CN106053440A - Mycoplasma pneumoniae IgG chemiluminescence immunoassay kit and preparation method thereof - Google Patents
Mycoplasma pneumoniae IgG chemiluminescence immunoassay kit and preparation method thereof Download PDFInfo
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- CN106053440A CN106053440A CN201610503653.0A CN201610503653A CN106053440A CN 106053440 A CN106053440 A CN 106053440A CN 201610503653 A CN201610503653 A CN 201610503653A CN 106053440 A CN106053440 A CN 106053440A
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- mycoplasma pneumoniae
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- acridinium ester
- test kit
- chemiluminescence
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a mycoplasma pneumoniae IgG chemiluminescence immunoassay kit. The kit comprises magnetic particles enveloped by mycoplasma pneumoniae antigens, a mouse-anti-human IgG antibody marked by acridinium ester, a mycoplasma pneumoniae IgG calibration product, pre-excitation liquid and excitation liquid. In addition, the invention further discloses a preparation method of the mycoplasma pneumoniae IgG chemiluminescence immunoassay kit. Compared with the existing kit, the kit has the advantages of being simple and convenient to operate, high in sensitivity, wide in detection range and the like.
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 mycoplasma pneumoniae IgG test kit and preparation method thereof.
Background technology
Mycoplasma pneumoniae (mycoplasma pneumoniae, MP) and it is a kind of ultra-filtration between antibacterial and virus
Sexually transmitted disease (STD) pathogenic microorganism, the main respiratory tract spittle that passes through is propagated with the form of aerosol particles.1962 first from human primary
The sputum of atypical pneumonia (primary atypical pneumonia, PAP) patient separates and cultivates.20th century
Since the 90's, along with the etiologic transition of pneumonia, MP has become the important pathogen body of infantile pneumonia, and MP infects and not only causes
Pulmonary lesion, and other organs such as the heart, brain, liver, kidney can be invaded, cause multiple Pulmonary hypofuntion.MP or community acquired pneumonia
(CAP) important pathogen, especially more than 5 years old children's, MP accounts for CAP cause of disease 10 ~ 20% in non-popular year.Because MP growth is slow
Slowly, incubation period and carriagable time long, forming the intermission falls ill the epidemic characteristic propagated slowly, over a long time, popular up to the several months extremely
Several years.In the last few years, influenza crowd's ratio increases year by year, not only invades teenager and child, and infant morbidity is also in increasing
Trend, and often show without specific clinical owing to MP infects, easily obscure mutually with general viral influenza, therefore, it is possible in time
Making a definite diagnosis which kind of cause of disease to cause a disease, accomplish early to find early treatment, sb.'s illness took a turn for the worse to reduce children acute pneumonia, therefore early diagnosis is the heaviest
Want.
Mycoplasma pneumoniae antibody is divided into IgG antibody and IgM antibody two kinds, because being 2 the incubation period of mycoplasma pneumoniae infection
In week in week-3, when patient occurs that symptom is gone to a doctor, IgM antibody has reached at a relatively high level, and therefore, the IgM antibody positive can be made
Diagnosis index for acute HIV infection.As IgM antibody is negative, can not negate mycoplasma pneumoniae infection, also need to detect IgG and resist
Body.There is evening in IgG relatively IgM, need to dynamically observe.The mensuration of mycoplasma pneumoniae IgG antibody, resists in combination with mycoplasma pneumoniae IgM
The detection of body, may be used for the diseases such as auxiliary diagnosis mycoplasma pneumonia.
The main method of Clinical detection mycoplasma pneumoniae 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
Mycoplasma pneumoniae 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
Mycoplasma pneumoniae 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 mycoplasma pneumoniae IgG monoclonal antibody coated magnetic granule, pneumonia
The coated acridinium ester of mycoplasma IgG monoclonal antibody and mycoplasma pneumoniae IgG calibrate 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 mycoplasma pneumoniae 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 2.0 AU/mL；
3, the acridinium ester chemiluminescent immunoassay system range of linearity width that the present invention selects, can reach 2.0-300.0 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 mycoplasma pneumoniae IgG canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Embodiment 1: mycoplasma pneumoniae IgG chemiluminescence immunoassay kit preparation method
(1) prepared by the coated nanometer magnetic bead of mycoplasma pneumoniae 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 mycoplasma pneumoniae 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 mycoplasma pneumoniae IgG monoclonal antibody coated magnetic granule, often
Bottle 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of mycoplasma pneumoniae IgG derivant labelling:
Take the mycoplasma pneumoniae 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 mycoplasma pneumoniae IgG derivant labelling obtained, and collects the liquid in centrifuge tube to preserving
Be in control the acridinium ester of mycoplasma pneumoniae IgG derivant labelling, every bottle of 5 mL subpackage be stored in 4 DEG C standby.
(3) preparation of mycoplasma pneumoniae IgG calibration product:
With standard substance buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0), mycoplasma pneumoniae 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: mycoplasma pneumoniae 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 mycoplasma pneumoniae IgG monoclonal antibody coated magnetic granule of 50 uL and 50 uL's
The coated acridinium ester of mycoplasma pneumoniae 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 mycoplasma pneumoniae IgG content of sample.
Embodiment 3: mycoplasma pneumoniae IgG chemiluminescence immunoassay 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 mycoplasma pneumoniae 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=0.9990, it addition, the range of linearity that this test kit is to mycoplasma pneumoniae IgG sample detection
For 1.5-300 AU/mL.
Precision measures:
Taking concentration is two mycoplasma pneumoniae IgG samples of 50 AU/mL and 200AU/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 mycoplasma pneumoniae IgG concentration.
Embodiment 4: the sensitivity experiment of mycoplasma pneumoniae IgG chemiluminescence immunoassay kit
Respectively by chemical luminescence detection method and traditional enzyme linked immunosorbent assay to the calibration object that concentration is 0 AU/mL or sample
Diluent is that sample detects, replication 20 times, draws the RLU value (relative light unit) of 20 measurement results, calculates it
Meansigma methods (M) and standard deviation (SD), draw M-2SD, this luminous value substitution calibration curve is calculated corresponding concentration value little
In 2.0 AU/mL.
Claims (10)
1. a mycoplasma pneumoniae IgG chemiluminescence immunoassay kit, described test kit includes: mycoplasma pneumoniae antigen bag
The nano magnetic microsphere of quilt, the mouse-anti human IgG antibody of acridinium ester label, Chemoluminescent substrate, mycoplasma pneumoniae IgG calibrate product.
Test kit the most according to claim 1, it is characterised in that the antigen coated solid phase carrier of described mycoplasma 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 mycoplasma 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 mycoplasma pneumoniae IgG calibration product are for using standard substance
Buffer mycoplasma pneumoniae 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 mycoplasma pneumoniae, the preparation of mouse-anti human IgG antibody of acridinium ester label, chemiluminescence
The preparation of thing liquid, the preparation of mycoplasma pneumoniae 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 mycoplasma 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 mycoplasma pneumoniae antigen, suspendible 2-10 h under room temperature, Magneto separate, removes supernatant, Tris buffer weight
Outstanding, obtain the magnetic particle that mycoplasma 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 mycoplasma pneumoniae IgG calibration product:
With standard substance buffer mycoplasma pneumoniae 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, Tween 80, Triton X-
100、Triton X-405。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645756A (en) * | 2016-12-30 | 2017-05-10 | 广州市达瑞生物技术股份有限公司 | Kit for detecting NMP22 (Nuclear Matrix Protein 22) and preparation method thereof |
| CN107478827A (en) * | 2017-08-23 | 2017-12-15 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of fumonisin and preparation method thereof |
| CN109387629A (en) * | 2017-08-04 | 2019-02-26 | 河北艾驰生物科技有限公司 | A kind of detection reagent detecting thyroid peroxidase antibody |
| CN111044724A (en) * | 2019-12-22 | 2020-04-21 | 上海复星长征医学科学有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
| CN111579773A (en) * | 2020-05-29 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Method for coating magnetic beads with mycoplasma pneumoniae membrane protein antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106645756A (en) * | 2016-12-30 | 2017-05-10 | 广州市达瑞生物技术股份有限公司 | Kit for detecting NMP22 (Nuclear Matrix Protein 22) and preparation method thereof |
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| CN111044724A (en) * | 2019-12-22 | 2020-04-21 | 上海复星长征医学科学有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
| CN111044724B (en) * | 2019-12-22 | 2023-11-10 | 复星诊断科技(上海)有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
| CN111579773A (en) * | 2020-05-29 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Method for coating magnetic beads with mycoplasma pneumoniae membrane protein antigen |
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