CN101699290A - Method for testing mycoplasma pneumoniae antibody, gold-labeled quick test card and preparation method - Google Patents
Method for testing mycoplasma pneumoniae antibody, gold-labeled quick test card and preparation method Download PDFInfo
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- CN101699290A CN101699290A CN200910022139A CN200910022139A CN101699290A CN 101699290 A CN101699290 A CN 101699290A CN 200910022139 A CN200910022139 A CN 200910022139A CN 200910022139 A CN200910022139 A CN 200910022139A CN 101699290 A CN101699290 A CN 101699290A
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Abstract
The invention relates to a method for testing mycoplasma pneumoniae antibody (IgG/IgM), a quick test card and a preparation method. The test method comprises the following steps that: (1) colloidal gold-labeled anti-human globulin Ig (IgG/IgM) is carried on a carrier, and a test line formed by gene engineering expressed mycoplasma pneumoniae P1 proteantigen and a control line formed by anti-mouse IgG antibody are coated on a test carrier connected with a colloidal gold carrier; and (2) human serum is dropped on the colloidal gold-labeled carrier, and a test line is formed on the test carrier when the serum contains pneumoniae antibody (IgG/IgM) and both the test line and the control line on the test carrier are positive, otherwise, negative. The test card comprises a sample-applying end water-absorbing layer, a test layer and a water-absorbing end water-absorbing layer, wherein a gold-labeled antihuman Ig (IgG/IgM) antibody layer is arranged between the test layer and the sample-applying end water-absorbing layer; and the test layer is coated with the test line formed by gene engineering expressed mycoplasma pneumoniae P1 proteantigen and the control line formed by anti-mouse IgG antibody. The method is a one-step quick test method and has high sensitivity and strong specificity; and the method is quick and simple and does not require specialized facilities.
Description
Technical field
The present invention relates to a kind of method that is used for diagnosis, detects mycoplasma pneumoniae antibody (IgG/IgM), and the mycoplasma pneumoniae gold-labeled quick test card and its preparation method that are used for this method.
Background technology
The gold-marking immunity chromatographic technique is a fast detecting diagnostic techniques that grows up nineteen nineties, because that this technology has is quick, easy, do not need any instrument and equipment, result to read simply, has become the various exploitation focuses that detect centuries.Up to the present, this technology has been applied in the detection diagnosis of multiple disease.
Mycoplasma pneumoniae (MP) mainly causes people's SARS (Severe Acute Respiratory Syndrome), tracheitis and bronchitis, and approach such as many per os and respiratory tract are propagated, and are mainly in children and young people.MP infects and has accounted for infantile respiratory disease more than 30%, and increase trend is arranged.The MP pneumonia is not obviously distinguished with the clinical symptoms of general respiratory tract infection and pneumonia, and MP infects the treatment that early diagnosis helps the MP pneumonia.Though the MP Infect And Diagnose has several different methods, studies show that at present it is one of the most desirable method of early diagnosis that infected person anteserum MPP1 protein antibodies detects.
Of the present invention open
First purpose of the present invention is for the detection method of a kind of mycoplasma pneumoniae antibody (IgG/IgM) is provided, this method is " single stage method " method for quick, highly sensitive, high specificity: and speed is fast, easy and simple to handle, need not specialized facilities, be applicable to clinical detection, epidemiology survey etc.
Second purpose of the present invention is for a kind of mycoplasma pneumoniae antibody (IgG/IgM) rapid detection card, this test card detects at mycoplasma pneumoniae antibody (IgG/IgM) a kind of easy, quick and direct " single stage method " test card is provided, and utilizes this test card to detect highly sensitive, the high specificity of mycoplasma pneumoniae antibody (IgG/IgM); And speed is fast, and is easy and simple to handle, need not specialized facilities.
The 3rd purpose of the present invention is for the preparation method of a kind of mycoplasma pneumoniae antibody (IgG/IgM) gold-labeled quick test card is provided, and this method is easy, good stability, good reproducibility.
First purpose of the present invention can realize by following measure:
A kind of mycoplasma pneumoniae antibody (IgG/IgM) detection method, comprise the steps: that (1) is stated from the antihuman globulin Ig (IgG/IgM) of colloid gold label on glass fibre or the nonwoven fabrics carrier, and on nitrocellulose membrane (NC) carrier that links to each other with the collaurum carrier, wrap detection line that is formed by gene engineering expression mycoplasma pneumoniae p1 protein antigen and the control line that forms by anti-mouse IgG antibody; (2) get human serum and drip on the carrier of colloid gold label, then form detection line detecting on the carrier, detect detection line on the carrier, the control line two-wire is positive as mycoplasma pneumoniae antibody (IgG/IgM) is arranged in the serum, otherwise negative.
Second purpose of the present invention can realize by following measure:
A kind of mycoplasma pneumoniae antibody (IgG/IgM) gold-labeled quick test card is made up of detector bar and plastics cartridge, and detector bar is installed in the plastic clip.
Described detector bar is to be provided with application of sample end water accepting layer, detection layers and suction side water accepting layer on liner plate, is provided with gold mark anti-people Ig (IgG/IgM) antibody layer between detection layers and application of sample end water accepting layer; On detection layers, be coated with detection line that forms by gene engineering expression mycoplasma pneumoniae p1 protein antigen and the control line that forms by anti-mouse IgG antibody.
The 3rd purpose of the present invention can realize by following measure:
The preparation method of a kind of mycoplasma pneumoniae antibody (IgG/IgM) gold mark test card mainly comprises golden labeling antibody layer and detection layers and goes up the preparation of wrapping Be Controlled line and detection line, and the assembling of detector bar and test card.
The preparation method of described golden labeling antibody layer comprises the steps: the preparation of i collaurum: concentration be add in 0.004%～0.012% the gold chloride its volume 1.3%～2%, concentration is 1%～3% trisodium citrate, boiled 10～20 minutes, and can obtain the colloidal gold solution of 15～50 nanometers; The anti-human IgG of ii colloid gold label/IgM monoclonal antibody, in colloidal gold solution, transfer PH8.0～8.4 with the 0.2m solution of potassium carbonate, add anti-human IgG/IgM monoclonal antibody by 4～12mg/100ml, after the stirring, add animal blood serum albumen by 0.1～0.6g/100ml again in solution, 4 ℃ left standstill 2～4 hours; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10～15 minutes, remove sediment, supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60～80 minutes centrifugal sediments; V is dissolved in the 0.02MPh7.4Tris-Hcl damping fluid with sediment by 4～10ml/100ml and gets colloidal gold solution, contains 0.2%～0.6% animal blood serum albumen and 0.01%～0.06% Sodium azide in this damping fluid; Till vi immersed above-mentioned colloidal gold solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ of dry filters formed golden labeling antibody layer.
The preparation of described detection layers adds fixing agent in the pulmonary branches antigen MPP1 of the purified rabbit for preparing or sheep anti-mouse igg antibody and gene engineering expression, form control line and detection line with special-purpose Membrane jetter on cellulose membrane; Its preparation method is to be the fixing agent that adds 0.1%-0.3% in the antigen of 0.8-2.4mg/ml or the antibody-solutions in concentration, utilize special-purpose Membrane jetter that antigen or antibody are sprayed line on the NC film, 37 ℃ of dryings behind the spray film, closed 30 minutes 0.01mPH7.0PBS rinsing, 37 ℃ of dry detection layers that get again with the 0.01mPH7.0PBS fluid-tight that contains 10% calf serum.
Described fixing agent is selected from a kind of in formaldehyde, the acetone.
The present invention has following advantage compared to existing technology:
1, the method for detection mycoplasma pneumoniae antibody of the present invention (IgG/IgM) is a single stage method, has the specificity and the high sensitivity of height, and it detects fast, and result's judgement was finished in 3～15 minutes, need not specialized facilities.
The specificity and the high sensitivity that have height when 2, test card of the present invention detects mycoplasma pneumoniae antibody (IgG/IgM), the testing process operation rapidly, fast, and is convenient, is fit to mycoplasma pneumoniae infection crowd's clinical diagnosis and epidemiology survey.
3, easy, the good stability of the preparation method of test card of the present invention, good reproducibility.
The drawing explanation
Fig. 1 is the structural representation of test card of the present invention
1-test card 2-well 3-viewport
Fig. 2 is the inner structure synoptic diagram of test card of the present invention
4-application of sample end water accepting layer 5-gold labeling antibody layer 6-control line
7-detection line 8-detection layers 9-suction side water accepting layer
The 10-liner plate
Embodiments of the present invention
The present invention also will be described in further detail in conjunction with the embodiments:
Embodiment one:
A kind of mycoplasma pneumoniae antibody IgG detection method, comprise the steps: that (1) is stated from the antihuman globulin Ig (IgG) of colloid gold label on glass fibre or the nonwoven fabrics carrier, and on nitrocellulose membrane (NC) carrier that links to each other with the collaurum carrier, wrap detection line that is formed by gene engineering expression mycoplasma pneumoniae p1 protein antigen and the control line that forms by anti-mouse IgG antibody; (2) get human serum and drip on the carrier of colloid gold label, then form detection line detecting on the carrier, detect detection line on the carrier, the control line two-wire is positive as mycoplasma pneumoniae antibody IgG is arranged in the serum, otherwise negative.
With reference to Fig. 1, a kind of mycoplasma pneumoniae antibody IgG gold mark test card 1
With reference to Fig. 2, described mycoplasma pneumoniae antibody IgG gold mark test card 1 is to be provided with application of sample end water accepting layer 4, detection layers 8 and suction side water accepting layer 9 on liner plate 10, is provided with the anti-human IgG antibody's layer 5 of gold mark between detection layers 8 and suction side water accepting layer 9; On detection layers 8, be coated with detection line 7 and control line 6.Wherein application of sample end water accepting layer 4 is made by compound glass fiber or nonwoven fabrics; Suction side water accepting layer 9 is made by multilayer filter paper; Detection layers 8 is a cellulose membrane, and this cellulose membrane can be nitrocellulose filter, cellulose acetate film; Gold labeling antibody layer is that glass fibre or nonwoven fabrics leaching colloid gold label antibody are made.
The preparation method of a kind of mycoplasma pneumoniae antibody IgG gold mark test card comprises the bag quilt of detection line 7 and control line 6 on golden labeling antibody layer 5 and the detection layers 8, combination mycoplasma pneumoniae antibody IgG gold mark test card 1 on liner plate 10 then.Paste application of sample end water accepting layer 4 and suction side water accepting layer 9 respectively at the two ends of plastic base plate 10; Disconnected therein detection layers 8 of pasting tested survey line of bag and control line at the handing-over position of application of sample end water accepting layer 4 with detection layers 8, is pressed from both sides labeling antibody layer 5 fiber carrier of gilding, and fiber carrier 4/5 part is clipped in the middle of the application of sample water accepting layer 4, and 1/5 part is stacked on the detection layers 8.Press 4-5 mm wide, 8 centimeter length specification slittings then, in being assembled in the plastics cartridge, well 2 is over against detector bar application of sample end water accepting layer 4 on the lid, and viewport 3 is over against detection layers 8.
The preparation method of described golden labeling antibody layer 5 comprises the steps: the preparation of i collaurum, getting the 100mg gold chloride is dissolved in the 1000ml tri-distilled water, add 15ml, be 1% trisodium citrate, boiled 15 minutes, can obtain the colloidal gold solution of 15～50 nanometers in concentration; The anti-human IgG monoclonal antibody of ii colloid gold label is got the 100ml colloidal gold solution, transfers PH8.4 with the 0.2m solution of potassium carbonate, go into the anti-human IgG monoclonal antibody of 6mg, stirred 20 minutes, add 240mg bovine serum albumin(BSA) (BSA) again, continue to stir 5 minutes, 4 ℃ left standstill 2～4 hours; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10～15 minutes, remove sediment, supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60 minutes centrifugal sediments; V is dissolved in 4ml 0.02M PH7.4Tris-Hcl damping fluid with sediment and gets colloidal gold solution, contains 0.25%BSA and 0.02% Sodium azide in this damping fluid; Till vi immersed colloidal gold solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ of dryings formed golden labeling antibody layer 5 in 2 hours.
Described colloid gold label antibody is anti-human IgG monoclonal antibody.
Described detection layers 8 is to be provided with gene engineering expression mycoplasma pneumoniae p1 protein antigen detection line 7 that constitutes and the control line 6 that has sheep, rabbit anti-mouse igg antibody to form on nitrocellulose membrane (NC).Its preparation method is to get gene engineering expression mycoplasma pneumoniae p1 protein antigen, and accent concentration is 1mg/ml, adds 0.2% formaldehyde, interrupts spray detection line 7 with special-purpose Membrane jetter at the NC film; Get sheep, rabbit anti-mouse igg antibody, accent concentration is 2mg/ml, add 0.2% formaldehyde, interrupt at the NC film with special-purpose Membrane jetter, apart from detection line 0.5cm place spray control line 6, detection line and control line all are provided with discharge rate by 20ul/ml, and the spray film was 37 ℃ of dryings 2 hours, again with the 0.01mPH7.0PBS fluid-tight that contains 10% calf serum close 37 ℃ 30 minutes, 0.01mPH7.0PBS rinsing, 37 ℃ of dryings.
Embodiment two:
Mycoplasma pneumoniae antibody detection method and gold-labeled quick test card thereof and example are together.
It is that anti-human IgG monoclonal anti is external that the preparation method of this test card removes preparation colloid gold label antibody, and other conditions and example are together.
Claims (8)
1. a mycoplasma pneumoniae antibody (IgG/IgM) detection method, comprise the steps: that (1) is stated from the antihuman globulin Ig (IgG/IgM) of colloid gold label on glass fibre or the nonwoven fabrics carrier, and on nitrocellulose membrane (NC) carrier that links to each other with the collaurum carrier, wrap detection line that is formed by gene engineering expression mycoplasma pneumoniae p1 protein antigen and the control line that forms by anti-mouse IgG antibody; (2) get human serum and drip on the carrier of colloid gold label, then form detection line detecting on the carrier, detect detection line on the carrier, the control line two-wire is positive as mycoplasma pneumoniae antibody (IgG/IgM) is arranged in the serum, otherwise negative.
2. a mycoplasma pneumoniae antibody (IgG/IgM) gold-labeled quick test card (1) is characterized in that this test card is made up of detector bar and plastics cartridge, and detector bar is installed in the plastic clip.
3. mycoplasma pneumoniae antibody as claimed in claim 2 (IgG/IgM) gold-labeled quick test card, it is characterized in that described mycoplasma pneumoniae antibody (IgG/IgM) gold-labeled quick test card (1) is to be provided with application of sample end water accepting layer (4), detection layers (8) and suction side water accepting layer (9) on liner plate (10), between detection layers (8) and suction side water accepting layer (9), be provided with gold mark anti-human IgG antibody's layer (5); On detection layers (8), be coated with detection line (7) and control line (6).
4. the preparation method of mycoplasma pneumoniae antibody as claimed in claim 2 (IgG/IgM) gold mark inspection card, it is characterized in that comprising golden labeling antibody layer (5) and detection layers (8) and go up the preparation of wrapping tested survey line (7) and control line (6), go up combination mycoplasma pneumoniae antibody (IgG/IgM) gold mark detector bar and test card (1) at liner plate (10).
5. the preparation method of mycoplasma pneumoniae antibody as claimed in claim 4 (IgG/IgM) gold mark test card, the preparation method who it is characterized in that described golden labeling antibody layer (5) comprises the following steps: the preparation of i collaurum: in concentration is 0.004%～0.012% gold chloride, add its volume 1.3%～2%, concentration is 1%～3% trisodium citrate, boiled 10～20 minutes, and can obtain the colloidal gold solution of 15～50 nanometers; The anti-human IgG of ii colloid gold label/IgM monoclonal antibody, in colloidal gold solution, transfer PH8.0～8.4 with the 0.2m solution of potassium carbonate, add anti-human IgG/IgM monoclonal antibody by 4～12mg/100ml, after the stirring, add animal blood serum albumen by 0.1～0.6g/100ml again in solution, 4 ℃ left standstill 2～4 hours; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10～15 minutes, remove sediment, supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60～80 minutes centrifugal sediments; V is dissolved in the 0.02MPh7.4Tris-Hcl damping fluid with sediment by 4～10ml/100ml and gets colloidal gold solution, contains 0.2%～0.6% animal blood serum albumen and 0.01%～0.06% Sodium azide in this damping fluid; Till vi immersed above-mentioned colloidal gold solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ of dry filters formed golden labeling antibody layer.
6. obtain the preparation method of 5 described mycoplasma pneumoniae antibodies (IgG/IgM) gold mark test card as claim 4, be characterised in that described colloid gold label antibody is anti-people Ig (IgG, IgM).
7. the preparation method of mycoplasma pneumoniae antibody as claimed in claim 4 (IgG/IgM) gold mark test card is characterized in that described detection layers (8) is to be provided with detection line (7) that the mycoplasma pneumoniae p1 protein antigen of gene engineering expression constitutes and the control line (6) that is formed by sheep, rabbit anti-mouse igg antibody on nitrocellulose membrane.
8. the preparation method of mycoplasma pneumoniae antibody gold mark detection kit as claimed in claim 7 is characterized in that described mycoplasma pneumoniae antigen is the mycoplasma pneumoniae p1 protein of gene engineering expression.
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| CN111505313A (en) * | 2020-05-07 | 2020-08-07 | 厦门万渤生物技术有限公司 | Diagnostic kit for combined diagnosis of mycoplasma pneumonia and application thereof |
| CN111505313B (en) * | 2020-05-07 | 2022-07-05 | 厦门万渤生物技术有限公司 | Diagnostic kit for combined diagnosis of mycoplasma pneumonia and application thereof |
| CN113817032A (en) * | 2021-09-28 | 2021-12-21 | 山东硕景生物科技有限公司 | Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody and preparation method and application thereof |
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