CN106248944A - A kind of CPn IgG chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
A kind of CPn IgG chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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- CN106248944A CN106248944A CN201610506944.5A CN201610506944A CN106248944A CN 106248944 A CN106248944 A CN 106248944A CN 201610506944 A CN201610506944 A CN 201610506944A CN 106248944 A CN106248944 A CN 106248944A
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- preparation
- acridinium ester
- test kit
- cpn
- chemiluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Abstract
The invention discloses a kind of CPn IgG chemiluminescence immune detection reagent kit, described test kit includes: the antigen coated magnetic particle of Chlamydia pneumoniae, the mouse-anti human IgG antibody of acridinium ester label, CPn IgG calibration product, preexciting liquid, exciting liquid.Additionally the invention also discloses the preparation method of a kind of CPn IgG chemiluminescence immune detection reagent kit.The advantages such as test kit of the present invention is easy and simple to handle compared with available reagent box, highly sensitive, and detection range is wide.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of chemiluminescence immunoassay inspection
Survey CPn IgG test kit and preparation method thereof.
Background technology
Chlamydia pneumoniae (Chlamydia pneumoniae, Cpn) be the strict eukaryotic cell of the one determined for 1989 in
Parasitic prokaryotic cell microorganism, according to the primary Jie Shi systematic bacteriology handbook of announcement in 2004, is renamed as pneumonia addicted to clothing
Substance, with Chlamydophila psittaci, C. abortus, Cavia porcellus addicted to chlamydia, cat addicted to chlamydia, beasts addicted to common group of chlamydia
Become addicted to chlamydiaceae.CPn Genome Size about 1.2Mbp, by 21 Pmp genes, a type III secretion virulence factor system, 3
Individual serine/threonine protein kitase, 2 phospholipase-D sample protein gene compositions.
The infection of Cpn is extremely widespread, in global distribution, mainly causes the respiratory tract infection of the mankind, with population density
Being proportionate, many clinically with recessive, persistent infection form existence, delay causes the complication being difficult to treat repeatedly, with
Sending out of the cardiovascular and cerebrovascular diseases such as asthma, bronchitis, chronic obstructive pulmonary disease, atherosclerosis, myocardial infarction, coronary heart disease
Give birth to and develop closely related.Childhood infection rate is about 20%, along with the increase infection rate at age rises rapidly, between twenty and fifty up to
50-60%, old 70-80%.Chlamydia pneumoniae carries out person to person's propagation, family, school, army by respiratory secretions
And the popular of little scope can be there is in the working region of other population concentrations.At present, Chlamydia pneumoniae has been streptococcus pneumoniae of continuing
With cause the main of community acquired pneumonia (community acquired pneumonia, CAP) after hemophilus influenza
Pathogen, Chlamydia pneumoniae actute infection rate accounts for 23%, and pneumonia patient only accounts for the fraction of the infected, and 70-90% is subclinical
Performance.The incubation period of infection involving chlamydia pneumoniae is 15-23 days, can cause upper respiratory tract infection, such as sinusitis, otitis media and pharynx
Scorching, it is possible to cause lower respiratory infection, such as bronchitis and pneumonia.Respiratory tract infection majority shows as pharyngalgia, generates heat, coughs,
The state of an illness is the lightest, has self limiting.Pneumonia in Older Patients chlamydia pneumonia patient symptom may be more serious, the most lethal, especially
When it is to merge bacterial infection or there is chronic obstructive pulmonary disease etc..Therefore, Cpn infects early diagnosis for finding as early as possible
Extremely important with treatment Cpn infectious disease.
At present, the most unanimously think that its specific antibody determination such as Cpn IgM, IgG are that clinical infection refers to the most reliably
One of mark, and high specificity, highly sensitive.The general beginning for about 7-9 days after infection of Cpn IgM raises, and 3-4 week reaches peak,
Continue 4-6 month.There is relatively Cpn IgM a little later in Cpn IgG, and the persistent period is longer, and specificity is higher.It is reported, after infection
Convalescent period, Cpn IgG antibody titre substantially rises, and high titre or the paired sera sample IgG being separated by about two weeks substantially rise
Height, can determine that and be in infection period of expansion.
The main method of Clinical detection CPn IgG is enzyme linked immunosorbent assay, but the method also exists following
Weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen coated apparatus with anti-
Answer container, 12 batches, 6 batches, 8 batches or imposite first use can only be divided in use, it is impossible to carry out independent, single part
Detection；
(2) reagent type used by quantitative determination is more, and each detectable will contain with reagent bottle, and often makes
Being required for changing imbibition nozzle during with a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, filling
The operation of reagent is the most loaded down with trivial details；
(3) lack the corresponding mark to detection information, can only just will appreciate that by the mark checking test kit external packing box or know
Know product batch number and the effect duration information of detectable, and the information known is uncontrolled during detection, has the biggest
Randomness；
(4) detectable is in open space during detection, easily causes the cross-contamination between various reagent and shadow
Ring the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, and operating process is the most loaded down with trivial details and multiple
Miscellaneous, it is susceptible to bust, accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, if needing inspection
Survey 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if the most a sample needs to detect 10
Different project, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, the shortcoming that there is inadequate economical rationality.
Summary of the invention
CPn IgG detection technique has the disadvantage in that testing cost is high, detection sensitivity is low, detection line at present
Property narrow range, repeatability low, can not quantitatively, operation complicated etc..
The present invention, precisely in order to overcome the above shortcoming, discloses that a kind of testing cost is low, highly sensitive, detection is linear
CPn IgG test kit that scope is wide, repeatability is high, can be quantitative, simple to operate and preparation method thereof.The present invention is first
Prepare chemical luminescence immune analysis reagent box, specifically include that CPn IgG monoclonal antibody coated magnetic granule, pneumonia
The coated acridinium ester of chlamydia IgG monoclonal antibody and CPn IgG calibration product；Then Full-automatic chemiluminescence is utilized
Calibration product are detected by immunity analysis instrument, draw standard curve, are built in computer software, test actual sample, according to sample
Luminous value calculates concentration of specimens；Finally CPn IgG automatic chemiluminescence immunoassay system is carried out performance (sensitive
Degree, linear, precision, interference) evaluation.
The present invention, compared with current technology, has the advantage that
1, the present invention selects acridinium ester as marker material, and is applied to chemiluminescence immunoassay system, and this luminescence system is
Directly chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more cost-effective；
2, the acridinium ester chemiluminescent immunoassay system detection sensitivity that the present invention selects is high, it is possible to reach 3.0 AU/mL；
3, the acridinium ester chemiluminescent immunoassay system range of linearity width that the present invention selects, can reach 3.0-200.0 AU/mL；
4, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention selects is high, in batch and difference between batch is all within 5%,
This is that other chemiluminescence immunoassay system is unapproachable；
5, the chemiluminescence immunoassay system of the present invention has realized the quantitative of sample, soft to test by built-in standard curve
Part, only needs test sample just can directly obtain the concentration value of sample；
6, the chemiluminescence immunoassay system of the present invention has realized the interpolation of full-automation, reagent and sample has instrument complete entirely
Become, operate easier, decrease artificial error.
Accompanying drawing explanation
Fig. 1 is the CPn IgG canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Embodiment 1: CPn IgG chemiluminescence immune detection reagent kit preparation method
(1) prepared by the coated nanometer magnetic bead of CPn IgG monoclonal antibody:
Taking magnetic particle (particle diameter is 0.05-1um) suspension carboxylated for 50mg, Magneto separate removes supernatant, uses 0.02 M, and pH is 5.5
MES buffer is resuspended, adds the EDC aqueous solution of 10 newly configured for 0.5-2mL mg/mL, activated magnetic beads surface carboxyl groups, adds 3-5
Mg CPn IgG monoclonal antibody, suspendible 2-10 h under room temperature, Magneto separate, remove supernatant, with containing the 0.1 of 2% BSA
M pH be 8.0 Tris buffer be resuspended to 1mg/mL, obtain CPn IgG monoclonal antibody coated magnetic granule, often
Bottle 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of CPn IgG derivant labelling:
Take the CPn IgG derivant of 50 uL 25mg/mL, add the carbon of 150 uL 0.1-0.2 M pH 9.0-9.5
Phthalate buffer, mixing, it is subsequently adding the acridinium ester mixing of 1-2 uL 5 mg/mL, under room temperature, lucifuge reaction, takes after 1-2 h
Go out, be centrifuged desalting column desalting processing with the zeba of 2 mL, the most respectively with at pure water and TBS buffer in desalination processes
Reason, is eventually adding the acridinium ester solution of the CPn IgG derivant labelling obtained, and collects the liquid in centrifuge tube to preserving
Be in control the acridinium ester of CPn IgG derivant labelling, every bottle of 5 mL subpackage be stored in 4 DEG C standby.
(3) preparation of CPn IgG calibration product:
With standard substance buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0), CPn IgG is configured
One-tenth concentration is 0 AU/mL, 20AU/mL, 40 AU/mL, 80 AU/mL, 160 AU/mL, 320 AU/mL, every bottle of 0.5 mL subpackage
Lyophilizing, 4 DEG C save backup.
(4) preparation of chemiluminescence preexciting liquid:
Measure 1.0 liters of purified water, be sequentially added into the hydrogen peroxide (H that 80uL mass fraction is 20%2O2), 1.0 grams of Hydrazoic acid,sodium salt, 1.5
Gram polysorbas20, shakes up rear lucifuge and deposits.
(5) preparation of chemiluminescence exciting liquid:
Measure 1.0 liters of purified water, be sequentially added into 0.6 gram of sodium hydroxide, 0.5 gram of PC300,0.5g Hydrazoic acid,sodium salt, 1.5 grams of Triton
X-405, shakes up rear lucifuge and deposits.
Embodiment 2: CPn IgG chemical luminous immune detection method:
The present invention is with Full-automatic chemiluminescence immunoassay analysis meter for detection instrument, and the methodology pattern of the present invention is competition law, i.e.
Instrument is sequentially added into the sample of 20 uL, the CPn IgG monoclonal antibody coated magnetic granule of 50 uL and 50 uL's
The coated acridinium ester of CPn IgG, after reacting 10 min, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, depends on
Secondary addition 50uL chemiluminescence preexciting liquid, 50uL chemiluminescence exciting liquid carry out luminescence-producing reaction, finally record luminous intensity, from
Standard curve calculates the CPn IgG content of sample.
Embodiment 3: CPn IgG chemiluminescence immune detection reagent kit performance evaluation
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate CPn IgG chemiluminescence immunoassay chromatography test kit
Sensitivity, the sensitivity tried to achieve is 1ng/ mL.
Linear detection:
Be 0 AU/mL to concentration, 20AU/mL, 40 AU/mL, 80 AU/mL, 160 AU/mL, 320 AU/mL standard substance do linearly
Analyze, calculating linearly dependent coefficient, r=0.9985, it addition, the range of linearity that this test kit is to CPn IgG sample detection
For 3-200 AU/mL.
Precision measures:
Taking concentration is two CPn IgG samples of 40 AU/mL and 100AU/mL, and each concentration of each sample is respectively done 3 and put down
OK, detecting with three batches of test kits, calculate test kit and criticize interior and difference between batch, result shows that this test kit is criticized interior and difference between batch is equal
Less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, sweet
Grease, adding proportion is carried out according to 1:20, the survey of pooled serum after measuring pooled serum respectively and with the addition of various chaff interference
Value, calculates deviation therebetween, with ± 10% as tolerance interval.Result shows, interference all reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's CPn IgG concentration.
Claims (10)
1. a CPn IgG chemiluminescence immune detection reagent kit, described test kit includes: Chlamydia pneumoniae antigen bag
The nano magnetic microsphere of quilt, the mouse-anti human IgG antibody of acridinium ester label, Chemoluminescent substrate, CPn IgG calibration product.
Test kit the most according to claim 1, it is characterised in that the antigen coated solid phase carrier of described Chlamydia pneumoniae is
Magnetic particle.
Test kit the most according to claim 1, it is characterised in that the antigen coated solid phase carrier of described Chlamydia pneumoniae is
Carboxylated particle diameter is 0.05-1um magnetic particle.
Test kit the most according to claim 1, it is characterised in that described chemiluminescent labels is acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
Test kit the most according to claim 1, it is characterised in that the preferred acridinium ester of described chemiluminescent labels.
Test kit the most according to claim 1, it is characterised in that described Chemoluminescent substrate includes that chemiluminescence excites
Liquid, chemiluminescence preexciting liquid.
Test kit the most according to claim 1, it is characterised in that described chemiluminescence preexciting liquid is mass fraction
Hydrogen peroxide (the H of 0.005% ~ 0.5%2O2) solution, exciting liquid is the sodium hydroxide of 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
Test kit the most according to claim 1, it is characterised in that described CPn IgG calibration product are for using standard substance
Buffer CPn IgG is configured to concentration be 0 AU/mL, 20AU/mL, 40 AU/mL, 80 AU/mL, 160 AU/mL,
320 AU/mL, 4 DEG C save backup.
Test kit the most according to claim 1, it is characterised in that the preparation method of described test kit, it is characterised in that bag
Include at the bottom of the preparation of the antigen coated magnetic particle of Chlamydia pneumoniae, the preparation of mouse-anti human IgG antibody of acridinium ester label, chemiluminescence
The preparation of thing liquid, the preparation of CPn IgG calibration product.
10. according to the preparation method of test kit described in claim 1 and claim 9, it is characterised in that comprise the following steps:
1) preparation of the magnetic particle that Chlamydia pneumoniae is antigen coated:
Taking carboxylated nanometer magnetic bead suspension, Magneto separate removes supernatant, and MES buffer is resuspended, adds EDC aqueous solution, activates magnetic
Bead surface carboxyl, adds Chlamydia pneumoniae antigen, suspendible 2-10 h under room temperature, Magneto separate, removes supernatant, Tris buffer weight
Outstanding, obtain the magnetic particle that Chlamydia pneumoniae is antigen coated；Optionally, a diameter of 0.1 μm ~ 2.0 μm of carboxylated nanometer magnetic bead；
MES buffer concentration is 10mM ~ 100mM, pH 5.5 ~ 8.5；
2) preparation of the mouse-anti human IgG antibody of acridinium ester label:
Take mouse-anti human IgG antibody, add carbonate buffer solution, mixing, be subsequently adding acridinium ester mixing, lucifuge reaction under room temperature,
Take out after 1-2 h, centrifugal desalting column desalting processing, desalination processes process with pure water and TBS buffer the most respectively,
It is eventually adding the acridinium ester solution of the mouse-anti human IgG antibody's derivant labelling obtained, collects the liquid in centrifuge tube to preserving pipe
Obtain the acridinium ester of mouse-anti human IgG antibody's derivant labelling；
3) preparation of CPn IgG calibration product:
With standard substance buffer CPn IgG is configured to concentration be 0 AU/mL, 20AU/mL, 40 AU/mL, 80 AU/
ML, 160 AU/mL, 320 AU/mL, subpackage lyophilizing, 4 DEG C save backup；
4) preparation of chemiluminescence preexciting liquid:
Measure 1.0 liters of purified water, be sequentially added into the hydrogen peroxide (H that 0.5 ~ 100uL mass fraction is 20%2O2), 0.5 ~ 5 gram prevent
Rotten agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge and deposit；
Optionally, preservative is commercialization Hydrazoic acid,sodium salt, PC300, and surfactant is polysorbas20, Tween 80, Triton X-
100、Triton X-405；
5) preparation of chemiluminescence exciting liquid:
Measure 1.0 liters of purified water, be sequentially added into 0.2 ~ 1 gram of sodium hydroxide, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surface work
Property agent, shakes up rear lucifuge and deposits；Optionally, preservative is commercialization Hydrazoic acid,sodium salt, PC300, and surfactant is polysorbas20, tells
Temperature 80, Triton X-100, Triton X-405.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107356753A (en) * | 2017-07-21 | 2017-11-17 | 苏州华益美生物科技有限公司 | Human T lymphotropic virus's (HTLV) antibody assay kit and its application and preparation |
| CN109521196A (en) * | 2018-12-27 | 2019-03-26 | 正元盛邦(天津)生物科技有限公司 | A kind of magnetic test strips of chlamydia pneumoniae (cp) detection and corresponding paper box |
| CN110244063A (en) * | 2019-07-25 | 2019-09-17 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of serum amyloid A protein detection kit, preparation method and serum amyloid A protein |
| CN110286236A (en) * | 2019-07-25 | 2019-09-27 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of mycoplasma pneumoniae detection kit, preparation method and mycoplasma pneumoniae |
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| CN107356753A (en) * | 2017-07-21 | 2017-11-17 | 苏州华益美生物科技有限公司 | Human T lymphotropic virus's (HTLV) antibody assay kit and its application and preparation |
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| CN110244063A (en) * | 2019-07-25 | 2019-09-17 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of serum amyloid A protein detection kit, preparation method and serum amyloid A protein |
| CN110286236A (en) * | 2019-07-25 | 2019-09-27 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of mycoplasma pneumoniae detection kit, preparation method and mycoplasma pneumoniae |
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Application publication date: 20161221 |