product), purify polyclonal antibody IgG strictly according to the operation manual, use KGI Braford protein content detection kit to measure antibody concentration and use phosphate buffer saline (8g/LNaCL, 0.2g/L KCl, 0.24g/L KH ₂ PO ₄ , 1.44g/L Na ₂ HPO ₄ pH=7.4) adjusted to 2mg/mL, set aside, so far the mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG. Western blot test showed that this polyclonal antibody IgG can specifically recognize the full-length P6 protein of human Haemophilus influenzae.

(3) Preparation of quantum dot-labeled anti-Haemophilus influenzae nanoprobes

1. Optimization of reaction conditions for rabbit polyclonal antibody IgG against Haemophilus influenzae P6 protein labeled with nano carboxyl quantum dots:

1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, and 9 respectively, and the fluorescence intensity of the labeled product was measured with a full spectrometer, and the influence of different pH values on the coupling reaction was observed, and the quantum dot labeled polyclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.

1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

Set the ratio of quantum dot molar concentration to polyclonal antibody concentration to 1:1, 1:2, 1:3, and 1:4 respectively. After the labeling reaction, use a full spectrometer to measure the fluorescence intensity of the labeled product, and observe the difference between the two. The influence of concentration ratio on the coupling reaction, the optimal molar concentration ratio of quantum dot-labeled rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG reaction was determined as the molar ratio of quantum dots to antibody was 1:3. In this experiment, the optimal concentration ratio was selected to determine the amount of labeling.

1.3. Determination of the best blocking agent for carboxy quantum dot-labeled antibody probes

Use ethanolamine, Tris, PEG2000-NH ₂ or BSA as the blocking agent, carry out the labeling reaction according to the conditions determined in steps 1.1 and 1.2, measure the fluorescence intensity of the labeled product with a full spectrometer, and observe the effects of different blocking agents on the labeling reaction. It was found that PEG2000-NH ₂ was the best blocking agent, which could significantly improve the colloidal stability and immune activity of the labeled complex.

2. Marking process:

Add 2nmol of carboxyl water-soluble quantum dots, 300nmol of N-hydroxysuccinimide (sulfo-NHS) and 300nmol of carbodiimide (EDC) to a microcentrifuge tube in turn, and then add phosphate buffer (2.9g/L dihydrogen phosphate Sodium, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, pH 7.4) was fixed to 2ml, kept mixing the solution, after reacting at 37°C for 30min, dialyzed to remove excess sulfo-NHS and EDC. In the activated quantum dots, add 6 nmol of the rabbit anti-Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step (2), react in the dark for 2 hours, and add single-terminal aminated polyethylene glycol (PEG2000-NH ₂ ) to a final concentration of 1%, block unreacted activated carboxyl sites, and continue to react in the dark for 1 h. Use a 0.2 μm PES filter to remove antibody aggregates, then transfer the filtrate to a 50,000 MW ultrafiltration centrifuge tube, and centrifuge at 4°C for 15 min at 8,000 g to remove antibodies that have not undergone coupling reactions and by-products in the reaction. Collect the quantum dot-antibody conjugate solution on the upper layer of the filter membrane of the ultrafiltration tube, dissolve it in 2ml phosphate washing solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml /L Tween-20, 0.3g/L sodium azide, pH 7.4), then transfer this solution to a 50000MW ultrafiltration centrifuge tube, centrifuge at 4°C for 15min with a centrifugal force of 8000g, and collect the upper layer of the filter membrane of the ultrafiltration tube Quantum dot-antibody conjugate solution, dissolved in 1ml phosphate preservation solution (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L BSA, 0.3g/L L sodium azide, pH 7.4), so far the quantum dot-labeled anti-Haemophilus influenzae nanoprobe was prepared, and stored at 4°C for future use.

(4) Loading of quantum dot-labeled anti-Haemophilus influenzae nanoprobes

Immerse the polyester fiber membrane in the quantum dot-labeled anti-Haemophilus influenzae nanoprobe solution obtained in step (3) for 1 hour, take it out, dry it at 25°C and cut it into a size of 4cm*0.6cm/strip, 4 ℃ sealed and stored for later use, so far to prepare the conjugate pad.

Embodiment 2 (preparation embodiment)

Preparation of sample pads

Prepare sample pad treatment solutions with different formulations, observe the release effect of quantum dot-labeled antibodies, and optimize through multiple orthogonal experiments to obtain the optimal sample pad treatment solution formulation (that is, described in the present invention). Take a piece of glass cellulose membrane, soak it in the sample pad treatment solution for at least 3 hours, then place it in a biological safety cabinet at 37°C, ventilate and dry it, and cut it into a size of 4cm*2.5cm/strip to prepare the sample pad , Store in airtight and dry place at 25°C. Tests have proved that the use of the sample pad greatly improves the release rate of the quantum dot-labeled antibody on the binding pad and achieves a better application effect.

Embodiment 3 (preparation embodiment)

Preparation of detection layer

Cut the nitrocellulose membrane into 4cm*4cm size. The mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG and anti-rabbit IgG prepared in Example 1 were adjusted with phosphate buffer to a final concentration of 2.0 mg/mL and 1.0 mg/mL, respectively. Put the diluted mouse anti-Haemophilus influenzae P6 protein polyclonal antibody IgG into the nozzle of the BIODOT membrane device, set the amount of 1.0μl/cm and spray it on the nitrocellulose membrane to form a detection line; the diluted antibody Put the rabbit IgG into the nozzle of the BIODOT film-drawing instrument, set the amount of 1.0 μl/cm to spray on the nitrocellulose membrane as the quality control line, and the distance between it and the detection line is 0.7cm. Dry the sprayed nitrocellulose membrane at 37°C for 2 hours, cut it into a size of 4cm*4cm, and store it sealed and dry at 4°C. So far the detection layer is prepared.

Embodiment 4 (preparation embodiment)

Assembling the test card

Below in conjunction with accompanying drawing 1 and accompanying drawing 2 the assembly of detection card is described further.

Cut the bottom plate into a size of 4cm*7.3cm and set it aside.

Cut the absorbent filter paper into a size of 4cm*3cm, and use it as an absorbent pad for later use.

The assembly work is performed in a biological safety cabinet. First, the adhesive protective film on the bottom plate 7 is removed, and the detection layer 3 described in Example 3, that is, the nitrocellulose membrane with the quality control line 5 and the detection line 4 is pasted on the bottom plate. 7 The specific area indicated in Figure 1 above, and carefully smooth the film surface. Next, assemble the pre-cut absorbent pad 6 on the base plate 7 so that the left side overlaps with the right end of the detection layer by 0.2 cm, and its right edge is aligned with the right edge of the base plate 7 and carefully smoothed. Then, the bonding pad 2 described in Example 1 is overlapped on the left edge of the detection layer 3 by 0.3 cm, and glued to the bottom plate 7 by 0.3 cm. Finally, the sample pad 1 described in Example 2 overlaps the left edge of the bonding pad 2 by 0.3 cm on one side, and aligns with the left edge of the bottom plate 7 on the other side, sticks it on the bottom plate 7 and smoothes it carefully. The assembled test board was cut into 4.0mm wide test cards under a strip cutter, and stored in a sealed and dry place at 4°C.

Embodiment 5 (application embodiment)

How to use the test card

Obtain the subject's throat swab according to the conventional method, insert it into a soft plastic tube containing 500 μl of phosphate buffered saline (PBST) added with 1% Tritonx-100 (volume percentage), and squeeze the plastic tube wall After fully dissolving the sample on the swab, take out 120 μL and drop it on the sample pad of the detection card, and observe and detect it under the ultraviolet analyzer (model WD-9403A, produced by Beijing Liuyi Instrument Factory, ultraviolet excitation wavelength 365nm) after 15 minutes result. If the throat swab contains human Haemophilus influenzae antigen, it will combine with the quantum dot-labeled anti-human Haemophilus influenzae nanoprobe in the binding pad, and first combine with the mouse anti-human influenza on the nitrocellulose membrane through chromatography. After combining with the Haemophilus P6 protein polyclonal antibody, a fluorescent detection line visible to the naked eye will be formed at the detection line under ultraviolet excitation, and the unbound quantum dot-labeled antibody continues to be chromatographically combined with anti-rabbit IgG to form under ultraviolet excitation The second fluorescent quality control line visible to the naked eye; if there is no relevant antigen in the throat swab to be tested, only one fluorescent quality control line will appear. If the fluorescent quality control line does not appear, the test card is invalid.

Embodiment 6 (application embodiment)

Examples of application effects of the present invention

For the method of using the human Haemophilus influenzae quantum dot immunochromatographic detection card referred to in this example, refer to the operation steps described in Example 5.

1) Specificity test

Common respiratory pathogens such as human respiratory syncytial virus (Long strain, ATCC number VR26), human mycoplasma pneumoniae (ATCC number 15531), human Chlamydia pneumoniae (AR-39 strain, ATCC number 53592), human adenovirus type 3 (GB strain , ATCC number VR-3), human adenovirus type 7 (Gomen strain, ATCC number VR-7), human influenza A virus (H1N1, ATCC number VR-1743), human influenza B virus (ATCC number VR-790 ), Moraxella catarrhalis (ATCC No. 25238), Streptococcus pneumoniae (ATCC No. 700670), etc. instead of Haemophilus influenzae, the PBST dilutions containing these microorganisms were all negative.

2) Sensitivity test

Sensitivity studies were conducted by measuring the dilution of the human Haemophilus influenzae culture, and it was determined that the lower limit of detection of the human Haemophilus influenzae strain (ATCC No. 53781) detected by the test card described in Example 4 was 2×10 ⁴ CFU/ml.

3) Clinical test case

With Haemophilus influenzae detection gold standard-cultivation method as reference, get the throat swab specimen of 88 routine respiratory department respiratory infection patients and detect with the detection card described in embodiment 4, the positive rate of culture method is 20.5% (18/88 ), the test card was 21.6% (19/88), and the coincidence rate of the two methods was 96.6% (85/88). The specific results are shown in Table 1.

Table 1 Test results of clinical specimens

It should be pointed out that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the present invention within the scope of protection.

Claims (2)

1. A preparation method based on human Haemophilus influenzae quantum dot immunochromatography detection card, characterized in that: the preparation method comprises the following steps: 1) Preparation of binding pad: 1.1) Preparation and purification of recombinant P6-His fusion protein: 1.1.1) Bioinformatics analysis was performed on the surface protein P6 of Haemophilus influenzae to obtain the peptide with the most abundant antigenic epitope in its extracellular domain; 1.1.2) Find the gene coding sequence corresponding to the peptide obtained in step 1.1.1), and introduce restriction sites at the 5' end and 3' end of the sequence and chemically synthesize the entire gene sequence, and mark it as P6; The full gene sequence of the P6 is: CATATG TACAACACCGTATATTTTGGTTTTGATAAATACGACATCACCGGTGAATACGTTCAAATCTT Nd AGATGCGCACGCAGCATATTTAAATGCAACGCCAGCTGCTAAAGTATTAGTAGAAGGTAATACTGATGAACGTGGTACACCAGAATACAACATCGCATTAGGACAACGTCGTGCAGATGCAGTTAAAGGTTATTTTAGCAGGTAAAGGTGTTGATGCTGGTAAATTAGGCACAGTATCTTACGGTGAAGAAAAACCTGCAGTATTAGGTCACGTGTGAAGCTGCATATGCTAAACAGTA wxya 1.1.3) The P6 obtained in step 1.1.2) is cloned into the expression vector pET-28a(+) according to molecular biology methods and then transferred into Escherichia coli to express the recombinant P6-His fusion protein; the recombinant P6-His The fusion protein exists in the bacteria in the form of soluble expression; 1.1.4) Purify the recombinant protein obtained in step 1.1.3) with a nickel column. After the purity is detected by SDS-PAGE, the protein concentration is determined by the Bradford method, and the protein concentration is adjusted to 0.2 mg/mL for later use; 1.2) Preparation of rabbit and mouse anti-Haemophilus influenzae P6 protein polyclonal antibody IgG: 1.2.1) Using the recombinant P6-His fusion protein obtained in step 1.1.4) as the complete antigen, immunize New Zealand white rabbits and guinea pigs respectively; prepare rabbit anti-Hemophilus influenzae P6 protein antiserum and mouse anti-human influenza respectively Haemophilus P6 protein antiserum; the indirect ELISA titers of the rabbit anti-human Haemophilus influenzae P6 protein antiserum and the mouse anti-human Haemophilus influenzae P6 protein antiserum are both greater than 1×10 ⁵ ; 1.2.2) Purify the polyclonal antibody IgG in the rabbit anti-Hemophilus influenzae P6 protein antiserum and the mouse anti-Hemophilus influenzae P6 protein antiserum respectively by using Protein G affinity chromatography column; 1.2.3) Measure the concentration of the two polyclonal antibody IgG obtained in step 1.2.2) with the KGI Braford protein content detection kit, and adjust the protein concentration to 3 mg/mL for later use; 1.3) Preparation and purification of quantum dot-labeled anti-Haemophilus influenzae nanoprobes: 1.3.1) Add 2nmol of quantum dots, 300nmol of N-hydroxysuccinimide and 300nmol of carbodiimide to the microcentrifuge tube successively, and make the volume to 2ml with phosphate buffer solution, and set the temperature in a rotary mixer at 15rpm, 37 After reacting at ℃ for 30 minutes, dialyze to remove excess N-hydroxysuccinimide and carbodiimide; the contents of each component in the phosphate buffer are: 2.9g/L disodium hydrogen phosphate, 0.295g/L phosphoric acid Sodium dihydrogen and 2g/L sodium chloride, the pH=7.3 of the phosphate buffer; 1.3.2) Add 4-12nmol of the rabbit anti-Haemophilus influenzae P6 protein polyclonal antibody IgG prepared in step 1.2) to the activated quantum dots, react in the dark for 2 hours, and add terminal aminated polyethylene glycol to The final concentration was 1.5%, and the unreacted activated carboxyl sites were blocked, and the reaction was continued in the dark for 1 hour; the antibody aggregates were removed by filtration with a 0.2 μm PES filter, and then the filtrate was transferred to a 50,000 MW ultrafiltration centrifuge tube, at 4°C with a centrifugal force of 8,000g Centrifuge for 15 minutes to remove the antibody that has not undergone coupling reaction and the by-products in the reaction; collect the quantum dot-antibody conjugate solution on the upper layer of the ultrafiltration tube filter, dissolve it in 2ml of phosphate washing solution, and then transfer the solution to In a 50,000MW ultrafiltration centrifuge tube, centrifuge at 4°C for 15 minutes with a centrifugal force of 8,000g, collect the quantum dot-antibody conjugate solution on the upper layer of the ultrafiltration tube membrane, dissolve it in 1ml of phosphate preservation solution, and thus prepare the quantum dot-labeled antibody Human Haemophilus influenzae nanoprobe; The contents of each component in the phosphate washing liquid are respectively: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml/L Tween-20 and 1g/L Sodium azide; the pH=7.3 of the phosphate washing solution; the contents of each component in the phosphate preservation solution are: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L Sodium chloride, 10g/LBSA and 1g/L sodium azide; pH=7.3 of the phosphate preservation solution; 1.4) Loading of quantum dot-labeled antibody: Immerse the polyester fiber membrane in the quantum dot-labeled anti-Haemophilus influenzae nanoprobe solution obtained in step 1.3.2) for 1 hour, take it out, dry it at 25°C, and seal it at 4°C for later use, so as to prepare the conjugate pad; 2) Preparation of sample pad: Take a piece of glass cellulose membrane, soak the glass cellulose membrane in the sample pad treatment solution for at least 3 hours, and then place it in a biological safety cabinet at 37°C to ventilate and dry, then store it in a sealed and dry place at 25°C; thus the sample pad is prepared; The contents of each component in the sample pad treatment solution are: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 20g/L bovine serum albumin, 10ml/L Tween-20, 20g/L sucrose and 5g/L polyvinylpyrrolidone, the pH of the sample pad treatment solution=7.3; 3) Preparation of detection layer: 3.1) Adjust the mouse anti-Haemophilus influenzae P6 protein polyclonal antibody prepared in step 1.2) and the anti-rabbit IgG with phosphate buffer to a final concentration of 0.5-2.5 mg/mL for later use; the phosphate buffer The contents of each component are: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, and the pH of the phosphate buffer is 7.3; 3.2) Put the diluted mouse anti-Haemophilus influenzae P6 protein polyclonal antibody into the nozzle of the BIODOT film-drawing instrument, set the amount of 0.8-2.5 μl/cm and spray it on the nitrocellulose membrane to form a detection line; Put the good anti-rabbit IgG into the nozzle of the BIODOT film-drawing instrument, set the amount of 0.8-2.5μl/cm and spray it on the nitrocellulose membrane at an interval of 0.5-0.8cm from the detection line as the quality control line; 3.3) Dry the nitrocellulose membrane sprayed with the test line and the quality control line at 37°C for 2 hours, and store in a sealed and dry place at 4°C; so far the detection layer is prepared; 4) Preparation of the bottom plate Cut the bottom plate of PVC material according to the actual requirements and reserve it for later use; 5) Preparation of absorbent pad Cut the water-absorbing filter paper according to the actual requirements for later use; 6) Assembly of human Haemophilus influenzae quantum dot immunochromatography detection card: 6.1) peel off the adhesive protective film on the bottom plate prepared in step 4); 6.2) Paste the detection layer prepared in step 3) to the middle area of the bottom plate, and smooth the film surface; 6.3) Assemble the water-absorbing pad prepared in step 5) on the bottom plate, so that the left side of the water-absorbing pad partially overlaps with the detection layer, and at the same time, align and glue the right edge of the water-absorbing pad to the right edge of the bottom plate and smooth it; 6.4) Overlay the binding pads prepared in step 1) on the left edge of the nitrocellulose membrane in a partially overlapping manner, and stick the binding pads on the bottom plate at the same time; 6.5) The sample pad prepared in step 2) is partially overlapped on the left edge of the bonding pad, and the other side is aligned with the left edge of the base plate, glued to the base plate and smoothed; 6.6) Cut the assembled Haemophilus influenzae quantum dot immunochromatography detection card, and store it in a sealed and dry place at 4°C; The steps 6.1) to 6.6) are all performed in a biological safety cabinet. 2. The method according to claim 1, characterized in that: the rabbit anti-human Haemophilus influenzae P6 protein polyclonal antibody IgG prepared by adding 6nmol of step 1.2) in the step 1.3.2); In the step 3.1), adjust the mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody and the anti-rabbit IgG prepared in step 1.2) to a final concentration of 1.5-2.0mg/mL and 0.5-1.5mg respectively with phosphate buffer /mL; In the step 3.2), put the diluted mouse anti-human Haemophilus influenzae P6 protein polyclonal antibody into the nozzle of the BIODOT filming instrument, set the amount of 1.0-2.0 μl/cm and spray it on the nitrocellulose membrane to form Detection line: put the diluted anti-rabbit IgG into the nozzle of BIODOT film-drawing instrument, set the amount of 1.0-2.0μl/cm and spray it on the nitrocellulose membrane at an interval of 0.5-0.8cm from the detection line as the quality control line.