CN102368068B - Kit for detecting chlamydia pneumoniae IgM antibody - Google Patents
Kit for detecting chlamydia pneumoniae IgM antibody Download PDFInfo
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Abstract
The invention relates to a kit for detecting Chlamydia pneumoniae IgM antibody, and belongs to a medical detection reagent. The kit provided by the invention comprises a microplate coated with avidin, a biotinylated chlamydia pneumoniae antigen, horseradish peroxidase labelled anti-human IgM and a chemiluminescence substrate. According to the invention, the microplate is coated with avidin and the biotinylated antigen is indirectly coated through an avidin-biotin system. The invention provides convenience to amplify production and monitor the production process, avoids defects of big activity loss and cockamamie process optimization by directly coating a solid phase, and simultaneously ensures the stability among each batch.
Description
Technical field
The present invention relates to medical science and detect reagent, particularly a kind of kit that detects chlamydia pneumoniae IgM antibody.
Background technology
Chlamydia pneumoniae (Chlamydia pneumoniae, Cpn) be the entozoic prokaryotic microorganism of definite a kind of strict eukaryotic in 1989, according to the uncle Jie Shi systematic bacteriology handbook of announcement in 2004, renamed as Chlamydophila pneumoniae (Chlamydophila pneumoniae, Cpn), have a liking for Chlamydia, cavy with Chlamydophila psittaci, miscarriage and have a liking for that Chlamydia, cat are had a liking for Chlamydia, beasts are had a liking for Chlamydia and have a liking for chlamydiaceae common composition.The about 1.2Mbp of CPn Genome Size, by 21 Pmp genes, an III type secretion virulence factor system, 3 serine/threonine protein kitases, 2 phosphatidase-D sample protein gene form.
The infection of Cpn is very general, be global distribution, mainly cause the mankind's respiratory tract infection, be proportionate with the density of population, mainly with recessive, persistent infection form, exist clinically, delay causes being difficult to the complication for the treatment of repeatedly, with the generation of the cardiovascular and cerebrovascular disease such as asthma, bronchitis, chronic obstructive pulmonary disease, atherosclerotic, miocardial infarction, coronary heart disease with develop closely related.Childhood infection rate is in 20% left and right, and along with the increase infection rate at age rises rapidly, person between twenty and fifty can reach 50-60%, old 70-80%.Chlamydia pneumoniae is carried out person to person's propagation by respiratory secretions, and the perform region of family, school, army and other population concentrations can exist among a small circle popular.At present, Chlamydia pneumoniae has been after streptococcus pneumonia and haemophilus influenzae, to cause community acquired pneumonia (community acquired pneumonia, CAP) main pathogens, Chlamydia pneumoniae acute infection rate accounts for 23%, pneumonia patient only accounts for the infected's fraction, and 70-90% is subclinical performance.Be 15-23 days the latent period of infection involving chlamydia pneumoniae, can cause the infection of the upper respiratory tract, as nasosinusitis, tympanitis and pharyngitis, also can cause lower respiratory infection, as bronchitis and pneumonia.Respiratory tract infection majority shows as pharyngalgia, heating, cough, and the state of an illness is conventionally lighter, has self limiting.Pneumonia in Older Patients chlamydia pneumonia patient symptom may be comparatively serious, sometimes even lethal, when especially merging bacterial infection or having chronic obstructive pulmonary disease etc.Therefore it is extremely important for finding as early as possible and treat Cpn infectious diseases that, Cpn infects early diagnosis.
At present the laboratory diagnostic method of Cpn is mainly contained to the methods such as pathogen isolation culture technique, nucleic acid detection technique, serodiagnosis, there is no in the world the recognized standard method.Pathogen isolation culture technique wherein, due to the resistibility of CPn a little less than, to room temperature or freezing sensitivity, be difficult to separated cultivation successfully from clinical samples, common laboratory is difficult to carry out, and is replaced gradually by other method for quick.Although complement fixation test (CFT) Test Condition Requirements is low, do not need specific apparatus, there is the defects such as influence factor complexity, operation steps be cumbersome.Microimmunofluorescence (micro-immunofluorescence, MIF) is the most frequently used and the most responsive serological method of current CPn Infect And Diagnose, and its result is considered to detect " goldstandard " that CPn infects.Its shortcoming is to waste time and energy very much, and the reliability of MIF testing result depends on the preparation and determination methods person's of antigen personal experience to a great extent, and the subjectivity of experimental implementation is very large, only limits to a few studies laboratory and uses.Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) have quick, responsive, easy, be easy to the advantages such as standardization, it is developed and widespread use rapidly, become one of detection method of widespread use.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is a kind of highly sensitive microdetermination technology growing up after radiommunoassay and EIA enzyme immunoassay in recent years.Having wide, the easy and simple to handle advantage such as fast of high sensitivity, sensing range, as the replacer of radioimmunoassay, EIA enzyme immunoassay, is current optimal immune analysis method.
The application of Avidin-Biotin system in immunoassay has various ways, comprising forms such as signal amplify, is indirectly coated with.Signal amplification refers to biotinylated antigen or antibody and enzyme mark Avidin reaction system, its advantage is to utilize biotin-avidin enlarge-effect to improve detection sensitivity, shortcoming is that the isoelectric point of Avidin is higher, the solid phase carriers such as p-poly-phenyl ethene have higher non-specific adsorption, thereby cause background higher.
Summary of the invention
The present invention, according to the blank of the existence in above-mentioned field and demand, provides a kind of kit that detects chlamydia pneumoniae IgM antibody, experiment showed, have easy and simple to handle, highly sensitive, the advantage that specificity is good.
Detect the kit of chlamydia pneumoniae IgM antibody, the microwell plate that comprises coated Avidin, anti-human IgM and the chemical luminous substrate of biotinylated Chlamydia pneumoniae antigen, horseradish peroxidase-labeled, described chemical luminous substrate comprises substrate solution A and substrate solution B, what wherein A liquid was consists of: pH value is 7.2～8.5, concentration is that in the phosphate buffer of 10～30mM, to add final concentration (W/V) be 0.02～0.1%Proclin300,0.1～1.0% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.02～0.1%Proclin300, the 4-iodophenol of 1.0～3.0mM, 0.1%～1% BSA, 0.1～1% cetrimonium bromide, 10～50mM luminol solution.
Described biotinylated Chlamydia pneumoniae antigen is adopted and is prepared with the following method and obtain: it is 1mg/mL that (1) biotin is dissolved in DMF final concentration; (2) use the NaHCO of 0.1mol/L pH 9.0
3chlamydia antigen dilution is 1～2mg/mL; (3) press 1: 7 left and right of biotin and antigen weight ratio and mix, under stirring at room, react 2～4h; (4) pack bag filter into, with the PBS of 0.05mol/L pH7.2,4 ℃ of dialysed overnight obtain biotinylated antigen, and dialysis product adds equal-volume glycerine and dissolves standby.
The microwell plate preparation method of described coated Avidin is: the coated microwell plate of Avidin coating buffer that is mixed with 2ug/ml with the carbonate buffer solution of pH 9.6.
Described kit also comprises dilution, cleansing solution, chlamydial antibody IgM negative control, positive control, calibration object, the anti-human IgM of horseradish peroxidase-labeled.
Described anti-human IgM is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibody.
Kit of the present invention adopts the coated microwell plate of Avidin, form Solid-phase avidin-bioid antigen-antibody complex with Chlamydia pneumoniae IgM in biotinylated antigen and sample to be tested, form the anti-sandwich complex of Solid-phase avidin-biotinylated antigen-antibody-mark two with the anti-human IgM of mark again, then add chemical luminous substrate, utilize Chemiluminescence Apparatus to detect relative luminous intensity, chlamydia pneumoniae IgM antibody in qualitative or half-quantitative detection sample to be tested, thereby avoided enzyme to be excused from an examination, sensitivity that agent exists is low, the shortcomings such as part substrate is poisonous or carcinogenic, it is fast that this kit has reaction velocity, light signal is not subject to influence of temperature change, cost is low, the advantages such as easy storage, can be the acute respiratory disease auxiliary diagnosis that clinical Chlamydia pneumoniae is relevant and provide more special, fast, reliable diagnosis basis.
A main contribution of the present invention has been to provide a kind of new Chemoluminescent substrate formula, compares with adopting the kit of existing conventional substrate solution formula, has the advantages such as highly sensitive, good stability.
Embodiment
Embodiment 1 kit preparation method of the present invention
1, kit of the present invention comprises:
1) Chlamydia pneumoniae IgM positive control; 2) Chlamydia pneumoniae IgM negative control; 3) Chlamydia pneumoniae IgM calibration object; 4) microwell plate of coated Avidin; 5) biotinylated Chlamydia pneumoniae antigen; 6) anti-human IgM enzyme labeling thing; 7) 20 times of concentrated cleaning solutions; And 8) Chemoluminescent substrate.
Wherein, microwell plate is the micropore lath in 24,48 or 96 holes;
Anti-human IgM is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibody;
Enzyme is horseradish peroxidase;
Chemoluminescent substrate: A liquid and B liquid.
2, the preparation method of I kit of the present invention:
With Chlamydia pneumoniae IgM positive serum preparation positive control, with Healthy Human Serum preparation negative control, with Chlamydia pneumoniae IgM positive serum preparation calibration object, coated in microporous plate Avidin, make Chlamydia pneumoniae antigen biotinylation, the anti-human IgM of mark, preparation chemical luminous substrate, the above-mentioned component of packing, and be assembled into finished product.
2.1 coated in microporous plate Avidins:
2.1.1 coated
With the carbonate buffer solution that pH value is 9.6, be mixed with the Avidin coating buffer of desired concn, and coating buffer is carried on to microwell plate (100ul/ hole)
Avidin coating buffer formula
2.1.2 sealing is with containing BSA, and pH value is 7.0-7.5, and the Tris damping fluid that concentration is 10mM seals the solid phase carrier after above-mentioned washing as confining liquid.
Confining liquid formula
The preparation of the anti-human IgM antibody of 2.2 enzyme mark adopts the sodium periodate method of improvement.Concrete steps are referring to " Acta Biochimica et Biophysica Sinica " 16 volume the 6th phase in 1984, Luo Jiali etc. " the some problems in sodium periodate oxidation horseradish peroxidase (HRP) and the labeling method of high effect ".
The preparation of 2.3 biotin coupling Chlamydia pneumoniae antigens
(1) biotin BNHS (purchased from U.S. Sigma) is dissolved in to DMF (DMF) and is made into 1mg/mL;
(2) NaHCO that is 9.0 by 0.1mol/L pH value
3by the Chlamydia antigen of purifying (purchased from the expensive Xiang in Guangzhou) dilution, be 1～2mg/mL; (3) by BNHS and antigen volumetric ratio, be that mix 1: 8 or 1: 7 left and right of weight ratio, under stirring at room, react 2-4h; (4) pack the PBS that bag filter is 7.2 to 0.05mol/L pH value into, 4 ℃ of dialysed overnight, bond adds equal-volume glycerine, packing in a small amount ,-20 ℃ save backup below.
The preparation of 2.4 biotinylated antigens, enzymic-labelled antibody dilution
The preparation of the anti-human IgM working fluid of 2.5 biotinylated antigens and enzyme labeling
The biotinylated antigen of preparation, the anti-human IgM antibody of enzyme labeling are diluted to respectively to variable concentrations with dilution, use chessboard titrimetry to select best dilutability for being respectively 1: 3000,1: 7000 (V/V).
2.620 cleansing solution formula doubly
During use, distilled water dilution is 20 times.
2.7HRP chemical luminous substrate
Each 1 bottle of A liquid and B liquid, each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, and in the phosphate buffer that concentration is 10mM, adding final concentration (V/V) is 0.02%Proclin300,1.0% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.02%Proclin300, the 4-iodophenol of 2mM, 0.1% BSA, 0.1% cetrimonium bromide, 10Mm luminol solution.
II kit:
Other is with I kit, the formula of HRP chemical luminous substrate is: each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, concentration is that in the phosphate buffer of 30mM, to add final concentration (V/V) be 0.05%Proclin300,0.5% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.05%Proclin300, the 4-iodophenol of 3mM, 1% BSA, 1% cetrimonium bromide, 50mM luminol solution.
III kit:
Other is with I kit, the formula of HRP chemical luminous substrate is: each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, concentration is that in the phosphate buffer of 20mM, to add final concentration (V/V) be 0.04%Proclin300,0.1% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.04%Proclin300, the 4-iodophenol of 2mM, 0.7% BSA, 0.4% cetrimonium bromide, 30mM luminol solution.
Contrast agents box: other is with I kit, and chemical reflective substrate is commercially available chemical luminous substrate (A liquid is urea peroxide solution, and B liquid is luminol solution, purchased from Huzhou Yingcheng Biological Technology Co., Ltd.).
3, the using method of kit of the present invention
1) in 2-8 ℃ of refrigerator, take out kit, equilibrium at room temperature 15 minutes.
2) take out coated slab, insert on grillage.
3) negative control, negative control, each 2 every hole 50ul in hole of calibration object are established in each test, the sample to be tested 50ul of pre-dilution (1: 100), and then every hole adds 50ul biotinylated antigen, and vibration mixes rear 37 ℃ of incubations 30 minutes.
4) get rid of dereaction liquid, the cleansing solution after dilution is filled it up with in every hole, washes plate 5 times, finally on clean thieving paper, buckles dry.
5) every hole adds respectively 100 μ L enzyme labeling things, 37 ℃ of incubations 20 minutes.
6) get rid of dereaction liquid, the cleansing solution after dilution is filled it up with in every hole, washes plate 5 times, finally on clean thieving paper, buckles dry.
7) each hole adds each 50 μ L of Chemoluminescent substrate A, B, fully vibrates and mixes, room temperature (20～25 ℃) lucifuge reaction 5 minutes with micro-oscillator.
8) must in 5th～30 minutes after adding Chemoluminescent substrate, measure, on chemiluminescence measuring instrument, sequentially measure the luminous intensity (RLU) in each hole, 1 second/hole of Measuring Time.
9), with calibration object critical value, qualitative or sxemiquantitative judges in sample to be tested whether have chlamydia pneumoniae IgM antibody or its content
4, the parallel contrast test of I kit of the present invention and ELISA reagent
To normal check sample 358 examples, respiratory tract infection sample 254 examples (being derived from Wuzhou treatment and prevention of tumour research institute) adopt kit of the present invention (chemoluminescence method) and chlamydia pneumoniae IgM antibody detection reagent (euzymelinked immunosorbent assay (ELISA)) to carry out Parallel testing, and trace routine and result judgement are carried out in strict accordance with each reagent instructions.
Table 1. chlamydia pneumoniae IgM antibody detects reagent (euzymelinked immunosorbent assay (ELISA)) measurement result
Table 2. I kit measurement of the present invention result
The comparison of table 3. I kit of the present invention and chlamydia pneumoniae IgM antibody detection kit (euzymelinked immunosorbent assay (ELISA))
Described in table 1-3, data show, kit of the present invention is 89.9% with the positive coincidence rate of contrast enzyme-linked immunologic detecting kit, and negative match-rate is that the coincidence rate of 99.8%, two kind of method of inspection is 98%.Empirical tests, enzyme-linked immunologic detecting kit detects 1 part of false positive, 11 parts of false negatives, and kit testing result of the present invention conforms to completely with clinical effectiveness.
II kit, III kit contrast test experience result conform to I kit.
Embodiment 2. kit chemical luminous substrate of the present invention and market sale chemical luminous substrate contrast test
Contrast agents box: identical with the kit of embodiment 1, difference is that chemical luminous substrate adopts the Chemoluminescent substrate (purchased from Huzhou Yingcheng Biological Technology Co., Ltd.) of market sale.
It is 358 parts of respiratory tract infection sample 254 examples and Healthy People blood samples in embodiment 1 that step 1 detects sample.
Detect operation steps with the using method of embodiment 1 step 3.
Result is as shown in table 4.
The chemical luminous substrate of table 4. I kit of the present invention chemical luminous substrate and market sale is compared
Described in table 4, data show, I kit chemical luminous substrate of the present invention is 89.9% with the positive coincidence rate of contrast chemical luminous substrate, and negative match-rate is that the coincidence rate of 100%, two kind of method of inspection is 98.2%.Empirical tests, contrast luminous substrate detects 11 parts of false negatives, and kit testing result of the present invention conforms to completely with clinical effectiveness.
II kit, III kit contrast test experience result conform to I kit, the present invention are described than adopting the kit of contrast luminous substrate and have higher detection sensitivity.
2-8 ℃ of step 2. I kit of the present invention, II kit, III kit were preserved after 6 months, for detection of the same lot sample of step 1 this, result shows, positive rate does not change, the rate that conforms to Clinical results is 100%.And adopt the kit that contrasts luminous substrate, and to preserve after 6 months for 2-8 ℃, false negative ratio brings up to 14/109 by 11/109, illustrates in kit of the present invention that chemical luminous substrate has good stability, is suitable for a large amount of production in batches, and testing result is reliable and stable.
Claims (5)
1. detect the kit of chlamydia pneumoniae IgM antibody, the microwell plate that comprises coated Avidin, anti-human IgM and the chemical luminous substrate of biotinylated Chlamydia pneumoniae antigen, horseradish peroxidase-labeled, described chemical luminous substrate comprises substrate solution A and substrate solution B, wherein
Consisting of of A liquid: pH value is 8.0, in the phosphate buffer that concentration is 10～30mM, adding final concentration is the Proclin300 of 0.02～0.05% (W/V), the Tween-20 of 0.1～1.0% (W/V) and 10mM urea peroxide solution;
B liquid is that in the Tris-HCl damping fluid of 50mM, to add final concentration be 0.02～0.05% (W/V) Proclin300, the 4-iodophenol of 2.0～3.0mM, the BSA of 0.1%～1% (W/V), the cetrimonium bromide of 0.1～1% (W/V), 10～50mM luminol solution.
2. kit according to claim 1, described biotinylated Chlamydia pneumoniae antigen is adopted and is prepared with the following method and obtain: it is 1mg/mL that (1) biotin is dissolved in DMF final concentration; (2) use the NaHCO of 0.1mol/L pH9.0
3by Chlamydia antigen dilution, be 1～2mg/mL; (3) press biotin and mix with antigen weight ratio 1:7 left and right, under stirring at room, react 2～4h; (4) pack bag filter into, with the PBS of 0.05mol/L pH7.2,4 ℃ of dialysed overnight obtain biotinylated antigen, and dialysis product adds equal-volume glycerine and dissolves standby.
3. kit according to claim 1, the microwell plate preparation method of described coated Avidin is: with the carbonate buffer solution that pH value is 9.6, be mixed with the coated microwell plate of 2 μ g/mL Avidin coating buffers.
4. kit according to claim 1, described kit also comprises dilution, cleansing solution, chlamydia pneumoniae (cp) IgM negative control, chlamydia pneumoniae (cp) IgM positive control, chlamydia pneumoniae (cp) IgM calibration object, the anti-human IgM of horseradish peroxidase-labeled.
5. according to the arbitrary described kit of claim 1～4, the anti-human IgM of horseradish peroxidase-labeled is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibody.
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| CN201110183271.1A CN102368068B (en) | 2011-06-30 | 2011-06-30 | Kit for detecting chlamydia pneumoniae IgM antibody |
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| CN201110183271.1A CN102368068B (en) | 2011-06-30 | 2011-06-30 | Kit for detecting chlamydia pneumoniae IgM antibody |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102749446A (en) * | 2012-07-21 | 2012-10-24 | 北京中检安泰诊断科技有限公司 | Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof |
| CN103048465B (en) * | 2012-11-28 | 2015-07-22 | 同昕生物技术（北京）有限公司 | IL-6 (Inter Leukin-6) micro-pore plate type chemiluminescent detection kit and manufacturing method thereof |
| CN106404754A (en) * | 2016-06-30 | 2017-02-15 | 深圳市亚辉龙生物科技股份有限公司 | A chlamydiae pneumoniae IgM chemiluminescent immunoassay kit and a preparing method thereof |
| CN106248944A (en) * | 2016-06-30 | 2016-12-21 | 深圳市亚辉龙生物科技股份有限公司 | A kind of CPn IgG chemiluminescence immune detection reagent kit and preparation method thereof |
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