CN105859884B - Anti-human mycoplasma pneumoniae P30 protein antibody and immunochromatography kit using same - Google Patents
Anti-human mycoplasma pneumoniae P30 protein antibody and immunochromatography kit using same Download PDFInfo
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- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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Abstract
The invention relates to an anti-human mycoplasma pneumoniae P30 protein antibody and an immunochromatography kit for detecting human mycoplasma pneumoniae by applying the antibody, wherein the anti-human mycoplasma pneumoniae P30 protein antibody is an antibody for recognizing a linear epitope consisting of 56-69 amino acids of a human mycoplasma pneumoniae P30 protein, and the GenBank sequence number of the human mycoplasma pneumoniae P30 protein is ABR 09215.1; the amino acid sequence of 56-69 of Mycoplasma pneumoniae P30 protein is AEEDTVQIQGKPIT. The rabbit anti-human mycoplasma pneumoniae P30 protein antibody provided by the invention has the characteristics of good specificity, high purity, high titer and low preparation cost.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to an anti-human mycoplasma pneumoniae P30 protein antibody and an immunochromatography kit for detecting human mycoplasma pneumoniae by using the antibody.
Background
Mycoplasma pneumoniae (m.pneumonia) is the causative agent of mycoplasma pneumoniae in humans. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchopneumonia, and are called primary atypical pneumonia. Mainly through droplet infection, the incubation period is 2-3 weeks, and the incidence rate is the highest for teenagers. The clinical symptoms are general respiratory symptoms such as headache, pharyngalgia, fever, cough and the like, which can occur all the year round, but are mostly in autumn and winter. The incidence rate of Mp infection in children pneumonia pathogen is as high as 10-30%, and in recent years, Mp infection gradually becomes one of the main pathogens of children infected respiratory diseases. The disease is very easy to cause respiratory tract infection such as pharyngitis, tonsillitis and the like, even can cause secondary meningitis, hepatitis, myocarditis and other multi-organ injuries at the same time, and can also cause death of children patients in severe cases.
Since Mp infection is similar to the symptoms of respiratory infections caused by other pathogens, it is difficult to distinguish Mp from respiratory infections caused by other pathogens without making a pathogenic examination. Mp has no cell wall and is not effective by commonly used beta-lactam drugs, so the treatment of infection caused by Mp is completely different from the treatment scheme of other bacterial and viral infections, and therefore, it is necessary to establish a simple, rapid and feasible method capable of early diagnosing mycoplasma pneumoniae infection.
Currently, there are 3 main types of Mp detection methods: the first method is a separation culture method, which is a 'gold standard' for confirming infection, but because the growth cycle of Mp is extremely slow, the culture cycle is long, the method can not carry out rapid diagnosis clinically; the other is a serology method, namely, an enzyme-linked immunosorbent assay, a colloidal gold immunoassay, a micro immunofluorescence assay, an indirect hemagglutination assay and the like are adopted to detect the level of the Mp antibody in the serum of a detected person, so that the existence of Mp infection can be indirectly prompted. However, serological tests can only provide a retrospective diagnosis and sometimes require duplicate sera. In addition, the occurrence time of the antibody is not easy to master, the difference of the Mp-specific antibody exists between children, adolescents and adults, and the glycolipid antigen on the Mp cell membrane has nonspecific cross reaction with other microorganisms and body tissues, so the detection quality of the existing serological method is limited to a certain extent; thirdly, the existence of the Mp DNA is detected by utilizing a molecular biology technology, wherein the most common method is Polymerase Chain Reaction (PCR), the method is rapid, sensitive and specific, and is an important means for researching Mp infection at present, but the PCR has higher requirements on experimental equipment and operation, and is easy to generate false positive, so the method cannot be used as a common clinical diagnosis method in China. Therefore, it is necessary to establish a diagnostic method for Mp-specific antigens. At present, the methods for detecting Mp antigens which are publicly reported mainly include a double-antibody sandwich ELISA method, an indirect immunofluorescence method, a quantum dot labeling immunochromatography method and the like, but none of the methods can carry out bedside detection, and a specific instrument (such as an enzyme labeling instrument, a fluorescence instrument and the like) is required to be used for detection in a specific occasion, so that the method is not convenient and fast enough, has long time and is inconvenient for clinical application.
Therefore, it is necessary to establish a rapid diagnostic method for mycoplasma pneumoniae-specific antigens. The human influenza mycoplasma pneumoniae P30 is an important detection target due to high conservation in protein sequence. Therefore, it is an important work to obtain a high-specificity anti-human mycoplasma pneumoniae P30 protein antibody. Currently, the most reported antibodies against human mycoplasma pneumoniae P30 protein are the corresponding monoclonal and polyclonal antibodies. The monoclonal antibody has the greatest advantage of high specificity, but the preparation method is complicated, the production cost is high, and the application of the monoclonal antibody is limited. The polyclonal antibody is mainly prepared by immunizing animals such as rabbits with P30 protein expressed by genetic engineering. The preparation method is simple and low in cost, but the P30 protein expressed by genetic engineering is of an inclusion body structure and does not have a natural protein structure, and the antibody prepared by using the protein has the defects of low specificity, low titer and the like. Therefore, it is very important to prepare the anti-mycoplasma pneumoniae P30 protein antibody with high specificity and high titer at low cost.
Disclosure of Invention
In view of the technical problems in the prior art, the invention aims to provide an antibody for identifying a linear epitope consisting of amino acids 56 to 69 of Mycoplasma pneumoniae P30 protein and an immunochromatographic kit using the antibody.
An anti-human mycoplasma pneumoniae P30 protein antibody, which is characterized in that: the anti-human mycoplasma pneumoniae P30 protein antibody is an antibody for recognizing a linear epitope consisting of 56-69 amino acids of human mycoplasma pneumoniae P30 protein, and the sequence number of the human mycoplasma pneumoniae P30 protein in GenBank is ABR 09215.1; the 14 amino acid sequences of the 56-69 positions of the protein of the mycoplasma pneumoniae P30 are AEEDTVQIQGKPIT; the 14 amino acid sequence of the 56-69 sites of the protein of the mycoplasma pneumoniae P30 is named as P30 Line; the anti-human mycoplasma pneumoniae P30 protein antibody is AbP30 Line.
An immunochromatography kit formed on the basis of the anti-human mycoplasma pneumoniae P30 protein antibody, which is characterized in that: the kit is an immunochromatography kit based on a quantum dot labeling technology or an immunochromatography kit based on a colloidal gold labeling technology.
Preferably, when the kit provided by the invention is an immunochromatography kit based on a quantum dot labeling technology, the preparation method of the kit is as follows:
1) quantum dot labeled antibody AbP30 Line:
sequentially adding 0.4nmol of carboxyl water-soluble quantum dots and 800nmol of carbodiimide EDC into a microcentrifuge tube, fixing the volume to 1ml by MES buffer solution, mixing the solution, reacting for 5min at 37 ℃, then adding 0.34mg of prepared antibody AbP30Line, reacting for 2h in a dark place, adding single-ended amino polyethylene glycol PEG2000-NH2 until the final concentration is 1% (m/v), sealing unreacted activated carboxyl sites, and continuing the reaction for 1h in the dark place; centrifuging the reacted sample by using an ultrafiltration tube (the cut-off molecular weight is 100k), centrifuging 6500g for 5min to the volume of 200ul, transferring the ultrafiltered sample into a common EP tube, centrifuging to remove agglomeration to obtain an upper clear liquid and a lower precipitate, and centrifuging for 3min under the condition of 10000 g; purifying the supernatant on a separation column Superdex-200, naturally flowing the supernatant into a column, washing with PBS, irradiating the column with ultraviolet light to observe the position of the sample, collecting when the sample begins to flow out from the lower part, and stopping collecting 1 ml; centrifuging and concentrating the purified sample to 200ul with an ultrafiltration tube (molecular weight cut-off of 100k) at 6500g, transferring to a common EP tube, centrifuging to remove agglomeration, wherein the condition for centrifuging the common EP tube is 10000g and 3 min; obtaining supernatant, diluting by 200 times with phosphate preservation solution, and preserving at 4 ℃ for later use; thus preparing a solution containing the quantum dot labeled antibody AbP30 Line;
the MES buffer solution comprises the following components in percentage by weight: 10.66g/L MES and 0.74g/L EDTA, the pH of the MES buffer being 7.4;
the phosphate preservation solution is prepared by weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin BSA and 0.1g NaN3Dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L NaOH, and then fixing the volume to 100ml by using the deionized water;
2) preparation of the bonding pad
Immersing the polyester fiber membrane into the solution containing the quantum dot labeled antibody AbP30Line obtained in the step 1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain a bonding pad;
3) preparation of sample pad
Taking one glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 3h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 2.5cm, and preparing the sample pad for sealed storage at 25 ℃;
the preparation method of the sample pad treatment solution comprises the steps of weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate, 0.2g of sodium chloride, 2g of bovine serum albumin BSA, 1ml of Tween-20, 2g of sucrose and 0.5g of polyvinylpyrrolidone PVP-10, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
4) preparation of detection layer
The invention discloses a rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG in the contents from paragraph [ 0134 ] to paragraph [ 0158 ] in the specification with the name of 'human mycoplasma pneumoniae gold-labeled silver immunochromatography detection kit and a preparation method and application thereof' of application number 201410405275.3.
Cutting the nitrocellulose membrane into 4cm × 4 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein and the goat anti-rabbit IgG prepared by the method to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer solution; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.7 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 2h, cutting into a specification of 4cm multiplied by 4cm, sealing at 4 ℃, drying and storing; thus, preparing a detection layer;
the preparation method of the phosphate buffer solution comprises the following steps: weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate and 0.2g of sodium chloride, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
5) assembling detection card
5.1) cutting the bottom plate into 4cm multiplied by 7.3cm for standby;
5.2) cutting the absorbent filter paper into 4cm multiplied by 3cm to be used as an absorbent pad for standby;
5.3) removing the adhesive protective film on the bottom plate prepared in the step 5.1), sticking the detection layer in the step 4), namely the nitrocellulose membrane with a quality control line and a detection line, to the bottom plate, and leveling the membrane surface;
5.4) assembling the water absorption pad prepared in the step 5.2) on the bottom plate, so that the left side of the water absorption pad is overlapped with the right tail end of the detection layer by 0.2cm, and the right edge of the water absorption pad is aligned with and adhered to and leveled with the right edge of the bottom plate; overlapping the bonding pad in the step 2) at the left edge of the detection layer according to 0.3cm, and adhering the bonding pad on the bottom plate according to 0.3 cm;
5.5) overlapping the sample pad in the step 3) on the left edge of the bonding pad by 0.3cm on one side, aligning the other side with the left edge of the bottom plate, adhering the sample pad on the bottom plate and leveling; cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing and drying at 4 ℃ and storing in dark place.
Preferably, when the kit provided by the invention is an immunochromatographic kit based on a colloidal gold labeling technology, the preparation method of the kit is as follows:
1) colloidal gold labeled antibody AbP30 Line:
1.1) preparation of a 30nm colloidal gold solution:
a siliconized 250ml triangular flask was taken, 99ml of ultrapure water was added, and 1ml of 1% (m/v) HAuCl was added4Adding the solution into a 250ml triangular flask, uniformly mixing with ultrapure water, heating in an oil bath, and stirring until the solution is boiled; rapidly adding 2ml of 1% (m/v) trisodium citrate aqueous solution into a 250ml triangular flask, continuously boiling the solution for 10min, stopping heating when the solution in the 250ml triangular flask is changed from blue to red, naturally cooling the solution in the 250ml triangular flask to room temperature, and then adding ultrapure water into the 250ml triangular flask to supplement to 100 ml;
1.2) colloidal gold labeled antibody AbP30 Line:
1.2.1) taking a siliconized 50ml triangular flask, adding 10ml of the colloidal gold solution prepared in the step 1.1), and adding 240ul of 0.2mol/L K to the colloidal gold solution2CO3Adjusting the pH to 8.5;
1.2.2) adding AbP30Line into the colloidal gold solution under the stirring of an electromagnetic stirrer until the final concentration of the antibody is 10ug/ml, dropwise adding the antibody when adding the antibody, and continuing stirring for 45-60 min after the addition is finished;
1.2.3) after the reaction is finished, adding 5% (m/v) bovine serum albumin BSA to a final concentration of 1% (m/v), stirring for 15-30 minutes, and storing at 4 ℃ for later use;
1.2.4) taking out the marked antibody AbP30Line, then putting the Line into a 50ml centrifuge tube, centrifuging for 5 minutes at 4 ℃ at 2500g to obtain a lower-layer precipitate and a supernatant, discarding the lower-layer precipitate, transferring the supernatant to another 50ml centrifuge tube, centrifuging for 30 minutes at 4 ℃ at 12000g to obtain a lower-layer precipitate and a supernatant, discarding the supernatant, re-suspending the lower-layer precipitate with 10ml of colloidal gold buffer solution, then centrifuging for 30 minutes at 4 ℃ at 12000g again to obtain a lower-layer precipitate and a supernatant again, finally re-suspending the lower-layer precipitate with 3ml of colloidal gold buffer solution, and preserving at 4 ℃ for later use to obtain a solution containing the colloidal gold marked antibody AbP30 Line;
the colloidal gold buffer solution comprises the following components in percentage by weight: 10mM Tris, 1% m/vBSA, 1% v/v Tween-20, 5% m/v sucrose and 3% o/v polyvinylpyrrolidone PVP-10, wherein the pH value of the colloidal gold buffer solution is 10.5;
2) preparation of the bonding pad
Immersing a polyester fiber membrane into the solution containing the colloidal gold labeled antibody AbP30Line obtained in the step 1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain a bonding pad;
3) preparation of sample pad
Taking one glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 2h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 1.5cm, and preparing the sample pad for sealed storage at 25 ℃;
the preparation method of the sample pad treatment solution comprises the steps of weighing 0.242g of Tris, 1g of bovine serum albumin BSA, 1ml of Tween-20, 5g of sucrose and 0.3g of polyvinylpyrrolidone PVP-10, dissolving in 90ml of deionized water, adjusting the pH value to 11 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
4) preparation of detection layer
The invention discloses a rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG in the contents from paragraph [ 0134 ] to paragraph [ 0158 ] in the specification with the name of 'human mycoplasma pneumoniae gold-labeled silver immunochromatography detection kit and a preparation method and application thereof' of application number 201410405275.3.
Cutting the nitrocellulose membrane into 4cm × 2 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein and the goat anti-rabbit IgG prepared by the method to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer solution; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.5 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 18h, cutting into a specification of 4cm multiplied by 2cm, sealing at 4 ℃, drying and storing; thus, preparing a detection layer;
the preparation method of the phosphate buffer solution comprises the following steps: weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate and 0.2g of sodium chloride, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
5) assembling detection card
5.1) cutting the bottom plate into 4cm multiplied by 6cm for later use;
5.2) cutting the absorbent filter paper into 4cm multiplied by 2.5cm to be used as an absorbent pad for standby;
5.3) removing the adhesive protective film on the bottom plate prepared in the step 5.1), sticking the detection layer in the step 4), namely the nitrocellulose membrane with a quality control line and a detection line, to the bottom plate, and leveling the membrane surface;
5.4) assembling the water absorption pad prepared in the step 5.2) on the bottom plate, so that the left side of the water absorption pad is overlapped with the right tail end of the detection layer by 0.2cm, and the right edge of the water absorption pad is aligned with and adhered to and leveled with the right edge of the bottom plate; overlapping the bonding pad in the step 2) at the left edge of the detection layer according to 0.2cm, and adhering the bonding pad on the bottom plate according to 0.4 cm;
5.5) overlapping the sample pad in the step 3) on the left edge of the bonding pad by 0.2cm on one side, aligning the other side with the left edge of the bottom plate, adhering the sample pad on the bottom plate and leveling; cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing and drying at 4 ℃ and storing in dark place.
The invention has the advantages that:
the invention provides an anti-human mycoplasma pneumoniae P30 protein antibody, the anti-human mycoplasma pneumoniae P30 protein antibody recognizes a linear epitope consisting of 14 amino acids at positions 56-69 of a human mycoplasma pneumoniae P30 protein, and the GenBank sequence number of the human mycoplasma pneumoniae P30 protein is ABR 09215.1; the amino acid sequence of 14 amino acids at positions 56-69 of the protein of the mycoplasma pneumoniae P30 is AEEDTVQIQGKPIT; the 14 amino acid sequence of the 56-69 sites of the protein of the mycoplasma pneumoniae P30 is named as P30 Line; the anti-human mycoplasma pneumoniae P30 protein antibody is AbP30 Line. The rabbit anti-human mycoplasma pneumoniae P30 protein antibody prepared based on the single linear epitope of mycoplasma pneumoniae has the characteristics of good specificity, high purity, high titer and low preparation cost, and can be used for producing various detection kits for detecting mycoplasma pneumoniae with high sensitivity based on the principle of antigen-antibody reaction. Meanwhile, two different immunochromatography kits are prepared based on the anti-human mycoplasma pneumoniae P30 protein antibody. Two different immunochromatography kits can quickly and accurately detect the mycoplasma pneumoniae in a biological sample, and both comprise the antibody; the two immunochromatography kits can be used for auxiliary diagnosis of mycoplasma pneumoniae infection, have higher sensitivity and specificity, simultaneously have the advantages of simplicity, rapidness, stability, low manufacturing cost and the like, are suitable for examination of clinical specimens, and are also suitable for epidemiological investigation due to the fact that large-batch rapid examination can be carried out. Therefore, the rabbit anti-human mycoplasma pneumoniae P30 protein antibody and the two immunochromatographic kits have wide application prospects and practical values.
Detailed Description
In order to assist those skilled in the art to further understand the present invention, the following detailed description will provide preferred embodiments.
It is common knowledge in the art that "m/v" in concentration units in the present document, when m is g, v is mL.
Formulation of sources of various materials and associated reagents used or employed in the invention
1. Sample pad treatment solution: 0.242g Tris, 1g Bovine Serum Albumin (BSA), 1ml Tween-20, 5g sucrose and 0.3g polyvinylpyrrolidone (PVP-10) are weighed and dissolved in 90ml deionized water, the pH value is adjusted to 11 by 1mol/L NaOH, and then the volume is adjusted to 100ml by deionized water.
2. Phosphate preservation solution: 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate, 0.2g of sodium chloride, 1g of bovine serum albumin BSA, and 0.1g of NaN were weighed3Dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L NaOH, and then fixing the volume to 100ml by using the deionized water;
3. phosphate Buffered Saline (PBS): 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate and 0.2g of sodium chloride are weighed and dissolved in 90ml of deionized water, the pH value is adjusted to 7.3 by 1mol/L of NaOH, and then the volume is adjusted to 100ml by using the deionized water.
4. Sample treatment solution: 0.0121g of tris (hydroxymethyl) aminomethane, 0.17g of sodium chloride and 0.025g of lysozyme are weighed and dissolved in 90ml of deionized water, the pH is adjusted to 8.0 by hydrochloric acid, and then the volume is adjusted to 100ml by deionized water.
5. Antibody AbP30 Line: for the invention, the antibody is prepared by diluting with PBS and shaking up to make the concentration of the antibody in the solution be 3 mg/ml.
6. Rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG: for the invention, the antibody is prepared by diluting with PBS and shaking up to make the concentration of the antibody in the solution be 3 mg/ml.
7. Goat anti-rabbit IgG: is a product of biological engineering Co., Ltd, and is diluted with PBS and shaken to make the concentration of the polyclonal antibody in the solution 1 mg/ml.
8. Quantum dot: the quantum dots used in the invention are water-soluble CdSe/ZnS quantum dots modified by carboxylated amphiphilic polymers, the emission wavelength of the quantum dots is 565nm, the quantum dots are purchased from Wuhan Jia source quantum dot technology development limited company, and the product name is carboxyl water-soluble quantum dots-565.
9. Glass cellulose membrane: the thickness is 0.4mm, and the water absorption capacity is 42mg/cm2The glass fiber has a diameter of 0.6-3 μm and good hydrophilicity, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number BT 40).
10. Polyester fiber film: has a thickness of 0.48mm and a water absorption rate of 18s/4cm, has excellent hydrophilicity, and is used for the preparation of a conjugate pad, which is available from Shanghai gold-labeled Biotech Co., Ltd (model No. DL 42).
11. Cellulose nitrate membrane: model number Millipore Corp SHF135, with liner plates, was purchased from Millipore corporation.
12. Water-absorbing filter paper: the thickness is 0.95mm, the water absorption speed is 60s/4cm, and the water absorption capacity is 700mg/cm2Has good water absorption and is used as a material for manufacturing the water absorption pad. Purchased from Shanghai gold Biotech, Inc. (model CH 37K).
13. A bottom plate: is made of high-whiteness PVC material, and is coated with a single-layer high-polymer pressure-sensitive adhesive SM31-40, which is purchased from Shanghai gold-labeled Biotech Co.
14. Mycoplasma pneumoniae: purchased from the American Type Culture Collection (ATCC) and numbered ATCC 15531.
15. The microorganism samples used in the present invention were purchased from the American Type Culture Collection (ATCC).
The technical scheme provided by the invention is explained in detail by combining the embodiment as follows:
EXAMPLE 1 preparation of Rabbit anti-human Mycoplasma pneumoniae P30 protein antibody
The preparation method of the rabbit anti-human mycoplasma pneumoniae P30 protein antibody comprises the following steps:
1) after structural biological analysis and related experimental research, 14 amino acids at positions 56-69 of the human mycoplasma pneumoniae P30 protein (GenBank sequence ABR09215.1) are selected as a linear epitope for preparing a rabbit anti-human mycoplasma pneumoniae P30 protein antibody, the amino acid sequence of the segment is AEEDTVQIQGKPIT, and the sequence is named as P30 Line;
2) adding cysteine to the C end of the amino acid sequence P30Line in the step 1), synthesizing and purifying the polypeptide by using an automatic polypeptide synthesizer, and coupling the purified polypeptide with a carrier protein KLH to form a P30Line-KLH composite protein;
3) emulsifying the compound protein synthesized in the step 2), and injecting the emulsified compound protein subcutaneously at multiple points at the abdomen of the rabbit for three times in sequence, wherein the interval is 7-10 days each time;
4) collecting and separating serum containing rabbit anti-human mycoplasma pneumoniae P30 protein antibody after 10-12 days of third injection, and detecting the titer of the rabbit anti-human mycoplasma pneumoniae P30 protein antibody in the serum by ELISA, wherein the titer of the rabbit anti-human mycoplasma pneumoniae P30 protein antibody is 1: more than 60000;
5) coupling the polypeptide synthesized and purified in the step 2) with Sepharose 4B activated by cyanogen bromide to form a polypeptide affinity chromatographic column;
6) adding the serum containing the rabbit anti-human mycoplasma pneumoniae P30 protein antibody obtained in the step 4) into the affinity chromatography column prepared in the step 5), incubating overnight at 4 ℃, and eluting the antibody to obtain the rabbit anti-human mycoplasma pneumoniae P30 protein antibody; the purity of the antibody is over 97 percent through SDS-PAGE identification, and the purified antibody is named as AbP30 Line.
In this embodiment, steps 2) -6) are conventional technologies, and various biotechnology companies can provide programmed technical services. In this embodiment, the implementation of the relevant experimental steps in the above steps is completed by the company of bioscience, jinsley, south beijing.
The term "antibody" as used herein should be construed to encompass any specific binding member having a binding domain with the desired specificity. Thus, this term encompasses antibody fragments, derivatives, humanized antibodies, and functional equivalents and homologs of the antibodies to which it is homologous. Examples of antibodies are immunoglobulin subtypes (e.g., IgG, IgE, IgM, IgD, and IgA) and subtypes subclasses thereof; fragments comprising antigen binding domains such as Fab, scFv, Fv, dAb, Fd and diabodies are also possible.
Example 2 preparation and application of immunochromatography kit based on quantum dot labeling technology
1. Quantum dot labeled antibody AbP30Line
Sequentially adding 0.4nmol of carboxyl water-soluble quantum dots and 800nmol of carbodiimide (EDC) into a microcentrifuge tube, keeping the volume constant to be 1ml by MES buffer solution (10.66g/L MES, 0.74g/L EDTA pH 7.4), continuously mixing the solution, reacting at 37 ℃ for 5min, then adding 0.34mg of the antibody AbP30Line prepared in example 1, reacting for 2h in a dark place, adding single-ended amino polyethylene glycol (PEG2000-NH2) until the final concentration is 1% (m/v), sealing unreacted activated carboxyl sites, and continuing to react for 1h in a dark place. Centrifuging the reacted sample by using an ultrafiltration tube (the cut-off molecular weight is 100k), centrifuging 6500g for 5min to the volume of 200ul, transferring the ultrafiltered sample to a common EP tube, and centrifuging to remove agglomeration (10000g, 3 min). The supernatant was purified by applying it to a separation column (Superdex-200) and allowed to flow into the column naturally, followed by washing with PBS (under natural flow of liquid), irradiating the column with ultraviolet light to observe the position of the sample at any time, starting collection when the sample started to flow out from the bottom, and stopping collection after 1ml was collected. The purified sample is centrifuged and concentrated to 200ul at 6500g by an ultrafiltration tube (molecular weight cut-off 100k), and then transferred to a common EP tube for centrifugation (10000g, 3min) to remove agglomeration. The supernatant was diluted 200 times with phosphate stock solution and stored at 4 ℃ for further use. Thus, a solution containing the quantum dot labeled antibody AbP30Line was prepared.
2. Preparation of the conjugate pad
And (3) immersing the polyester fiber membrane into the solution containing the quantum dot labeled antibody AbP30Line obtained in the step (1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain the bonding pad.
3. Preparation of sample pad
Taking a glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 3h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 2.5cm, and preparing the sample pad for sealed storage at 25 ℃.
4. Preparation of the detection layer
The invention discloses a rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG in the contents from paragraph [ 0134 ] to paragraph [ 0158 ] in the specification with the name of 'human mycoplasma pneumoniae gold-labeled silver immunochromatography detection kit and a preparation method and application thereof' of application number 201410405275.3.
Cutting the nitrocellulose membrane into 4cm × 4 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein and the goat anti-rabbit IgG prepared by the method to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer solution; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.7 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 2h, cutting into a specification of 4cm multiplied by 4cm, sealing at 4 ℃, drying and storing; thus, preparing a detection layer;
5. assembly of test card
Cutting the bottom plate into 4cm × 7.3cm size for use.
Cutting the water absorption filter paper into 4cm × 3cm to obtain water absorption pad.
And (4) assembling the components in a biological safety cabinet, firstly tearing off the adhesive protective film on the bottom plate, sticking the detection layer, namely the nitrocellulose membrane with the quality control line and the detection line, on the bottom plate, and carefully screeding the membrane surface. Next, the pre-cut absorbent pad was assembled to the base plate so that the left side of the pad overlapped the right end of the detection layer by 0.2cm and the right edge of the pad was aligned with and carefully smoothed by the right edge of the base plate. And overlapping the bonding pad in the step 2 at the left edge of the detection layer by 0.3cm, and adhering the bonding pad on the bottom plate 7 by 0.3 cm. Finally, the sample pad described in step 3 was overlaid 0.3cm on one side at the left edge of the conjugate pad, and the other side was aligned with the left edge of the base plate, adhered to the base plate and carefully smoothed. Cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing and drying at 4 ℃ and storing in dark place.
6. Composition of immunochromatography kit based on quantum dot labeling technology
The immunochromatography kit based on the quantum dot labeling technology is composed of the detection card and the sample treatment solution in the step 5.
7. Application method of immunochromatography kit based on quantum dot labeling technology
The pharyngeal swab of a person to be detected is obtained by a conventional method, the pharyngeal swab is inserted into a soft plastic pipe filled with 500 mul of sample treatment fluid, the wall of the plastic pipe is squeezed, 120 mul of the sample on the swab is taken out after being fully dissolved and is dripped on a sample pad of a detection card, and a detection result is observed under an ultraviolet analyzer (model WD-9403A, produced by six instruments factory in Beijing, and the ultraviolet excitation wavelength is 365nm) after 15 minutes. If the pharynx swab contains the mycoplasma pneumoniae antigen, the pharynx swab is combined with the quantum dot labeled antibody AbP30Line in the combination pad, firstly combined with the rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG on the nitrocellulose membrane through chromatography, and then a macroscopic fluorescence detection Line is formed at the detection Line under the excitation of ultraviolet rays, and the quantum dot labeled antibody which is not combined is continuously subjected to chromatography and combined with the goat anti-rabbit IgG, and then a macroscopic second fluorescence quality control Line is formed under the excitation of ultraviolet rays; if no related antigen exists in the throat swab to be detected, only one fluorescence quality control line appears. If the phosphor control line is not present, the test card is disabled.
8. Application effect example of immunochromatography kit based on quantum dot labeling technology
The method for using the immunochromatographic kit based on the quantum dot labeling technology referred to in this example refers to the operation steps described in step 7.
1) Specificity test
The detection is carried out by replacing human mycoplasma pneumoniae with common respiratory pathogens such as legionella pneumophila (ATCC 33152), human parainfluenza virus III (ATCCVR-93), human haemophilus influenzae (ATCC 53781), human chlamydia pneumoniae (AR-39 strain, ATCC No. 53592), human adenovirus type 3 (GB strain, ATCC No. VR-3), human adenovirus type 7 (Gomen strain, ATCC No. VR-7), human influenza A virus (H1N1, ATCC No. VR-1743), human influenza B virus (ATCC No. VR-790), human respiratory syncytial virus (ATCC No. VR26), human streptococcus pneumoniae (ATCC No. 700670) and the like, and the kit detects that phosphate buffer diluents containing the microorganisms are negative.
2) Clinical test example
100 samples of pharyngeal swabs of respiratory tract infected persons in the respiratory department were tested with the kit described in step 6, with the "gold standard" culture method for detection of human mycoplasma pneumoniae as a reference, and the culture method positive rate was 13% (13/100), the kit was 13% (13/100), and the compliance rate of 2 methods was 98% (98/100). Specific results are shown in table 1.
TABLE 1 test results of clinical specimens
Example 3 preparation and application of immunochromatography kit based on colloidal gold labeling technology
1. Colloidal gold labeled antibody AbP30Line
Preparation of a.30nm colloidal gold
A siliconized 250ml triangular flask was taken, 99ml of ultrapure water was added, 1ml of 1% (m/v) HAuCl4 solution was added thereto and mixed well, heated in an oil bath and stirred until boiling. 2ml of a 1% (m/v) aqueous solution of trisodium citrate are added rapidly and the solution is boiled for a further 10min (during which the solution turns from blue to red). The heating was stopped, the solution was allowed to cool to room temperature, and then ultrapure water was added thereto to make up to 100 ml.
b. Colloidal gold labeled antibody AbP30Line
1) Taking a silicified 50ml triangular flask, adding 10ml of the colloidal gold solution prepared in the step a, and adding 240ul of 0.2mol/L K into the gold solution2CO3Adjusting the pH to 8.5;
2) adding AbP30Line into the colloidal gold solution under the stirring of an electromagnetic stirrer until the final concentration of the antibody is 10ug/ml, dropwise adding the antibody when the antibody is added, and continuously stirring for 45-60 min after the addition is finished;
3) after the reaction is finished, 2.5ml of 5% (m/v) Bovine Serum Albumin (BSA) is added to the reaction solution until the final concentration is 1% (m/v), the mixture is stirred for 15-30 minutes, and the mixture is stored at 4 ℃ for later use.
4) Taking the marked antibody AbP Line out, putting the antibody AbP Line into a 50ml centrifuge tube, centrifuging the antibody at 2500g for 5 minutes at 4 ℃ to obtain a lower-layer precipitate and an upper-layer clear liquid, discarding the lower-layer precipitate, transferring the upper-layer clear liquid to another 50ml centrifuge tube, centrifuging the supernatant at 12000g for 30 minutes at 4 ℃ to obtain a lower-layer precipitate and an upper-layer clear liquid, discarding the upper-layer clear liquid, re-suspending the lower-layer precipitate by using 10ml of colloidal gold buffer solution, then, re-suspending the precipitate at 12000g and centrifuging the precipitate at 4 ℃ for 30 minutes to obtain a lower-layer precipitate and an upper-layer clear liquid again, finally re-suspending the lower-layer precipitate by using 3ml of colloidal gold buffer solution, and storing the precipitate at 4 ℃ for;
the colloidal gold buffer solution comprises the following components in percentage by weight: 10mM Tris, 1% (m/v) BSA, 1% (v/v) Tween-20, 5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone PVP-10, wherein the pH of the colloidal gold buffer is 10.5.
2. Preparation of the conjugate pad
And (3) immersing the polyester fiber membrane into the solution containing the colloidal gold labeled antibody AbP30Line obtained in the step (1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain the bonding pad.
3. Preparation of sample pad
Taking a glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 2h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 2.5cm, and preparing the sample pad for sealed storage at 25 ℃.
4. Preparation of the detection layer
The invention discloses a rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG in the contents from paragraph [ 0134 ] to paragraph [ 0158 ] in the specification with the name of 'human mycoplasma pneumoniae gold-labeled silver immunochromatography detection kit and a preparation method and application thereof' of application number 201410405275.3.
Cutting the nitrocellulose membrane into 4cm × 4 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein and the goat anti-rabbit IgG prepared by the method to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer solution; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.7 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 2h, cutting into a specification of 4cm multiplied by 4cm, sealing at 4 ℃, drying and storing; thus, a detection layer was prepared.
5. Assembly of test card
Cutting the bottom plate into 4cm × 6cm size for use.
Cutting the water-absorbing filter paper into 4cm × 2.5cm size to obtain water-absorbing pad.
And (4) assembling the components in a biological safety cabinet, firstly tearing off the adhesive protective film on the bottom plate, sticking the detection layer, namely the nitrocellulose membrane with the quality control line and the detection line, on the bottom plate, and carefully screeding the membrane surface. Next, the pre-cut absorbent pad was assembled to the base plate so that the left side of the pad overlapped the right end of the detection layer by 0.2cm and the right edge of the pad was aligned with and carefully smoothed by the right edge of the base plate. And overlapping the bonding pad in the step 2 at the left edge of the detection layer 3 by 0.2cm, and adhering the bonding pad on the bottom plate by 0.4 cm. Finally, the sample pad described in step 3 was overlaid 0.2cm on one side at the left edge of the conjugate pad, and the other side was aligned with the left edge of the base plate, adhered to the base plate and carefully smoothed. Cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing and drying at 4 ℃ and storing in dark place.
6. Composition of immunochromatography kit based on colloidal gold labeling technology
The immunochromatography kit based on the colloidal gold labeling technology is composed of the detection card and the sample treatment solution in the step 5.
7. Application method of immunochromatography kit based on colloidal gold labeling technology
Obtaining a throat swab of a person to be detected according to a conventional method, inserting the throat swab into a soft plastic pipe filled with 500 mu L of sample treatment liquid, extruding the wall of the plastic pipe to fully dissolve a sample on the swab, carrying out water bath at 50 ℃ for 20min, taking out 120 mu L of the throat swab, dripping the throat swab on a sample pad of a detection card, and observing a detection result by naked eyes after 15 min. If the pharynx swab contains the mycoplasma pneumoniae antigen, the pharynx swab is combined with the colloidal gold labeled antibody AbP30Line in the combination pad, firstly combined with the rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG on the nitrocellulose membrane through chromatography, and then a macroscopic red detection Line is formed at the detection Line, and the unbound colloidal gold labeled antibody is continuously chromatographed and combined with the goat anti-rabbit IgG to form a macroscopic second red quality control Line; if no related antigen exists in the throat swab to be detected, only one red quality control line appears. And if the red quality control line does not appear, the detection card fails.
8. Application effect example of immunochromatography kit based on colloidal gold labeling technology
The immunochromatography kit based on the colloidal gold labeling technique used in this example was used in accordance with the procedure described in step 7.
1) Specificity test
The detection is carried out by replacing the human mycoplasma pneumoniae with common respiratory pathogens such as legionella pneumophila (ATCC 33152), human parainfluenza virus type I (ATCC VR-94), human parainfluenza virus type II (ATCC VR-92), human parainfluenza virus type III (ATCC VR-93), human haemophilus influenzae (ATCC 53781), 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), human respiratory syncytial virus (ATCC number VR26), human streptococcus pneumoniae (ATCC number 700670) and the like, and the phosphate buffer diluent containing the microorganisms is negative by using the kit.
2) Clinical test example
100 samples of pharyngeal swabs of respiratory tract infected persons in the respiratory department were tested with the kit described in step 6, with the "gold standard" culture method for detection of human mycoplasma pneumoniae as a reference, and the culture method positive rate was 13% (13/100), the kit was 12% (12/100), and the compliance rate of 2 methods was 97% (97/100). Specific results are shown in table 1.
TABLE 1 test results of clinical specimens
It should be understood that the above description is only exemplary of the present invention, and is not intended to limit the present invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention should be included in the present invention.
Claims (1)
1. An immunochromatography kit formed on the basis of an anti-human mycoplasma pneumoniae P30 protein antibody, which is characterized in that: the kit is an immunochromatography kit based on a quantum dot labeling technology or an immunochromatography kit based on a colloidal gold labeling technology;
the preparation method of the anti-human mycoplasma pneumoniae P30 protein antibody AbP30Line, AbP30Line is as follows:
1) after structural biological analysis and related experimental research, 14 amino acids at positions 56-69 of the protein of the mycoplasma pneumoniae P30 are selected as linear epitope for preparing a rabbit anti-human mycoplasma pneumoniae P30 protein antibody, the amino acid sequence of the segment is AEEDTVQIQGKPIT, and the sequence is named as P30 Line;
2) adding cysteine to the C end of the amino acid sequence P30Line in the step 1), synthesizing and purifying the polypeptide by using an automatic polypeptide synthesizer, and coupling the purified polypeptide with a carrier protein KLH to form a P30Line-KLH composite protein;
3) emulsifying the compound protein synthesized in the step 2), and injecting the emulsified compound protein subcutaneously at multiple points at the abdomen of the rabbit for three times in sequence, wherein the interval is 7-10 days each time;
4) collecting and separating serum containing rabbit anti-human mycoplasma pneumoniae P30 protein antibody after 10-12 days of third injection, and detecting the titer of the rabbit anti-human mycoplasma pneumoniae P30 protein antibody in the serum by ELISA, wherein the titer of the rabbit anti-human mycoplasma pneumoniae P30 protein antibody is 1: more than 60000;
5) coupling the polypeptide synthesized and purified in the step 2) with Sepharose 4B activated by cyanogen bromide to form a polypeptide affinity chromatographic column;
6) adding the serum containing the rabbit anti-human mycoplasma pneumoniae P30 protein antibody obtained in the step 4) into the affinity chromatography column prepared in the step 5), incubating overnight at 4 ℃, and eluting the antibody to obtain the rabbit anti-human mycoplasma pneumoniae P30 protein antibody; the purity of the antibody is over 97 percent through SDS-PAGE identification, and the purified antibody is named as AbP30 Line;
when the kit is an immunochromatography kit based on a quantum dot labeling technology, the preparation method of the kit is as follows:
1) quantum dot labeled antibody AbP30 Line:
sequentially adding 0.4nmol of carboxyl water-soluble quantum dots and 800nmol of carbodiimide EDC into a microcentrifuge tube, fixing the volume to 1ml by MES buffer solution, mixing the solution, reacting for 5min at 37 ℃, then adding 0.34mg of prepared antibody AbP30Line, reacting for 2h in a dark place, adding single-ended amino polyethylene glycol PEG2000-NH2 until the final concentration is 1% m/v, sealing unreacted activated carboxyl sites, and continuing the reaction for 1h in the dark place; centrifuging the reacted sample by using an ultrafiltration tube, centrifuging 6500g for 5min until the volume is 200ul, transferring the ultrafiltered sample into a common EP tube, centrifuging to remove agglomeration to obtain an upper clear liquid and a lower precipitate, and centrifuging for 3min under the condition of 10000 g; purifying the supernatant on a separation column Superdex-200, naturally flowing the supernatant into a column, washing with PBS, irradiating the column with ultraviolet light to observe the position of the sample, collecting when the sample begins to flow out from the lower part, and stopping collecting 1 ml; centrifuging and concentrating the purified sample to 200ul at 6500g by using an ultrafiltration tube, transferring the sample into a common EP tube, centrifuging to remove aggregates, wherein the conditions for centrifuging the common EP tube are 10000g and 3 min; obtaining supernatant, diluting by 200 times with phosphate preservation solution, and preserving at 4 ℃ for later use; thus preparing a solution containing the quantum dot labeled antibody AbP30 Line;
the MES buffer solution comprises the following components in percentage by weight: 10.66g/L MES and 0.74g/L EDTA, the pH of the MES buffer being 7.4;
the phosphate preservation solution is prepared by weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin BSA and 0.1g NaN3Dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L NaOH, and then fixing the volume to 100ml by using the deionized water;
2) preparation of the bonding pad
Immersing the polyester fiber membrane into the solution containing the quantum dot labeled antibody AbP30Line obtained in the step 1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain a bonding pad;
3) preparation of sample pad
Taking one glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 3h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 2.5cm, and preparing the sample pad for sealed storage at 25 ℃;
the preparation method of the sample pad treatment solution comprises the steps of weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate, 0.2g of sodium chloride, 2g of bovine serum albumin BSA, 1ml of Tween-20, 2g of sucrose and 0.5g of polyvinylpyrrolidone PVP-10, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
4) preparation of detection layer
4.1) preparing rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG;
4.2) cutting the nitrocellulose membrane into 4cm multiplied by 4 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein prepared in the step 4.1) and the goat anti-rabbit IgG to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.7 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 2h, cutting into a specification of 4cm multiplied by 4cm, sealing at 4 ℃, drying and storing; thus, preparing a detection layer;
the preparation method of the phosphate buffer solution comprises the following steps: weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate and 0.2g of sodium chloride, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
5) assembling detection card
5.1) cutting the bottom plate into 4cm multiplied by 7.3cm for standby;
5.2) cutting the absorbent filter paper into 4cm multiplied by 3cm to be used as an absorbent pad for standby;
5.3) removing the adhesive protective film on the bottom plate prepared in the step 5.1), sticking the detection layer in the step 4), namely the nitrocellulose membrane with a quality control line and a detection line, to the bottom plate, and leveling the membrane surface;
5.4) assembling the water absorption pad prepared in the step 5.2) on the bottom plate, so that the left side of the water absorption pad is overlapped with the right tail end of the detection layer by 0.2cm, and the right edge of the water absorption pad is aligned with and adhered to and leveled with the right edge of the bottom plate; overlapping the bonding pad in the step 2) at the left edge of the detection layer according to 0.3cm, and adhering the bonding pad on the bottom plate according to 0.3 cm;
5.5) overlapping the sample pad in the step 3) on the left edge of the bonding pad by 0.3cm on one side, aligning the other side with the left edge of the bottom plate, adhering the sample pad on the bottom plate and leveling; cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing, drying and storing in dark at 4 ℃;
when the kit is an immunochromatography kit based on a colloidal gold labeling technology, the preparation method of the kit is as follows:
1) colloidal gold labeled antibody AbP30 Line:
1.1) preparation of a 30nm colloidal gold solution:
a siliconized 250ml triangular flask was taken, 99ml of ultrapure water was added, and 1ml of 1% (m/v) HAuCl was added4Adding the solution into a 250ml triangular flask, uniformly mixing with ultrapure water, heating in an oil bath, and stirring until the solution is boiled; quickly adding 2ml of 1% m/v trisodium citrate aqueous solution into a 250ml triangular flask, continuously boiling the solution for 10min, stopping heating when the solution in the 250ml triangular flask is changed from blue to red, naturally cooling the solution in the 250ml triangular flask to room temperature, and then adding ultrapure water into the 250ml triangular flask to supplement to 100 ml;
1.2) colloidal gold labeled antibody AbP30 Line:
1.2.1) taking a siliconized 50ml triangular flask, adding 10ml of the colloidal gold solution prepared in the step 1.1), and adding 240ul of 0.2mol/L K to the colloidal gold solution2CO3Adjusting the pHTo 8.5;
1.2.2) adding AbP30Line into the colloidal gold solution under the stirring of an electromagnetic stirrer until the final concentration of the antibody is 10ug/ml, dropwise adding the antibody when adding the antibody, and continuously stirring for 45-60 min after adding;
1.2.3) adding 5% m/v bovine serum albumin BSA after the reaction is finished until the final concentration is 1% m/v, stirring for 15-30 minutes, and storing at 4 ℃ for later use;
1.2.4) taking out the marked antibody AbP30Line, then putting the Line into a 50ml centrifuge tube, centrifuging for 5 minutes at 4 ℃ at 2500g to obtain a lower-layer precipitate and a supernatant, discarding the lower-layer precipitate, transferring the supernatant to another 50ml centrifuge tube, centrifuging for 30 minutes at 4 ℃ at 12000g to obtain a lower-layer precipitate and a supernatant, discarding the supernatant, re-suspending the lower-layer precipitate with 10ml of colloidal gold buffer solution, then centrifuging for 30 minutes at 4 ℃ at 12000g again to obtain a lower-layer precipitate and a supernatant again, finally re-suspending the lower-layer precipitate with 3ml of colloidal gold buffer solution, and preserving at 4 ℃ for later use to obtain a solution containing the colloidal gold marked antibody AbP30 Line;
the colloidal gold buffer solution comprises the following components in percentage by weight: 10mM Tris, 1% m/vBSA, 1% v/v Tween-20, 5% m/v sucrose and 3% o/v polyvinylpyrrolidone PVP-10, wherein the pH value of the colloidal gold buffer solution is 10.5;
2) preparation of the bonding pad
Immersing a polyester fiber membrane into the solution containing the colloidal gold labeled antibody AbP30Line obtained in the step 1) for 1h, taking out, drying at 25 ℃, cutting into strips with the specification of 4cm multiplied by 0.6 cm/strip, and sealing and storing at 4 ℃ for later use to obtain a bonding pad;
3) preparation of sample pad
Taking one glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 2h, placing the sample pad in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad into strips with the specification of 4cm multiplied by 1.5cm, and preparing the sample pad for sealed storage at 25 ℃;
the preparation method of the sample pad treatment solution comprises the steps of weighing 0.242g of Tris, 1g of bovine serum albumin BSA, 1ml of Tween-20, 5g of sucrose and 0.3g of polyvinylpyrrolidone PVP-10, dissolving in 90ml of deionized water, adjusting the pH value to 11 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
4) preparation of detection layer
4.1) preparing rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG;
4.2) cutting the nitrocellulose membrane into 4cm multiplied by 2 cm; adjusting the polyclonal antibody IgG of the rabbit anti-recombinant P1-His fusion protein prepared in the step 4.1) and the goat anti-rabbit IgG to final concentrations of 2.0mg/mL and 1.0mg/mL respectively by using a phosphate buffer; loading the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG into a BIODOT membrane scribing instrument nozzle, and spraying the diluted rabbit anti-recombinant P1-His fusion protein polyclonal antibody IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted anti-rabbit IgG into a sprayer of a BIODOT membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted anti-rabbit IgG on a nitrocellulose membrane as a quality control line, and setting the distance between the quality control line and a detection line to be 0.5 cm; drying the sprayed nitrocellulose membrane at 37 ℃ for 18h, cutting into a specification of 4cm multiplied by 2cm, sealing at 4 ℃, drying and storing; thus, preparing a detection layer;
the preparation method of the phosphate buffer solution comprises the following steps: weighing 0.29g of disodium hydrogen phosphate, 0.0295g of sodium dihydrogen phosphate and 0.2g of sodium chloride, dissolving in 90ml of deionized water, adjusting the pH to 7.3 by using 1mol/L of NaOH, and then fixing the volume to 100ml by using the deionized water;
5) assembling detection card
5.1) cutting the bottom plate into 4cm multiplied by 6cm for later use;
5.2) cutting the absorbent filter paper into 4cm multiplied by 2.5cm to be used as an absorbent pad for standby;
5.3) removing the adhesive protective film on the bottom plate prepared in the step 5.1), sticking the detection layer in the step 4), namely the nitrocellulose membrane with a quality control line and a detection line, to the bottom plate, and leveling the membrane surface;
5.4) assembling the water absorption pad prepared in the step 5.2) on the bottom plate, so that the left side of the water absorption pad is overlapped with the right tail end of the detection layer by 0.2cm, and the right edge of the water absorption pad is aligned with and adhered to and leveled with the right edge of the bottom plate; overlapping the bonding pad in the step 2) at the left edge of the detection layer according to 0.2cm, and adhering the bonding pad on the bottom plate according to 0.4 cm;
5.5) overlapping the sample pad in the step 3) on the left edge of the bonding pad by 0.2cm on one side, aligning the other side with the left edge of the bottom plate, adhering the sample pad on the bottom plate and leveling; cutting the assembled detection plate into detection cards with the width of 4.0mm under a slitter, sealing and drying at 4 ℃ and storing in dark place.
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