CN105259345A - Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method - Google Patents
Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method Download PDFInfo
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- CN105259345A CN105259345A CN201510801825.8A CN201510801825A CN105259345A CN 105259345 A CN105259345 A CN 105259345A CN 201510801825 A CN201510801825 A CN 201510801825A CN 105259345 A CN105259345 A CN 105259345A
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Abstract
The invention provides a colloidal gold test strip for detecting an IgM antibody. The IgM antibody is a specific IgM antibody for nine respiratory tract infection pathogens, and the colloidal gold test strip comprises a sample pad, a conjugate pad, a nitrocellulose film and a water absorption pad which are attached to a polyvinyl chloride base plate in sequence; the conjugate pad is a glass fiber film wrapped with a rabbit-anti-human IgM antibody-colloidal gold conjugate; the nitrocellulose film is wrapped with 9 detection lines and 1 quality control line in sequence; the 9 detection lines are respectively a mycoplasma pneumoniae recombined antigen, a chlamydia pneumoniae recombined antigen, an influenza a virus antigen, an influenza B virus antigen, a sendai virus antigen, a legionella pneumophila antigen, a Coxiella burnetii antigen, a respiratory syncytial virus antigen and an adenovirus antigen, and the quality control line is a second antibody. The invention further provides a colloidal gold test strip card comprising the colloidal gold test strip and a colloid gold kit, a preparation method of the colloidal gold test strip card, and a method for realizing detection by adopting the colloidal gold test strip, the test strip card or the kit.
Description
Technical field
The present invention relates to a kind of test strips, be specifically related to the colloidal gold strip of a kind of detection nine respiratory tract infection pathogen specific IgM antibody, and comprise colloidal gold test paper card and the colloidal gold kit of this colloidal gold strip, the preparation method of this colloidal gold strip, and use this colloidal gold strip, colloidal gold test paper card or colloidal gold kit to carry out the method detected.
Background technology
The current specific IgM antibodies by nine respiratory pathogens in immunofluorescent reagent box detection sample is a kind of proven technique, but required time is longer.Such as, Antu biotech firm nine respiratory tract infection pathogen IgM antibody detection kit adopt indirect immunofluorescence principle, on microslide, (10 holes/sheet) wraps quilt respectively: 1 hole bag is by legionella pneumophilia serum I type, 2 hole bags are by the mycoplasma pneumoniae in McCoy cell, 3 hole bags are by the hot Richettsia II phase of Q, 4 hole bags are by Chlamydia pneumoniae (elementary body), 5 hole bags are by the adenovirus in HEp-2 cell, 6 hole bags are by the Respiratory Syncytial Virus(RSV) in HEp-2 cell, 7 hole bags are by the influenza A virus in LLC-MK2 cell, 8 hole bags are by the influenza B virus in LLC-MK2 cell, 9 hole bags are by the parainfluenza virus 1 in LLC-MK2 cell, 2 and 3 types, 10 holes are Quality Control hole.Except legionella pneumophilia, in microslide, all antigen is all obtain from cell culture.Easy steps is: dilute serum sample (5min) → add anti-human igg sorbent treatment serum sample, and it is centrifugal, (20min) → in microslide, add detected serum sample, and 37 DEG C hatch 90min → clean and dry 15min → add the anti-human IgM-FITC bond of rabbit, and 7 DEG C hatch 30min → clean and dry 15min → add closed dielectric and be placed in basis of microscopic observation result 15min, so need more than about 3 hours detection time.
The kit of other detection respiratory pathogen is also had in prior art, but these kits and related detecting method often need the nucleic acid fragment of amplification sample, some need are cultivated or are prepared corresponding probe solution, what have also needs use large-scale instrument and equipment, as mass spectrometer, GeXP genetic analyzer etc. detect.This can pay extra incubation time and reaction time, and requires professional and expensive professional equipment and match and just can implement, and thus testing cost is high and efficiency is lower.
Colloidal gold strip is as a kind of rapid field detection technique, easy and simple to handle, can read result by naked eyes in general 15 minutes.But, yet there are no the report simultaneously detecting the specific IgM antibodies of nine respiratory pathogens on a colloidal gold strip.
Summary of the invention
Therefore, the object of the invention is to overcome above-mentioned defect of the prior art, provide a kind of high specificity, detect the colloidal gold strip of convenient, cheap detection nine respiratory tract infection pathogen specific IgM antibody, additionally provide the colloidal gold test paper card and colloidal gold kit that comprise this colloidal gold strip, and the preparation method of this colloidal gold strip and use this colloidal gold strip, test card or kit to carry out the method that detects.
For achieving the above object, the invention provides a kind of colloidal gold strip detecting IgM antibody, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, and described colloidal gold strip is included in sample pad, bond pad, nitrocellulose filter and adsorptive pads that Polyvinylchloride (PVC) base plate attaches successively; Described bond pad is the glass fibre membrane being coated with rabbit anti-human IgM antibodies-colloidal gold conjugate; Described nitrocellulose filter is coated with 9 detection lines and 1 nature controlling line successively, described 9 detection lines are respectively mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen, influenza A virus antigen, influenza B virus antigen, parainfluenza virus antigens, legionella pneumonia Ag, the hot rickettsial antigen of Q, respiratory syncytial viral antigens and Adenovirus Antigen (hereinafter can respectively referred to as T1 ~ T9 line), and described nature controlling line is two anti-(hereinafter can referred to as C line).Above 9 detection lines can not affect testing result with random order arrangement.
The related introduction of the respiratory tract infection pathogen that the present invention relates to is as follows:
Mycoplasma pneumoniae: the pneumonia that mycoplasma pneumoniae causes is the most common in Children and teenager, can hide by intracutaneous on respiratory mucosa, some patients non-evident sympton, but most of patient is apparent infection, below 3 years old, childrens respiratory tract infection is common, is grown up based on pneumonic.Mycoplasma pneumoniae pneumonia latent period is 14 ~ 21 days, and onset is slow, and IgM antibody is appearance in 1 week after infection generally, within 3 ~ 4 weeks, reaches peak period, sustainable 3 ~ 6 months.
Chlamydia pneumoniae: Chlamydia pneumoniae very easily causes infection in respiratory system, particularly bronchitis and pneumonia, in the elderly, the incidence of disease is higher, pneumonia caused by it accounts for 10% of all pneumonia cases, Chlamydia pneumoniae infect caused pneumonia, sings and symptoms is without specificity, most onset is slow, be generally about 30 days latent period, IgM antibody occurs in morbidity for 2 ~ 3 weeks, in general sustainable existence 2 ~ 6 months.
Influenza virus: it is the pathogen of influenza, can produce serious complication having in potential pathological patient.Because it is easy and other breathing problem is obscured, so very difficult in epidemic period clinical diagnosis, therefore laboratory diagnosis seems extremely important.The latent period of influenza infection is generally 1 ~ 7 day, IgM antibody in morbidity about 1 week appearance, sustainable 2 ~ 3 months.
Parainfluenza: parainfluenza virus can cause laryngotracheobronchitis in 2 ~ 4 years old children, is generally 2 ~ 7 days latent period, IgM antibody in morbidity about 1 week appearance, sustainable 2 ~ 3 months.
Legionella pneumophilia: most easy infection legionella pneumophilia 1 type of people, atypical pneumonia is often attended by constitutional symptom.The pneumonia of 10%, caused by legionella pneumophilia 1 type, is generally 2 ~ 10 days latent period, and IgM antibody is deposited and within 1 week, occurred after infection, and sustainable 3 ~ 6 months.
The hot Richettsia of Q: Q heat is the general disease caused by the hot Richettsia of Q, can cause heating, SARS (Severe Acute Respiratory Syndrome), hepatitis or endocarditis, latent period is 2 ~ 38 days, is generally 12 ~ 19 days, IgM antibody is general to be occurred after infection for 2 weeks, within 4 ~ 8 weeks, reaches peak, sustainable existence 3 ~ 4 months.
Respiratory Syncytial Virus(RSV): the main pathogens being child's respiratory tract infection in less than two years old, in the winter time eruption and prevalence.Latent period is 3 ~ 7 days, and IgM antibody is in morbidity about 1 week appearance.
Adenovirus: adenovirus is a kind of important respiratory pathogen, can cause upper respiratory disease, is attended by hurried heating and slight respiratory tract infection.The latent period of adenovirus infection is 2 ~ 14 days, and IgM antibody is in morbidity about 1 week appearance.Sustainable 2 ~ 3 months.
Immunoglobulin M (IgM) is most important a kind of immunoglobulin (Ig) in the mankind or animal body.When after organism infection pathogen, in vivo, what occur the earliest is the specific IgM antibody of this pathogen, and IgM concentration reduces afterwards, and specific IgG antibody raises.Therefore, by whether detecting the existence of the IgM antibody of pathogen specific in body, can diagnose people or animal to this pathogen whether have an early infection may.If detect the specific IgM antibody of pathogen in serum, prove that this body has the generation of recent infection, if early diagnosis, early diagnosis and treatment can be realized, undoubtedly clinician and patient are had great importance.
According to colloidal gold strip of the present invention, wherein, described sample pad is glass fibre membrane.Preferably, described adsorptive pads is thieving paper.
According to colloidal gold strip of the present invention, wherein, each adjacent lines of described detection line and nature controlling line be spaced apart 3 ~ 4mm, be preferably 3mm; Preferably, the width of described colloidal gold strip is 3 ~ 5mm, is preferably 4mm.
According to colloidal gold strip of the present invention, wherein, overlapping 1 ~ 2mm between described sample pad with bond pad, be preferably 1mm, described bond pad 1 ~ 2mm overlapping with between nitrocellulose filter, be preferably 1mm, described nitrocellulose filter 1 ~ 2mm overlapping with between adsorptive pads, is preferably 1mm.
According to colloidal gold strip of the present invention, wherein, select two resist for goat anti-rabbit igg antibody.
The Cleaning Principle of this colloidal gold strip is roughly: after sample pad is added dropwise to testing sample, and by capillary action, the rabbit anti-human IgM antibodies in bond pad-colloidal gold conjugate is dissolved and to be discharged and direction to adsorptive pads one end is moved by testing sample.If contained for mycoplasma pneumoniae in detected sample, Chlamydia, Flu-A, influenza B, parainfluenza, legionella pneumophilia, the hot Richettsia of Q, Respiratory Syncytial Virus(RSV), any one in adenovirus or specific IgM antibody corresponding to multiple pathogens, then there is specific reaction with gold nano grain-rabbit anti-human IgM antibodies, generate gold nano grain-rabbit anti-human IgM antibodies-specific IgM antibody compound, when sample continues to move to T1 ~ T9 line, wherein have for the specific IgM antibodies meeting of the pathogen infected and the antigen generation specific reaction of corresponding pathogen and caught by it, form the compound of antigen-specific IgM antibodies-rabbit anti-human IgM antibodies-collaurum, therefore a red stripes is formed at this p-wire place, when sample continues to move to C line place, unnecessary meeting is resisted by two catches, thus a red stripes is formed at C line place.The colour developing of C line proves the validity of this test, and C line does not develop the color, and is considered as test invalidation, need again detect.
Present invention also offers the preparation method of above-mentioned colloidal gold strip, the method comprises the following steps:
(1) rabbit anti-human IgM antibodies is processed: get 200 μ g rabbit anti-human IgM antibodies and be placed in bag filter and carry out dialysis 24h to 5mMTris-HCl, period changes a water every 2h; After having dialysed, antigen is taken out and is placed in centrifuge tube, add tri-distilled water to 2mL, after centrifugal, discard precipitated impurities, obtain treated rabbit anti-human IgM antibodies.
(2) adopt reduction of sodium citrate gold chloride legal system for colloidal gold solution: in beaker, add 1000mL tri-distilled water, then the sodium citrate solution of the 1 quality % of 10mL is added, after being heated to boiling, add the chlorauric acid solution of 15mL1 quality % again, to cooling after boiling 15min, be placed in 4 DEG C of preservations, obtain colloidal gold solution, wherein gold grain diameter is about 30 ~ 40nm; Preferably, the glass apparatus used in this step to spend the night immersion by washing lotion in advance, then rinses well.
(3) bond pad is prepared: get colloidal gold solution 20mL prepared by step (2) and be placed in beaker, add the K of 150 μ L0.1M
2cO
3solution regulates the pH to 7.0 of solution, stir, then the treated rabbit anti-human IgM antibodies of step (1) is added, stir 20min, then bovine serum albumin(BSA) (BSA) solution of 2mL10 quality % is added, carry out centrifugal, centrifugation time 40Min, rotating speed is 10000rpm, then abandoning supernatant, the BSA solution adding 1 quality % again continues centrifugal, abandoning supernatant, sediment is recovered, the composition recovering liquid is: the BSA of 1 quality %, the sucrose of 0.5 quality %, the casein-sodium of 0.5 quality %, above the glass fibre membrane being coated in 200 square centimeters of sizes,-45 DEG C freezing after, for subsequent use as the freeze-drying of bond pad, preferably, described bond pad at room temperature keeps in Dark Place for subsequent use after freeze drying.
(4) assemble test paper: bond pad, nitrocellulose filter and adsorptive pads prepared by sample pad, step (3) are attached to adjacent to one another on Polyvinylchloride base plate in turn, obtain assembling test paper; Preferably, the overlapping 1mm of adjacent part, make liquid sample can smooth flow to the direction of adsorptive pads or thieving paper.
(5) bag is by nitrocellulose filter: use and draw film instrument by mycoplasma pneumoniae recombination antigen solution, Chlamydia pneumoniae recombinant antigen solution, influenza A virus antigen solution, influenza B virus antigenic solution, parainfluenza virus antigens solution, legionella pneumonia Ag solution, the hot rickettsial antigen solution of Q, respiratory syncytial viral antigens solution, as 9 detection lines and 1 nature controlling line on the nitrocellulose filter that Adenovirus Antigen solution and two anti-solution are coated on the assembling test paper of step (4) respectively.
(6) the assembling test paper after step (5) being processed is cut into width 3 ~ 5mm, the test strips of preferred 4mm, namely obtains described colloidal gold strip.
Preparation in accordance with the present invention, wherein, in step (5), the concentration of the solution of described mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen solution, influenza A virus antigen solution, influenza B virus antigenic solution, parainfluenza virus antigens solution, legionella pneumonia Ag solution, the hot rickettsial antigen solution of Q, respiratory syncytial viral antigens solution and Adenovirus Antigen solution is 0.5mg/mL, and the concentration of described two anti-solution is 0.3mg/mL; Preferably, the setup parameter of described stroke of film instrument is: each adjacent lines of described detection line and nature controlling line be spaced apart 3 ~ 4mm, be preferably 3mm; Line concentration is 1 μ L/cm; More preferably, described stroke of film instrument is that three-dimensional planar draws film instrument.
Present invention also offers a kind of colloidal gold test paper card detecting IgM antibody, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, described colloidal gold test paper card comprises housing and the colloidal gold strip of the present invention being arranged in described housing or the colloidal gold strip prepared according to said method of the present invention, described housing is provided with well and colour developing view window, described well aims at the position of described sample pad, is coated with the position of the nitrocellulose filter of 9 detection lines and 1 nature controlling line described in described colour developing view window aligning.
The preparation method of this colloidal gold test paper card can be: (1) is prepared or bought described housing; (2) loaded in described housing by colloidal gold strip of the present invention, make well aim at the position of sample pad, the position of nitrocellulose filter aimed at by colour developing view window, to observe the color status of each detection line and nature controlling line.
Present invention also offers a kind of colloidal gold kit detecting IgM antibody, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, and described colloidal gold kit comprises aluminium foil bag and is sealed in drying agent, dropper and colloidal gold strip of the present invention in described aluminium foil bag or the colloidal gold strip prepared according to said method of the present invention or colloidal gold test paper card of the present invention.
The preparation method of this colloidal gold kit can be: be installed in aluminium foil bag by colloidal gold strip of the present invention or colloid gold test paper, reinstall drying agent and dropper, carries out sealing and at room temperature preserves.
Present invention also offers and use colloidal gold strip of the present invention or the colloidal gold strip prepared according to said method of the present invention or colloidal gold test paper card of the present invention or colloidal gold kit of the present invention to carry out the method detected, the method comprises the following steps:
(1) testing sample is collected: testing sample is whole blood or serum; Wherein, when serum exists muddy, high speed centrifugation is carried out to it and removes muddy impurity;
(2) colloidal gold strip or colloidal gold test paper card are lain on detection table top, drip after the dilution of the serum 10 times of step (1) in sample pad, in 10 ~ 20 minutes, preferably in 10 ~ 15 minutes, preferred colour developing result of observing detection line and nature controlling line 15 minutes time further; Colour developing result after 20 minutes and/or nature controlling line do not show red stripes, are considered as testing result invalid, need again detect;
(3) judge the situation of nine respiratory tract infection pathogen specific IgM antibody in testing sample according to following table, wherein "+" represents display red stripes, and "-" expression does not show red stripes:
| Detection line | Colour developing situation | Result of determination | Colour developing situation | Result of determination |
| Mycoplasma pneumoniae recombination antigen | + | Positive | - | Negative |
| Chlamydia pneumoniae recombinant antigen | + | Positive | - | Negative |
| Influenza A virus antigen | + | Positive | - | Negative |
| Influenza B virus antigen | + | Positive | - | Negative |
| Parainfluenza virus antigens | + | Positive | - | Negative |
| Legionella pneumonia Ag | + | Positive | - | Negative |
| The hot rickettsial antigen of Q | + | Positive | - | Negative |
| Respiratory syncytial viral antigens | + | Positive | - | Negative |
| Adenovirus Antigen | + | Positive | - | Negative |
Colloidal gold immunochromatographimethod technology and indirect immunization principle combine by the present inventor, by p-wire bag by nine corresponding recombinant antigens of respiratory pathogen institute, by immune indirect method principle, whether single stage method just can detect in sample fast nine corresponding specific IgM antibody of respiratory pathogen.Up to the present, the kit do not detected fast by colloidal gold strip about nine respiratory pathogen specific IgM antibodies.Although Antu biotech firm can realize detecting simultaneously, consuming time very long, be greater than 3 hours.More specifically, colloidal gold strip provided by the present invention and relevant test card and kit, preparation method and detection method can have following advantage relative to prior art:
(1) detection speed is fast, efficiency is high.Generally in 10 ~ 15 minutes, namely can read colour developing result and judge, not needing extra sample culturing time or reaction time.
(2) high specificity, coincidence rate are high.9 detection lines on colloidal gold strip can detect the specific IgM antibodies of nine respiratory pathogens that may exist pointedly respectively, and embody feminine gender or the positive of specific IgM antibodies by colour developing that can be visual (such as aobvious red or do not develop the color).According to the testing result of the present invention to actual sample, its to the detection coincidence rate of nine respiratory tract infection pathogen specific IgM antibody all more than 95%.
(3) simple to operate, cheap, do not need professional and technical personnel or large-scale instrument and equipment to get final product examinations, be particluarly suitable for remote poverty-stricken area and use.
(4) be applicable to early diagnosis, can read testing result by naked eyes, thus obtain more relevant information, assist clinicians gives correct medical diagnosis etc., and early discovery, early diagnosis, early treatment, early recovery from illness, have important clinical meaning.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 shows schematic structure and the schematic diagram of the colloidal gold strip of detection IgM antibody of the present invention;
Fig. 2 shows the structural representation of the colloidal gold test paper card of detection IgM antibody of the present invention.
Description of reference numerals:
1, sample pad; 11, nine respiratory tract infection pathogen specific IgM antibody;
2, bond pad; 21, rabbit anti-human IgM antibodies-colloidal gold conjugate;
3, nitrocellulose filter; 4, adsorptive pads;
5, detection line; 51,52,53,54,55,56,57,58,59 mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen, influenza A virus antigen, influenza B virus antigen, parainfluenza virus antigens, legionella pneumonia Ag, the hot rickettsial antigen of Q, respiratory syncytial viral antigens, the Adenovirus Antigen that can arrange according to any order is respectively;
6, nature controlling line; 61, two resist;
7, well; 8, develop the color view window; 9, housing.
Embodiment
Further illustrate the present invention below by specific embodiment, but should be understood to, these embodiments are only used for the use specifically described more in detail, and should not be construed as limiting the present invention in any form.
General description is carried out to the material used in the present invention's test and test method in this part.Although for realizing many materials that the object of the invention uses and method of operating is well known in the art, the present invention still describes in detail as far as possible at this.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and method of operating are well known in the art.
embodiment 1
The present embodiment is for illustration of the colloidal gold strip and preparation method thereof of detection IgM antibody of the present invention.
As shown in Figure 1, the colloidal gold strip of the present embodiment is included in sample pad, bond pad, nitrocellulose filter and adsorptive pads that Polyvinylchloride (PVC) base plate attaches successively; Described sample pad is glass fibre membrane; Described bond pad is the glass fibre membrane being coated with rabbit anti-human IgM antibodies-colloidal gold conjugate; Described nitrocellulose filter is coated with 9 detection lines and 1 nature controlling line successively, described 9 detection lines are respectively mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen, influenza A virus antigen, influenza B virus antigen, parainfluenza virus antigens, legionella pneumonia Ag, the hot rickettsial antigen of Q, respiratory syncytial viral antigens and Adenovirus Antigen (being respectively T1 ~ T9 line), and described nature controlling line is two anti-(C lines); Described adsorptive pads is thieving paper.
Wherein, the interval of each adjacent lines of detection line and nature controlling line is 3mm, and the width of colloidal gold strip is 4mm.Overlapping 1mm between described sample pad with bond pad, described bond pad 1mm overlapping with between nitrocellulose filter, described nitrocellulose filter 1mm overlapping with between adsorptive pads.Select two resist for goat anti-rabbit igg antibody (purchased from Beijing Mo Zhidong Bioisystech Co., Ltd).
The preparation method of above-mentioned colloidal gold strip is as follows:
(1) rabbit anti-human IgM antibodies is processed: get 200 μ g rabbit anti-human IgM antibodies (purchased from Beijing Mo Zhidong Bioisystech Co., Ltd) and be placed in bag filter and carry out dialysis 24h to 5mMTris-HCl, period changes a water every 2h; After having dialysed, antigen is taken out and is placed in centrifuge tube, add tri-distilled water to 2mL, after centrifugal, discard precipitated impurities, obtain treated rabbit anti-human IgM antibodies.
(2) adopt reduction of sodium citrate gold chloride legal system for colloidal gold solution: in beaker, add 1000mL tri-distilled water, then the sodium citrate solution of the 1 quality % of 10mL is added, after being heated to boiling, add the chlorauric acid solution of 15mL1 quality % again, extremely cool after boiling 15min, be placed in 4 DEG C of preservations, obtain colloidal gold solution, wherein gold grain diameter is about 30 ~ 40nm, and all prior washing lotion of the glass apparatus used in this step is spent the night immersion, then rinses well.
(3) bond pad is prepared: get colloidal gold solution 20mL prepared by step (2) and be placed in beaker, add the K of 150 μ L0.1M
2cO
3solution regulates the pH to 7.0 of solution, stir, then the treated rabbit anti-human IgM antibodies of step (1) is added, stir 20min, then the bovine serum albumin solution of 2mL10 quality % is added, carry out centrifugal, centrifugation time 40Min, rotating speed is 10000rpm, then abandoning supernatant, the solution adding the bovine serum albumin(BSA) of 1 quality % again continues centrifugal, abandoning supernatant, sediment is recovered, the composition recovering liquid is: the bovine serum albumin(BSA) of 1 quality %, the sucrose of 0.5 quality %, the casein-sodium of 0.5 quality %, above the glass fibre membrane (purchased from Hangzhou Han Gan Science and Technology Ltd.) being coated in 200 square centimeters of sizes,-45 DEG C freezing rear as bond pad, (freeze drier is purchased from Beijing Bo Yikang experimental apparatus company limited) at room temperature keeps in Dark Place for subsequent use after freeze drying.
(4) test paper is assembled: bond pad sample pad glass fibre membrane (purchased from Hangzhou Han Gan Science and Technology Ltd.), step (3) prepared, nitrocellulose filter and thieving paper (purchased from Hangzhou Han Gan Science and Technology Ltd.) are attached on Polyvinylchloride base plate (purchased from Hangzhou Han Gan Science and Technology Ltd.) in turn adjacent to one another, the equal overlapping 1mm of adjacent part, obtains assembling test paper.
(5) bag is by nitrocellulose filter: (antigen is all purchased from grand bio tech ltd of Xiamen Wanke by mycoplasma pneumoniae recombination antigen solution, Chlamydia pneumoniae recombinant antigen solution, influenza A virus antigen solution, influenza B virus antigenic solution, parainfluenza virus antigens solution, legionella pneumonia Ag solution, Q hot rickettsial antigen solution, respiratory syncytial viral antigens solution, Adenovirus Antigen solution to use three-dimensional planar to draw film instrument (or claiming three-dimensional planar gold spraying instrument, purchased from American Biodot).The concentration of these antigenic solutions is 0.5mg/mL) and two anti-solution (concentration is 0.3mg/mL) be coated on the assembling test paper of step (4) respectively nitrocellulose filter (purchased from Merck Mi Libo) on as 9 detection lines and 1 nature controlling line, the setup parameter wherein drawing film instrument is: line interval is 3mm, and line concentration is 1 μ L/cm.
(6) the assembling test paper after step (5) being processed is cut into the test strips (cutting cutter is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.) of width 4mm, namely obtains colloidal gold strip of the present invention.
embodiment 2
The present embodiment is for illustration of the colloidal gold test paper card of detection IgM antibody of the present invention and colloidal gold kit and their preparation method.
As shown in Figure 2, colloidal gold test paper card comprises housing and is arranged in the colloidal gold strip of described housing, and the colloidal gold strip of the present embodiment is from embodiment 1.Described housing is provided with well and colour developing view window, described well aims at the position of described sample pad, is coated with the position of the nitrocellulose filter of 9 detection lines and 1 nature controlling line described in described colour developing view window aligning.
The preparation method of this colloidal gold test paper card is: (1) is prepared or bought described housing (such as purchased from Hangzhou Han Gan Science and Technology Ltd.); (2) loaded in described housing by the colloidal gold strip of embodiment 1, make well aim at the position of sample pad, the position of nitrocellulose filter aimed at by colour developing view window, to observe the color status of each detection line and nature controlling line.
The present embodiment also can provide colloidal gold kit further, and it comprises aluminium foil bag and the colloidal gold strip of drying agent, dropper and embodiment 1 be sealed in described aluminium foil bag or the colloidal gold test paper card of the present embodiment.
The preparation method of this colloidal gold kit is: be installed in aluminium foil bag by the colloidal gold strip of embodiment 1 or the colloid gold test paper of the present embodiment, reinstalls a drying agent and dropper, carries out sealing and at room temperature preserves.
embodiment 3
The present embodiment is for illustration of using colloidal gold strip of the present invention, colloidal gold test paper card or colloidal gold kit for detecting the method for nine respiratory tract infection pathogen specific IgM antibody.
(1) collect testing sample: testing sample is serum, when serum exists muddy, high speed centrifugation is carried out to it and removes muddy impurity.
(2) colloidal gold strip or colloidal gold test paper card are lain on detection table top, drip after the dilution of the serum 10 times of step (1) in sample pad, in 10 ~ 15 minutes, observe the colour developing result of detection line and nature controlling line; Colour developing result after 20 minutes and/or nature controlling line do not show red stripes, are considered as testing result invalid, need again detect;
(3) judge the situation of nine respiratory tract infection pathogen specific IgM antibody in testing sample according to following table, wherein "+" represents display red stripes, and "-" expression does not show red stripes:
| Detection line | Colour developing situation | Result of determination | Colour developing situation | Result of determination |
| Mycoplasma pneumoniae recombination antigen | + | Positive | - | Negative |
| Chlamydia pneumoniae recombinant antigen | + | Positive | - | Negative |
| Influenza A virus antigen | + | Positive | - | Negative |
| Influenza B virus antigen | + | Positive | - | Negative |
| Parainfluenza virus antigens | + | Positive | - | Negative |
| Legionella pneumonia Ag | + | Positive | - | Negative |
| The hot rickettsial antigen of Q | + | Positive | - | Negative |
| Respiratory syncytial viral antigens | + | Positive | - | Negative |
| Adenovirus Antigen | + | Positive | - | Negative |
Adopt said method to detect the clinical positive serum made a definite diagnosis and negative serum, the results are shown in Table 2:
The verification and measurement ratio of table 2 actual sample
Although present invention has been description to a certain degree, significantly, under the condition not departing from the spirit and scope of the present invention, can carry out the suitable change of each condition.Be appreciated that and the invention is not restricted to described embodiment, and be attributed to the scope of claim, it comprises the equivalent replacement of described each factor.
Claims (10)
1. detect the colloidal gold strip of IgM antibody, it is characterized in that, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, and described colloidal gold strip is included in sample pad, bond pad, nitrocellulose filter and adsorptive pads that Polyvinylchloride base plate attaches successively; Described bond pad is the glass fibre membrane being coated with rabbit anti-human IgM antibodies-colloidal gold conjugate; Described nitrocellulose filter is coated with 9 detection lines and 1 nature controlling line successively, described 9 detection lines are respectively mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen, influenza A virus antigen, influenza B virus antigen, parainfluenza virus antigens, legionella pneumonia Ag, the hot rickettsial antigen of Q, respiratory syncytial viral antigens and Adenovirus Antigen, described nature controlling line be two resist.
2. colloidal gold strip according to claim 1, is characterized in that, described sample pad is glass fibre membrane; Preferably, described adsorptive pads is thieving paper.
3. colloidal gold strip according to claim 1 and 2, is characterized in that, each adjacent lines of described detection line and nature controlling line be spaced apart 3 ~ 4mm, be preferably 3mm; Preferably, the width of described colloidal gold strip is 3 ~ 5mm, is preferably 4mm.
4. colloidal gold strip according to any one of claim 1 to 3, it is characterized in that, overlapping 1 ~ 2mm between described sample pad with bond pad, be preferably 1mm, described bond pad 1 ~ 2mm overlapping with between nitrocellulose filter, be preferably 1mm, described nitrocellulose filter 1 ~ 2mm overlapping with between adsorptive pads, be preferably 1mm.
5. colloidal gold strip according to any one of claim 1 to 4, is characterized in that, select two resist for goat anti-rabbit igg antibody.
6. the preparation method of the colloidal gold strip according to any one of claim 1 to 5, is characterized in that, the method comprises the following steps:
(1) rabbit anti-human IgM antibodies is processed: get 200 μ g rabbit anti-human IgM antibodies and be placed in bag filter and carry out dialysis 24h to 5mMTris-HCl, period changes a water every 2h; After having dialysed, antigen is taken out and is placed in centrifuge tube, add tri-distilled water to 2mL, after centrifugal, discard precipitated impurities, obtain treated rabbit anti-human IgM antibodies;
(2) adopt reduction of sodium citrate gold chloride legal system for colloidal gold solution: in beaker, add 1000mL tri-distilled water, then the sodium citrate solution of the 1 quality % of 10mL is added, after being heated to boiling, add the chlorauric acid solution of 15mL1 quality % again, to cooling after boiling 15min, be placed in 4 DEG C of preservations, obtain colloidal gold solution, wherein gold grain diameter is about 30 ~ 40nm; Preferably, the glass apparatus used in this step to spend the night immersion by washing lotion in advance, then rinses well;
(3) bond pad is prepared: get colloidal gold solution 20mL prepared by step (2) and be placed in beaker, add the K of 150 μ L0.1M
2cO
3solution regulates the pH to 7.0 of solution, stir, then the treated rabbit anti-human IgM antibodies of step (1) is added, stir 20min, then the bovine serum albumin solution of 2mL10 quality % is added, carry out centrifugal, centrifugation time 40Min, rotating speed is 10000rpm, then abandoning supernatant, the solution adding the bovine serum albumin(BSA) of 1 quality % again continues centrifugal, abandoning supernatant, sediment is recovered, the composition recovering liquid is: the bovine serum albumin(BSA) of 1 quality %, the sucrose of 0.5 quality %, the casein-sodium of 0.5 quality %, above the glass fibre membrane being coated in 200 square centimeters of sizes,-45 DEG C freezing after, for subsequent use as the freeze-drying of bond pad, preferably, described bond pad at room temperature keeps in Dark Place for subsequent use after freeze drying,
(4) assemble test paper: bond pad, nitrocellulose filter and adsorptive pads prepared by sample pad, step (3) are attached to adjacent to one another on Polyvinylchloride base plate in turn, obtain assembling test paper;
(5) bag is by nitrocellulose filter: use and draw film instrument by mycoplasma pneumoniae recombination antigen solution, Chlamydia pneumoniae recombinant antigen solution, influenza A virus antigen solution, influenza B virus antigenic solution, parainfluenza virus antigens solution, legionella pneumonia Ag solution, the hot rickettsial antigen solution of Q, respiratory syncytial viral antigens solution, as 9 detection lines and 1 nature controlling line on the nitrocellulose filter that Adenovirus Antigen solution and two anti-solution are coated on the assembling test paper of step (4) respectively,
(6) the assembling test paper after step (5) being processed is cut into width 3 ~ 5mm, the test strips of preferred 4mm, namely obtains described colloidal gold strip.
7. preparation method according to claim 6, it is characterized in that, in step (5), the concentration of the solution of described mycoplasma pneumoniae recombination antigen, Chlamydia pneumoniae recombinant antigen solution, influenza A virus antigen solution, influenza B virus antigenic solution, parainfluenza virus antigens solution, legionella pneumonia Ag solution, the hot rickettsial antigen solution of Q, respiratory syncytial viral antigens solution and Adenovirus Antigen solution is 0.5mg/mL, and the concentration of described two anti-solution is 0.3mg/mL; Preferably, the setup parameter of described stroke of film instrument is: each adjacent lines of described detection line and nature controlling line be spaced apart 3 ~ 4mm, be preferably 3mm; Line concentration is 1 μ L/cm; More preferably, described stroke of film instrument is that three-dimensional planar draws film instrument.
8. detect the colloidal gold test paper card of IgM antibody, it is characterized in that, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, described colloidal gold test paper card comprises housing and the colloidal gold strip according to any one of Claims 1 to 5 being arranged in described housing or the colloidal gold strip prepared according to the method described in claim 6 or 7, described housing is provided with well and colour developing view window, described well aims at the position of described sample pad, the position of the nitrocellulose filter of 9 detection lines and 1 nature controlling line is coated with described in described colour developing view window aligning.
9. detect the colloidal gold kit of IgM antibody, it is characterized in that, described IgM antibody is nine respiratory tract infection pathogen specific IgM antibody, and described colloidal gold kit comprises aluminium foil bag and is sealed in the colloidal gold strip according to any one of drying agent, dropper and Claims 1 to 5 in described aluminium foil bag or colloidal gold test paper card described in the colloidal gold strip prepared according to the method described in claim 6 or 7 or claim 8.
10. described in the colloidal gold strip using colloidal gold strip according to any one of claim 1 ~ 5 or prepare according to the method described in claim 6 or 7 or claim 8, colloidal gold test paper card or colloidal gold kit according to claim 9 carry out the method detected, it is characterized in that, the method comprises the following steps:
(1) testing sample is collected: testing sample is whole blood or serum; Wherein, when serum exists muddy, high speed centrifugation is carried out to it and removes muddy impurity;
(2) colloidal gold strip or colloidal gold test paper card are lain on detection table top, drip after the dilution of the serum 10 times of step (1) in sample pad, in 10 ~ 20 minutes, preferably in 10 ~ 15 minutes, preferred colour developing result of observing detection line and nature controlling line 15 minutes time further; Colour developing result after 20 minutes and/or nature controlling line do not show red stripes, are considered as testing result invalid, need again detect;
(3) judge the situation of the specific IgM antibodies of nine respiratory tract infection pathogen in testing sample according to following table, wherein "+" represents display red stripes, and "-" expression does not show red stripes:
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| CN105823875A (en) * | 2016-03-15 | 2016-08-03 | 威尚生物技术（合肥）有限公司 | Mycoplasma pneumoniae antibody immunity colloid gold test strip and preparation method thereof |
| CN105866434A (en) * | 2016-05-17 | 2016-08-17 | 北京美康基因科学股份有限公司 | Immunochromatography kit for detecting IgM antibodies of nine respiratory tract infection pathogens and preparation method thereof |
| CN106841604A (en) * | 2017-03-22 | 2017-06-13 | 上海市疾病预防控制中心 | A kind of Respirovirus quick detection kit and preparation method thereof |
| CN107365364A (en) * | 2016-05-12 | 2017-11-21 | 兰州雅华生物技术有限公司 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
| CN110133255A (en) * | 2018-02-02 | 2019-08-16 | 中国人民解放军军事科学院军事医学研究院 | A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
| CN111289746A (en) * | 2020-04-03 | 2020-06-16 | 山东发现生物技术有限公司 | Novel coronavirus IgM + IgG antibody detection method |
| CN111351927A (en) * | 2020-03-17 | 2020-06-30 | 陈韬 | Antibody matrix detection method (MEGA method) aiming at pathogen antigen and multi-connected detection card |
| CN111521786A (en) * | 2020-04-28 | 2020-08-11 | 天津健博生物科技有限公司 | Respiratory tract pathogen IgM antibody joint detection kit and preparation method thereof |
| CN111537727A (en) * | 2020-06-04 | 2020-08-14 | 湖南康润药业股份有限公司 | Test strip for combined detection of influenza and COVID-19 and application thereof |
| CN111579792A (en) * | 2020-04-30 | 2020-08-25 | 杨小军 | Influenza virus and 2019 novel coronavirus antibody combined detection device and preparation method thereof |
| CN111665365A (en) * | 2020-05-22 | 2020-09-15 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection kit |
| CN111693713A (en) * | 2020-04-09 | 2020-09-22 | 泰州市人民医院 | Detection card for detecting novel coronavirus IgM by colloidal gold method and preparation method thereof |
| CN114034859A (en) * | 2021-10-29 | 2022-02-11 | 济南百博生物技术股份有限公司 | New corona and respiratory syncytial virus antibody joint detection test strip |
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| WO2023202271A1 (en) * | 2022-04-17 | 2023-10-26 | 吉林迅准生物技术有限公司 | Multiple pathogen combined detection apparatus and manufacturing method therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105823875A (en) * | 2016-03-15 | 2016-08-03 | 威尚生物技术（合肥）有限公司 | Mycoplasma pneumoniae antibody immunity colloid gold test strip and preparation method thereof |
| CN107365364A (en) * | 2016-05-12 | 2017-11-21 | 兰州雅华生物技术有限公司 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
| CN105866434A (en) * | 2016-05-17 | 2016-08-17 | 北京美康基因科学股份有限公司 | Immunochromatography kit for detecting IgM antibodies of nine respiratory tract infection pathogens and preparation method thereof |
| CN106841604A (en) * | 2017-03-22 | 2017-06-13 | 上海市疾病预防控制中心 | A kind of Respirovirus quick detection kit and preparation method thereof |
| CN110133255A (en) * | 2018-02-02 | 2019-08-16 | 中国人民解放军军事科学院军事医学研究院 | A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
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| CN111289746A (en) * | 2020-04-03 | 2020-06-16 | 山东发现生物技术有限公司 | Novel coronavirus IgM + IgG antibody detection method |
| CN111693713A (en) * | 2020-04-09 | 2020-09-22 | 泰州市人民医院 | Detection card for detecting novel coronavirus IgM by colloidal gold method and preparation method thereof |
| CN111521786A (en) * | 2020-04-28 | 2020-08-11 | 天津健博生物科技有限公司 | Respiratory tract pathogen IgM antibody joint detection kit and preparation method thereof |
| CN111579792A (en) * | 2020-04-30 | 2020-08-25 | 杨小军 | Influenza virus and 2019 novel coronavirus antibody combined detection device and preparation method thereof |
| CN111665365A (en) * | 2020-05-22 | 2020-09-15 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection kit |
| CN111537727A (en) * | 2020-06-04 | 2020-08-14 | 湖南康润药业股份有限公司 | Test strip for combined detection of influenza and COVID-19 and application thereof |
| CN114034859A (en) * | 2021-10-29 | 2022-02-11 | 济南百博生物技术股份有限公司 | New corona and respiratory syncytial virus antibody joint detection test strip |
| CN114113593A (en) * | 2021-11-22 | 2022-03-01 | 南京市吉诺思原精准医学科技有限公司 | Test paper for rapidly detecting various respiratory pathogens and preparation method thereof |
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| WO2023202271A1 (en) * | 2022-04-17 | 2023-10-26 | 吉林迅准生物技术有限公司 | Multiple pathogen combined detection apparatus and manufacturing method therefor |
| CN116660520A (en) * | 2023-03-07 | 2023-08-29 | 江苏一诺生物科技有限公司 | Four-in-one antigen detection card and kit |
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