CN101067626B - Colloidal gold chromatographic band for detecting IgM, IgG antibody by double-capture method and producing method thereof - Google Patents
Colloidal gold chromatographic band for detecting IgM, IgG antibody by double-capture method and producing method thereof Download PDFInfo
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Abstract
The invention provides a double capture method to detect colloidal gold chromatography strip of the IgM, IgG antibody and its preparation technology. The chromatography strip takes the colloid gold as the marking matter, one end of the PVC backing mutually builds the sample pad, the compound pad, the nitryl cellulose membrane, another end pastes the absorb pad; the states compound pad is the glass cellulose membrane which coated with the antigen - colloid gold compound, which characteristic lies in: it contains three peridium lines on the states nitryl cellulose membrane, separately peridium the anti- IgG gamma chain antibody, the anti- IgM mu chain antibody, and the antibody which corresponds to antigen. The invention chromatography strip uses the double capture method to detect the IgM antibody, IgG antibody, through once operation detects the specificity IgM, IgG antibody, which simplified the operating process, and the examination result of which conforms to the actual examinationwork need.
Description
Technical field
The present invention relates to a kind of chromatography strip and technology of preparing thereof of utilizing immunoassay detection specificity IgM, IgG antibody; Specifically, relate to a kind of immunochromatography reaction pair specific IgM, IgG antibody and carry out chromatography strip of joint-detection and preparation method thereof by the colloid gold label colour developing.
Background technology
Immunoglobulin M (Immunoglobulin M, IgM) and immunoglobulin G (Immunoglobulin G IgG) is most important two kinds of antibody in the human or animal body.After a kind of antigen (bacterium or virus etc.) got into body, the immunoglobulin (Ig) that occurs the earliest was an IgM antibody, occurs IgG antibody afterwards.Although also IgA antibody possibly occur in some cases, because its clinical meaning is basic identical with IgG, and IgA content is very low, and the practical significance in Clinical detection is little, thereby in Clinical detection, does not generally distinguish.Through detecting the existence of IgM, IgG antibody in the body, can diagnose the immune response state of human or animal to specific antigen.If detect specific IgM antibodies, show the generation that recent infection is arranged, but IgM is shorter antibody half life in the serum; The detection feminine gender of IgM can not prove that body is infected; Sometimes it is longer also need to detect the half life period, and the IgG antibody that content is higher is to clarify a diagnosis.
Detection specificity IgM, IgG antibody mainly adopt the independent detection method of analysis-by-synthesis more at present; Adopt conventional methods such as ELISA method, dual-antigen sandwich method or colloidal gold immunochromatographimethod that IgM and IgG antibody are detected respectively earlier; The testing result of twice of analysis-by-synthesis is to diagnose the illness then; The whole operation process is loaded down with trivial details, detects convenient and swift inadequately.
Summary of the invention
Technical matters to be solved by this invention provides a kind of colloidal gold chromatography strip, adopts the dual seizure method to get final product joint detecting specific IgM, IgG antibody through the single job process, thereby simplifies the operation course, and realizes the purpose of fast detecting.
Technical scheme of the present invention is following:
A kind of dual seizure method detects the colloidal gold chromatography strip of IgM, IgG antibody, and said chromatography strip end on the PVC backboard is pasted with sample pad, compound pad, NC Nitroncellulose film in order each other overlap joint, and the other end is pasted with absorption pad; Said compound pad is the plain film of the spun glass that is coated with antigen-colloidal gold composite, and it is characterized in that: comprising three on the said NC Nitroncellulose film encapsulates line, be coated with respectively anti-IgG γ chain antibody, anti-IgM μ chain antibody, with the antigen corresponding antibody.
The present invention also provides the preparation method of said colloidal gold chromatography strip, may further comprise the steps:
(1) preparation anti-IgM μ chain antibody, anti-IgG γ chain antibody, with the antigen corresponding antibody: adopt routine fashion prepare anti-IgM μ chain antibody, anti-IgG γ chain antibody and with the corresponding animal's antibody of known antigens, concentration is respectively 1mg/ml～3mg/ml, 3mg/ml～5mg/ml, 2mg/ml～6mg/ml;
(2) preparation antigen-colloidal gold composite: adopt trisodium citrate reduction method to prepare collaurum, in aurosol,, after sealing, centrifugal treating, get deposition and be diluted to A according to the known antigens of 20 μ g/ml～80 μ g/ml ratios adding purifying
530Value is 1～3, and is subsequent use;
(3) get the NC Nitroncellulose film and be attached in the middle of the PVC backboard, will resist IgM μ chain antibody, anti-IgG γ chain antibody, separate with the antigen corresponding antibody and be coated on the NC Nitroncellulose film; Antigen-colloidal gold composite is coated in preparation compound pad on the plain film of spun glass;
(4) end is pasted with sample pad, compound pad, NC Nitroncellulose film in order each other overlap joint on the PVC backboard, and the other end attaches absorption pad;
(5) material of PVC backboard and attaching thereof is cut into the chromatography strip of suitable width.
Prepared anti-IgM μ chain antibody can be monoclonal or polyclonal antibody.
Prepared anti-IgG γ chain antibody can be monoclonal or polyclonal antibody.
Said antigen---colloidal gold composite can spraying, the mode of spreading or immersion is coated on the plain film of spun glass.
Said NC Nitroncellulose film encapsulate anti-IgM μ chain antibody, anti-IgG γ chain antibody, with the antigen corresponding antibody after can seal processing, to reduce nonspecific reaction.
Said chromatography strip can be packed into after preparation is accomplished in the plastics integrated circuit board.
Colloidal gold chromatography strip of the present invention; With the collaurum thing that serves as a mark, adopt the dual seizure method to measure IgM antibody and IgG antibody, get final product joint-detection through single job and go out specific IgM, IgG antibody; Simplified operating process, and the overall coincidence rate of testing result is higher.
Embodiment
Embodiment 1: the IgM, the IgG antibody that detect anti-human cytomegalovirus (HCMV)
1, detects the preparation method of the colloidal gold chromatography strip of human cytomegalovirus (HCMV) IgM, IgG antibody
(1) adopt the animal immune legal system to be equipped with the anti-HCMV antibody of rabbit that concentration is 5mg/ml, adopt the Monoclonal Antibody method to prepare the mouse-anti people IgM μ chain monoclonal antibody that concentration is 2mg/ml, the mouse-anti human IgG γ chain monoclonal antibody that concentration is 4mg/ml respectively, subsequent use.
(2) preparation antigen-colloidal gold composite: adopt trisodium citrate reduction method to prepare collaurum, in aurosol,, after sealing, centrifugal treating, get deposition and be diluted to A according to the HCMV antigen of 60 μ g/ml ratios adding purifying
530Value 2.5, subsequent use.
(3) encapsulate mouse-anti people IgM μ chain monoclonal antibody, mouse-anti human IgG γ chain monoclonal antibody, the anti-HCMV antibody of rabbit: get the NC Nitroncellulose film and be affixed in the middle of the PVC backboard, setting and drawing film machine coating parameters is 1 μ l/cm.Get the anti-HCMV antibody of the rabbit 1.5ml of 5mg/ml, the mouse-anti human IgG γ chain monoclonal antibody 1.5ml of 4mg/ml, the mouse-anti people IgM μ chain monoclonal antibody 1.5ml of 2mg/ml, receive A, B, the C pipe joint of drawing the film machine respectively.The PVC backboard that posts the NC Nitroncellulose film is placed on the to-and-fro movement platform of drawing the film machine.Open to draw a film machine, on the NC Nitroncellulose film, apply the anti-HCMV antibody of rabbit, mouse-anti human IgG γ chain monoclonal antibody, mouse-anti people IgM μ chain monoclonal antibody, 37 ℃ of dryings 2 hours.Place 1% polyglycol solution to soak 30 minutes backboard, dry, 37 ℃ of dryings 2 hours.
(4) antigen-colloidal gold composite is coated on the plain film of spun glass: setting and drawing film machine spray parameters is 30.0 μ l/cm, antigen-colloidal gold composite is connected to the D pipe joint of drawing the film machine.Getting the plain film of spun glass of 30cm * 12cm places on the to-and-fro movement platform.Open to draw a film machine, on the plain film of spun glass, spray antigen-colloidal gold composite and process the compound pad, 37 ℃ of dryings 2 hours.
(5) end sticks compound pad and sample pad in order each other overlap joint on the PVC backboard, and the other end sticks absorption pad, and the material of PVC backboard and attaching thereof is cut into the wide chromatography strip of 4mm.Chromatography strip is put into the plastics integrated circuit board, use the card press machine compacting.
After adding sample to be detected, through the chromatography effect, sample and antigen-colloidal gold composite move to absorption pad one end.When sample encapsulated the place through anti-IgM μ chain antibody, IgM antibody combined with anti-IgM μ chain antibody, and the IgM antibody to specific antigen that wherein contains combines with antigen-colloidal gold composite, showed an aubergine band at this place.If contain IgG antibody in the sample to be detected to specific antigen; When moving to anti-IgG γ chain antibody, it encapsulates when place; Specific IgG antibodies both combined with anti-IgG γ chain antibody, also combined with antigen-colloidal gold composite, showed an aubergine band herein.Antigen-colloidal gold composite moves to when place of encapsulating with its corresponding antibodies, shows an aubergine band with antibodies.
During actual detected, show the IgM antibody that contains in the sample to be detected to specific antigen by the aubergine band, be judged to be positive findings as if showing at the anti-IgM μ chain antibody place of encapsulating; If demonstration aubergine band shows the IgG antibody that contains in the sample to be detected to specific antigen at the anti-IgG γ chain antibody place of encapsulating, be judged to be positive findings; If all show the aubergine band at anti-IgM μ chain antibody and the anti-IgG γ chain antibody place of encapsulating, show the IgM antibody and the IgG antibody that contain in the sample to be detected to specific antigen, be judged to be positive findings; Otherwise show the IgM antibody, the IgG antibody that do not contain in the sample to be detected to specific antigen, be judged to be negative findings.Antibody sandwich place to specific antigen shows the aubergine band, points out the testing result of this detection system effective; If this place does not show the aubergine band, show that this detection system lost efficacy, testing result is invalid.
2, detect anti-human cytomegalovirus (HCMV) IgM, IgG detection of antibodies result
The colloidal gold chromatography strip of using preparation detects for 2197 parts clinical diagnosis clear and definite 278 parts of anti-HCMV IgG positive serums and negative serum; And contrast with result that indirect method ELISA reagent detects separately anti-HCMV IgG, the result is shown in table 1 and table 2 respectively.
The testing result of the clinical serum of table 1 colloidal gold chromatography strip antagonism HCMV IgG
The result that table 2 collaurum and ELISA detect anti-HCMV IgG compares
Can calculate youden index by table 1 data is 261/ (261+17)+2195/ (2+2195)-1=0.94, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 2 data is: (262+2192)/2475 * 100%=99.2%, this shows that two kinds of detection methods detect anti-human cytomegalovirus (HCMV) IgG and have the height consistance.
The colloidal gold chromatography strip of using preparation detects for 1569 parts clinical diagnosis clear and definite 153 parts of anti-HCMV IgM positive serums and negative serum; And contrast with result that prize law ELISA reagent detects separately anti-HCMV IgM antibody, the result is shown in table 3 and table 4 respectively.
The testing result of the clinical serum of table 3 colloidal gold chromatography strip antagonism HCMV IgM
The result that table 4 collaurum and ELISA detect anti-HCMV IgM compares
Can calculate youden index by table 3 data is 141/ (141+12)+1568/ (1+1568)-1=0.92, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 4 data is: (143+1566)/1722 * 100%=99.2%, this shows that two kinds of detection methods detect anti-human cytomegalovirus (HCMV) IgM and have the height consistance.
Above analysis result shows; The colloidal gold chromatography strip of the anti-human cytomegalovirus IgM of joint-detection of the present invention, IgG antibody has similar testing result with the ELISA reagent that detects IgG or IgM antibody separately, and testing result of the present invention can meet the actual detected requirements of one's work.
Embodiment 2: the IgM, the IgG antibody that detect wind resistance exanthema virus (RV)
Adopt the colloidal gold chromatography strip that detects wind resistance exanthema virus (RV) IgM, IgG antibody with embodiment 1 similar method preparation.The colloidal gold chromatography strip of using preparation detects for 1563 parts clinical diagnosis clear and definite 568 parts of anti-RV IgG positive serums and negative serum; And contrast with result that indirect method ELISA reagent detects separately anti-RV IgG, the result is shown in table 5 and table 6 respectively.
The testing result of the clinical serum of table 5 colloidal gold chromatography strip antagonism RV IgG
The result that table 6 collaurum and ELISA detect anti-RV IgG compares
Can calculate youden index by table 5 data is 534/ (534+34)+1562/ (1+1562)-1=0.94, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 6 data is: (539+1560)/2131 * 100%=98.5%, this shows that two kinds of detection methods detect anti-RV IgG and have the height consistance.
The colloidal gold chromatography strip of using preparation detects for 1968 parts clinical diagnosis clear and definite 136 parts of anti-RV IgM positive serums and negative serum; And contrast with result that prize law ELISA reagent detects separately anti-RV IgM antibody, the result is shown in table 7 and table 8 respectively.
The testing result of the clinical serum of table 7 colloidal gold chromatography strip antagonism RV IgM
The result that table 8 collaurum and ELISA detect anti-RV IgM compares
Can calculate youden index by table 7 data is 127/ (127+9)+1966/ (2+1966)-1=0.93, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 8 data is: (129+1966)/2104 * 100%=99.6%, this shows that two kinds of detection methods detect anti-RV IgM and have the height consistance.
Above analysis result shows; The colloidal gold chromatography strip of joint-detection rubella virus IgM of the present invention, IgG antibody has similar testing result with the ELISA reagent that detects IgG or IgM antibody separately, and testing result of the present invention can meet the actual detected requirements of one's work.
Embodiment 3: the IgM, the IgG antibody that detect anti-herpes simplex virus (HV)
Adopt the colloidal gold chromatography strip that detects anti-herpes simplex virus (HV) IgM, IgG antibody with embodiment 1 similar method preparation.The colloidal gold chromatography strip of using preparation detects for 1863 parts clinical diagnosis clear and definite 326 parts of anti-HV IgG positive serums and negative serum; And contrast with result that indirect method ELISA reagent detects separately anti-HV IgG, the result is shown in table 9 and table 10 respectively.
The testing result of the clinical serum of table 9 colloidal gold chromatography strip antagonism HV IgG
The result that table 10 collaurum and ELISA detect anti-HV IgG compares
Can calculate youden index by table 9 data is 304/ (304+22)+1862/ (1+1862)-1=0.93, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 10 data is: (308+1856)/2189 * 100%=98.9%, this shows that two kinds of detection methods detect anti-HV IgG and have the height consistance.
The colloidal gold chromatography strip of using preparation detects for 1836 parts clinical diagnosis clear and definite 132 parts of anti-HV IgM positive serums and negative serum; And contrast with result that prize law ELISA reagent detects separately anti-HV IgM antibody, the result is shown in table 11 and table 12 respectively.
The testing result of the clinical serum of table 11 colloidal gold chromatography strip antagonism HV IgM
The result that table 12 collaurum and ELISA detect anti-HV IgM compares
Can calculate youden index by table 11 data is 123/ (123+9)+1835/ (1+1835)-1=0.93, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 12 data is: (125+1830)/1968 * 100%=99.3%, this shows that two kinds of detection methods detect anti-HV IgM and have the height consistance.
Above analysis result shows; The colloidal gold chromatography strip of joint-detection herpes simplex virus I gM of the present invention, IgG antibody has similar testing result with the ELISA reagent that detects IgG or IgM antibody separately, and testing result of the present invention can meet the actual detected requirements of one's work.
Embodiment 4: the IgM, the IgG antibody that detect anti-toxoplasm (Tox)
Adopt the colloidal gold chromatography strip that detects toxoplasm (Tox) IgM, IgG antibody with embodiment 1 similar method preparation.The colloidal gold chromatography strip of using preparation detects for 1769 parts clinical diagnosis clear and definite 368 parts of anti-Tox IgG positive serums and negative serum; And contrast with result that indirect method ELISA reagent detects separately anti-Tox IgG, the result is shown in table 13 and table 14 respectively.
The testing result of the clinical serum of table 13 colloidal gold chromatography strip antagonism Tox IgG
The result that table 14 collaurum and ELISA detect anti-Tox IgG compares
Can calculate youden index by table 13 data is 349/ (349+19)+1768/ (1+1768)-1=0.95, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 14 data is: (348+1766)/2137 * 100%=98.9%, this shows that two kinds of detection methods detect anti-Tox IgG and have the height consistance.
The colloidal gold chromatography strip of using preparation detects for 1836 parts clinical diagnosis clear and definite 132 parts of anti-Tox IgM positive serums and negative serum; And contrast with result that prize law ELISA reagent detects separately anti-Tox IgM antibody, the result is shown in table 15 and table 16 respectively.
The testing result of the clinical serum of table 15 colloidal gold chromatography strip antagonism Tox IgM
The result that table 16 collaurum and ELISA detect anti-Tox IgM compares
Can calculate youden index by table 15 data is 202/ (202+14)+1583/ (3+1583)-1=0.93, explains that the overall coincidence rate of testing result and sample truth is very high.The overall coincidence rate that can be calculated testing result of the present invention and ELISA testing result by table 16 data is: (205+1581)/1802 * 100%=99.1%, this shows that two kinds of detection methods detect anti-ToxIgM and have the height consistance.
Above analysis result shows; The colloidal gold chromatography strip of the anti-toxoplasm IgM of joint-detection of the present invention, IgG antibody has similar testing result with the ELISA reagent that detects IgG or IgM antibody separately, and testing result of the present invention can meet the actual detected requirements of one's work.
Claims (7)
1. a dual seizure method detects the colloidal gold chromatography strip of IgM, IgG antibody, and said chromatography strip end on the PVC backboard is pasted with sample pad, compound pad, NC Nitroncellulose film in order each other overlap joint, and the other end is pasted with absorption pad; Said compound pad is the plain film of the spun glass that is coated with antigen-colloidal gold composite, and it is characterized in that: comprising three on the said NC Nitroncellulose film encapsulates line, be coated with respectively anti-IgG γ chain antibody, anti-IgM μ chain antibody, with the antigen corresponding antibody.
2. method for preparing the said colloidal gold chromatography strip of claim 1 may further comprise the steps:
(1) preparation anti-IgM μ chain antibody, anti-IgG γ chain antibody, with the antigen corresponding antibody: adopt routine fashion prepare anti-IgM μ chain antibody, anti-IgG γ chain antibody and with the corresponding animal's antibody of known antigens, concentration is respectively 1mg/ml～3mg/ml, 3mg/ml～5mg/ml, 2mg/ml～6mg/ml:
(2) preparation antigen-colloidal gold composite: adopt trisodium citrate reduction method to prepare collaurum, in aurosol,, after sealing, centrifugal treating, get deposition and be diluted to A according to the known antigens of 20 μ g/ml～80 μ g/ml ratios adding purifying
530Value is 1～3, and is subsequent use;
(3) get the NC Nitroncellulose film and be attached in the middle of the PVC backboard, will resist IgM μ chain antibody, anti-IgG γ chain antibody, separate with the antigen corresponding antibody and be coated on the NC Nitroncellulose film; Antigen-colloidal gold composite is coated in preparation compound pad on the plain film of spun glass;
(4) end is pasted with sample pad, compound pad, NC Nitroncellulose film in order each other overlap joint on the PVC backboard, and the other end attaches absorption pad;
(5) material of PVC backboard and attaching thereof is cut into the chromatography strip of suitable width.
3. preparation method according to claim 2 is characterized in that: prepared anti-IgM μ chain antibody is monoclonal or polyclonal antibody.
4. preparation method according to claim 2 is characterized in that: prepared anti-IgG γ chain antibody is monoclonal or polyclonal antibody.
5. preparation method according to claim 2 is characterized in that: said antigen-colloidal gold composite is coated on the plain film of spun glass with the mode of spraying, spreading or immersion.
6. preparation method according to claim 2 is characterized in that: said NC Nitroncellulose film encapsulate anti-IgM μ chain antibody, anti-IgG γ chain antibody, with the antigen corresponding antibody after can also seal processing.
7. preparation method according to claim 2 is characterized in that: said chromatography strip is packed into after preparation is accomplished in the plastics integrated circuit board.
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| CN101825636B (en) * | 2010-05-19 | 2013-06-26 | 厦门大学附属中山医院 | Reagent strip for joint detection of syphilis specific IgM antibody and specific total antibody and preparation method thereof |
| CN101825635B (en) * | 2010-05-19 | 2014-07-16 | 厦门大学附属中山医院 | Reagent strip for joint detection of syphilis specific IgG antibody and specific total antibody and preparation method thereof |
| CN101825634A (en) * | 2010-05-19 | 2010-09-08 | 厦门大学附属中山医院 | Reagent strip for joint detection of syphilis specific IgM and IgG antibodies and preparation method thereof |
| CN104280543B (en) * | 2013-07-11 | 2016-08-24 | 国家纳米科学中心 | A kind of method of IgG and IgM in reagent paper and a kind of fluid sample of detection simultaneously |
| CN109596824A (en) * | 2019-01-04 | 2019-04-09 | 杭州奥泰生物技术股份有限公司 | A kind of Test paper and preparation method thereof of quick diagnosis Lyme disease |
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| CN1431509A (en) * | 2003-01-24 | 2003-07-23 | 湖州瑞泽生物技术有限公司 | Immunity colloidal gold reagent for detecting IgM antibody of rubella virus and its preparing method |
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