CN109490555A - A kind of kit, method and application based on chemoluminescence method detection Lp-PLA2 and CRP content - Google Patents
A kind of kit, method and application based on chemoluminescence method detection Lp-PLA2 and CRP content Download PDFInfo
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Abstract
The invention discloses a kind of kits based on chemoluminescence method detection Lp-PLA2 and CRP content, method and application, belong to technical field of immunoassay, including the anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody, the Lp-PLA2 monoclonal antibody of marked by fluorescein isothiocyanate, the CRP monoclonal antibody of marked by fluorescein isothiocyanate, the Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark, the CRP monoclonal antibody of alkali phosphatase enzyme mark and the Chemoluminescent substrate of alkaline phosphatase catalytic luminescence, dilution, cleaning solution, Lp-PLA2 series of calibration product and CRP series of calibration product.The present invention uses the reaction pattern of double antibody sandwich method, isolation technics principle is immunized using chemiluminescence detection technology combination magnetic particle, quantitative determines Lp-PLA2 and CRP content, it is ensured that the sensitivity of detection, quick, high-throughput gross sample can be detected, convenient for operation and produced.
Description
Technical field
The present invention relates to a kind of kit for immunoassay, method and applications, are based on chemiluminescence more particularly to one kind
Method detects kit, method and the application of Lp-PLA2 and CRP content, belongs to technical field of immunoassay.
Background technique
Cerebral arterial thrombosis is a kind of common disease, frequently-occurring disease clinically, and the common cause of disease is led for atherosclerosis
Luminal stenosis and cerebral thrombosis are caused, local cerebral blood supply area's blood flow is caused to interrupt, brain tissue ischemia, anoxic, softening necrosis occurs.
Numerous studies indicate that atherosclerosis itself is the process of an inflammation, and lasting low-level inflammation implies cardiovascular and cerebrovascular disease
Pathogenetic possibility.Lp-PLA2 is newly discovered marker of inflammation, belong to non-calcium ion in phospholipase A2 superfamily according to
Rely type phosphate, is mainly secreted by inflammatory cell (such as macrophage, lymphocyte), and by the adjusting of inflammatory mediator.CRP
It is the non-specific markers of the systemic inflammatory reaction acute stage synthesized by liver a kind of, is that cardiovascular and cerebrovascular disease is most strong
One of predictive factor, have best prediction and outcome to the cardiovascular, cerebrovascular and peripheral artery disease.
After occurring some researches show that, cerebral arterial thrombosis, patient's Lp-PLA2 and CRP level is all significantly raised, it is horizontal with
Neurological functional deficit increases and is in increasing trend, and difference is statistically significant, while serum CRP level is to prediction cerebral infarction
Volume is also of great significance.Detection helps to judge more fully hereinafter the severity of cerebral infarction simultaneously while Lp-PLA2 and CRP
It is prevented and treated and provides laboratory reference with prognosis.
The detection side of the diagnosis to cerebral arterial thrombosis is realized by joint-detection Lp-PLA2 and CRP two indices at present
Method mainly has latex enhancing immune turbidimetry, enzyme-linked immunosorbent assay (ELISA) etc., and latex enhancing immune turbidimetry has inspection
The disadvantages such as linear narrow range, positive rate be low；That there are sensitivity is low for ELISA method, the range of linearity is narrow, is not easy to realize full-automation etc.
The defects of；To limit popularization and use, clinical diagnosis and research work can not be widely used in.Chemiluminescence immunoassay at present
Analytical technology has many advantages, such as above-mentioned be widely used because of it.
Summary of the invention
The main object of the present invention be solve to detect in the prior art cumbersome, inconvenient, sensitivity is low, range of linearity width and
The problems such as stability is poor, and provide it is a kind of based on chemoluminescence method based on chemoluminescence method detection Lp-PLA2 and CRP content
Kit and methods and applications.
The purpose of the present invention can reach by using following technical solution:
A kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content, which includes anti-isothiocyanic acid
The coated magnetic particle of fluorescein polyclonal antibody, the Lp-PLA2 monoclonal antibody of marked by fluorescein isothiocyanate, isothiocyanic acid are glimmering
Light element label CRP monoclonal antibody, the Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark, alkali phosphatase enzyme mark CRP
The Chemoluminescent substrate of monoclonal antibody and alkaline phosphatase catalytic luminescence, dilution, cleaning solution, Lp-PLA2 series of calibration
Product and CRP series of calibration product.
The anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody by carboxyl modified magnetic particle surface covalent bond
It is 0.7~1 μm that magnetic particle partial size, which is prepared, in anti-fluorescein isothiocynate polyclonal antibody, and kernel is ferroso-ferric oxide surface packet
It is wrapped with carboxylic group.
The anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody is suspended in the dilution, is configured to 0.5~2
The magnetic particle solution of μ g/mL；The Lp-PLA2 monoclonal antibody and fluorescein isothiocynate mark of the marked by fluorescein isothiocyanate
The CRP monoclonal antibody of note is dissolved in the dilution, is configured to 0.25~0.5 μ g/mL fluorescein isothiocynate mark respectively
The CRP monoclonal antibody of the Lp-PLA2 monoclonal antibody solution of note and 0.25~0.5 μ g/mL marked by fluorescein isothiocyanate is molten
Liquid；The Lp-PLA2 monoclonal antibody of the alkali phosphatase enzyme mark and the CRP monoclonal antibody dissolution of alkali phosphatase enzyme mark
In the dilution, it is configured to the Lp-PLA2 monoclonal antibody solution of 0.25~0.5 μ g/mL alkali phosphatase enzyme mark respectively
With the CRP monoclonal antibody solution of alkali phosphatase enzyme mark.
The Lp-PLA2 monoclonal antibody solution of marked by fluorescein isothiocyanate and the CRP of marked by fluorescein isothiocyanate are mono-
Clonal antibody solution is prepared by dialysis.
The Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark and the CRP monoclonal antibody difference of alkali phosphatase enzyme mark
By after activation Lp-PLA2 monoclonal antibody, CRP monoclonal antibody and alkaline phosphatase enzyme solutions by the Lp-PLA2 after activation
Monoclonal antibody and CRP monoclonal antibody obtain after mixing with the alkaline phosphatase enzyme solutions after activation.
The Tris-HCl buffer that Chemoluminescent substrate is 0.1~0.3M, pH value is 8~10, Chemoluminescent substrate
Dioxane containing 0.2~0.4mg/ml.
The Tris-HCl buffer and the dilution that dilution is 0.05~0.5M, pH value is 7.0~8.0 containing 0.4~
The sodium azide of the bovine serum albumin(BSA) BSA and 0.05~0.3wt% of 0.6wt%；The cleaning solution is 0.1~0.2M, pH
The Tris-HCl buffer that value is 8~9, Tris-HCl buffer contain the polysorbas20 and 15~20wt% of 0.01~0.04wt%
Sodium chloride.
The concentration range of Lp-PLA2 series of calibration product and CRP series of calibration product is respectively 0~50ng/ml and 0~100ng/
ml。
A kind of method of kit detection Lp-PLA2 and CRP content, includes the following steps:
Step 1: room temperature is equilibrated to after all reagents in kit are taken out, using preceding by anti-FITC polyclonal antibody packet
The magnetic particle solution of quilt thoroughly mixes, it is ensured that magnetic particle suspends uniformly, with deionized water by cleaning solution dilution, mixes, sets
37 DEG C of water-baths, keep chemiluminescence detector in a state to be tested；
Step 2: the Lp- of sample to be tested or calibration object, the Lp-PLA2 monoclonal antibody solution of FITC label and ALP label
The mixing of PLA2 monoclonal antibody solution incubates, while the CRP monoclonal antibody solution that sample to be tested or calibration object, FITC are marked
Incubation is mixed with the CRP monoclonal antibody solution marked with ALP respectively, is vibrated and is mixed with multitube vortex mixer, set 37 ± 0.5 DEG C of water
Bath, the coated magnetic particle solution of anti-fluorescein isothiocynate polyclonal antibody is then added into each test tube, is mixed with multitube
Device oscillation mixes, and sets 37 ± 0.5 DEG C of water-baths, test tube frame linking is put to magnetic separator, it is ensured that test tube is contacted with magnetic sheet, precipitating,
Supernatant out, then plus the cleaning solution after dilution is into each test tube, sets the oscillation of multitube vortex mixer and mixes, and repeats above-mentioned cleaning behaviour
Make, cleans 3 times altogether；
Step 3: adding the Chemoluminescent substrate of alkaline phosphatase catalytic luminescence into each test tube, mix, in 5 minutes
It is detected with luminometer, obtains the regression equation of calibration object dose-response curve, then root using four parameter logistic fits
It can be from the concentration for returning determinand in calculating sample on regression curve according to the relative luminous intensity of sample to be tested.
A kind of application of the kit based on chemoluminescence method detection Lp-PLA2 and CRP content, is used to prepare for brain soldier
After middle generation in nervous function damage degree and the reagent of Infarction volume evaluation.
Advantageous effects of the invention:
Kit of the invention uses the reaction pattern of double antibody sandwich method, efficiently utilizes chemiluminescence detection technology
Isolation technics principle is immunized in conjunction with magnetic particle, Lp-PLA2 the and CRP content in quantitatively determining human blood sample, it is ensured that detection
Sensitivity, and indices reach similar import reagent box analytic approach it is horizontal.Moreover, the pre-treatment requirement to sample
Low, sample pretreatment process is simple and reliable, quick, high-throughput can detect gross sample, convenient for operation and produce.The present invention provides
Reagent cartridge configuration it is easy, easy to use, high sensitivity, high specificity, detection range is wide, and easy to operate, kit is at low cost,
"dead" pollution, clinical applicability is strong, is more suitable for China's clinical detection, and situation of all-level hospitals at home is suitble to promote the use of.
Detailed description of the invention
Fig. 1 is chemoluminescence method detection principle diagram；
Fig. 2 is Lp-PLA2 standard curve；
Fig. 3 is CRP standard curve.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, the present invention is made below further
Detailed description, embodiments of the present invention are not limited thereto.
Embodiment 1:
Detect the preparation of the kit of Lp-PLA2 and CRP:
(1) preparation of Lp-PLA2 and CRP calibration object:
Lp-PLA2 and CRP sterling is diluted to 6 horizontal freeze-dryings respectively with pH=7.5,0.1MTris-HCl buffer
Calibration object, the aimed concn after being redissolved with pure water are respectively 0,0.25,2,10,25 and 50ng/ml.Wherein, Lp-PLA2 and CRP
The raw material of calibration object is standard level, and purity is not less than 99%；
(2) preparation of the coated magnetic particle solution of anti-fluorescein isothiocynate (FITC) polyclonal antibody:
The magnetic particle that partial size is 1 μm is added into magnetic field, 15min is stood, pours out supernatant, the MES with the 25mM of pH=4.7 is slow
Fliud flushing is cleaned 3 times, and is suspended with the buffer, concentration 50mg/ml；Anti-FITC Anti-TNF-α is added in every ml suspension
Body 2mg, is uniformly mixed at room temperature；1- (3- the dimethylamino-propyl) -3- for being 10mg/ml with deionized water compound concentration
Ethyl carbodiimide (EDC) solution the EDC solution that 1ml concentration is 10mg/ml is added in every ml magnetic particle suspension, in room temperature
Under the conditions of be stirred to react 4 hours after obtain coating anti-FITC polyclonal antibody magnetic particle；Then plus magnetic field, 15min is stood,
Supernatant out is 0.5% bovine serum albumin(BSA) (BSA) and 0.2% sodium azide preservatives containing mass ratio with pH=8.0
0.1MTris-HCl buffer solution for cleaning 4 times, and with the buffer by be coated with anti-FITC polyclonal antibody magnetic particle be configured to 1
The working solution of μ g/ml.The magnetic particle solution of the coating anti-FITC polyclonal antibody is saved at 4 DEG C, should not be frozen, and the used time mixes.
Magnetic particle is 0.7 μm of partial size, ferroso-ferric oxide kernel, the polymer for being coated with active group；Wherein, active group is carboxylic
Base；
(3) the Lp-PLA2 monoclonal antibody solution of fluorescein isothiocynate (FITC) label and CRP monoclonal antibody are molten
The preparation of liquid:
Lp-PLA2 monoclonal antibody and CRP monoclonal antibody are respectively placed in bag filter, in the carbon with 0.2MpH=9.0
It is dialyzed overnight in phthalate buffer；The FITC solution for being 0.5mg/ml with the NaHCO3 solution compound concentration of 0.2MpH=9.0, often
The FITC solution that 0.15mL concentration is 0.5mg/ml is added in mgLp-PLA2 monoclonal antibody and CRP monoclonal antibody, and mixing is equal
It is even, after reacting 20 hours at room temperature, it is dialyzed overnight in the carbonate buffer solution of 0.2MpH=9.0 to get glimmering to isothiocyanic acid
The Lp-PLA2 monoclonal antibody of light element label and the CRP monoclonal antibody of marked by fluorescein isothiocyanate；With contain quality percentage
The 0.1MTris-HCl for the sodium azide preservatives that the bovine serum albumin(BSA) and mass percentage that content is 0.5% are 0.2%
Buffer dilutes the Lp-PLA2 monoclonal antibody of marked by fluorescein isothiocyanate and the CRP Dan Ke of marked by fluorescein isothiocyanate
Grand antibody, respectively obtain the marked by fluorescein isothiocyanate that concentration is 0.5 μ g/ml Lp-PLA2 monoclonal antibody and different sulphur
The fluorescein-labeled CRP monoclonal antibody of cyanic acid；
(4) preparation of the phylaxin monoclonal antibody solution of alkaline phosphatase (ALP) label:
The 2- imino group thiol heptane hydrochloride salting liquid that 14.00mg/ml is prepared with deionized water takes Lp-PLA2 monoclonal anti-
Body and CRP monoclonal antibody are pre-installed in respectively in sharp bottom test tube, and the 2- imino group thiol heptane hydrochloride salt of 14.00mg/ml is added
Solution (additional amount is antibody volume 1/100), mixes, places 30 minutes at room temperature to get the Lp-PLA2 monoclonal after activation
Antibody and CRP monoclonal antibody.Alkaline phosphatase is taken to be pre-installed in sharp bottom test tube (alkaline phosphatase and Lp-PLA2 monoclonal
Antibody and the mass ratio of CRP monoclonal antibody are respectively 1:1 and 1:1), prepare 6.69mg/ml's with anhydrous dimethyl formamide
Sulfo-SMCC solution, Sulfo-SMCC solution is added in the test tube containing alkaline phosphatase, and (additional amount is ALP volume 1/
20) it, mixes, places 15 minutes at room temperature to get the alkaline phosphatase enzyme solutions after activation.
The MgCl of 1M is added in the Lp-PLA2 monoclonal antibody and CRP monoclonal antibody after above-mentioned activation2Solution (adds
Entering amount is antibody volume 1/500), it is further continued for that the alkaline phosphatase enzyme solutions after activation are added, 14 under the conditions of test tube is placed on 4 DEG C~
16 hours molten to get the Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark and the CRP monoclonal antibody of alkali phosphatase enzyme mark
Liquid.
It installs protein purification system (AKTApurifier100), uses (or the phase of Superdex200 preparation scale 16/70
Closely) pillar, the triethanolamine solution that the mass percentage concentration prepared using pH=7.0 deionized water is 2% are balanced, flow velocity
1ml/min collects each eluting peak flow point, measures light absorption value at the 280nm of each eluting peak flow point, and it is big to collect light absorption value at 280nm
It is mono- in the CRP of 0.02 pipe part, the Lp-PLA2 monoclonal antibody and alkali phosphatase enzyme mark that calculate separately alkali phosphatase enzyme mark
The concentration of clonal antibody.It is 0.2% with the bovine serum albumin(BSA) and mass percentage concentration for being 0.5% containing mass percentage concentration
The 0.1MTris-HCl buffer of sodium azide preservatives dilutes the Lp-PLA2 monoclonal antibody of the alkali phosphatase enzyme mark respectively
With the CRP monoclonal antibody of alkali phosphatase enzyme mark, the Lp- that concentration is the alkali phosphatase enzyme mark of 0.5 μ g/ml is respectively obtained
The CRP monoclonal antibody solution of PLA2 monoclonal antibody solution and alkali phosphatase enzyme mark；
(5) preparation of the Chemoluminescent substrate of alkaline phosphatase catalytic luminescence:
It is final concentration of with the Tris-HCl buffer dioxane compound (APCL) of 0.2MpH=9.3
The solution of 0.3mg/ml；
(6) preparation of cleaning solution:
Polysorbas20 and sodium chloride are added into the Tris-HCl buffer of 0.1M, pH=8.0, wherein the quality of polysorbas20
Percentage concentration is 0.02%, and the mass percentage concentration of sodium chloride is 15%；
(7) each component prepared in aforementioned manners is assembled into kit after the assay was approved, is needed after being assembled into kit
It could dispatch from the factory after sampling observation is qualified.
Embodiment 2:
Detect the preparation of the kit of Lp-PLA2 and CRP:
It is essentially the same with embodiment 1, the difference is that the anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody is outstanding
Diluent preparing is floated on into the magnetic particle solution of 0.5 μ g/ml；The Lp-PLA2 monoclonal antibody of marked by fluorescein isothiocyanate is molten
Solution is configured to the Lp-PLA2 monoclonal antibody solution of 0.25 μ g/ml marked by fluorescein isothiocyanate in dilution；；Different sulphur cyanogen
The fluorescein-labeled CRP monoclonal antibody of acid, which is dissolved in, is configured to 0.25 μ g/ml marked by fluorescein isothiocyanate in dilution
CRP monoclonal antibody solution；The Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark, which is dissolved in dilution, is configured to 0.25 μ
The Lp-PLA2 monoclonal antibody solution of g/ml alkali phosphatase enzyme mark；The CRP monoclonal antibody of alkali phosphatase enzyme mark is dissolved in
The CRP monoclonal antibody solution of 0.25 μ g/ml alkali phosphatase enzyme mark is configured in dilution；Chemoluminescent substrate is
0.1M, the Tris-HCl buffer that pH value is 10, and the dioxane containing 0.2mg/ml；Cleaning solution is 0.2M, pH value
For in 9 Tris-HCl buffer be added mass percent be 0.01% polysorbas20 and 20% sodium chloride.Magnetic particle is 3 μm
Partial size, ferroso-ferric oxide kernel, the polymer for being coated with active group；Active group is carboxyl.
Embodiment 3:
The detection of kit (kit prepared by embodiment 2) prepared by the present invention
1, it samples: taking whole blood or serum 1ml；
2, sample detection；
Reagent in kit should equilibrate to room temperature after taking out under condition of storage and be used further to detect；Using preceding by anti-FITC
The coated magnetic particle solution of polyclonal antibody thoroughly mixes, it is ensured that magnetic particle suspends uniformly, but cannot use magnetic stirring apparatus
Stirring；Cleaning solution: it with deionized water by 15 times of cleaning solution dilution, mixes；Set 37 DEG C of water-baths；Make at chemiluminescence detector
In state to be measured；Prepare test tube as needed and marks.50 μ l calibration objects or sample to be tested and 50 μ lFITC are marked
The Lp-PLA2 monoclonal antibody solution of Lp-PLA2 monoclonal antibody solution and ALP label, which mixes, to be incubated, while 50 μ l being calibrated
The CRP monoclonal antibody solution of product or sample to be tested and FITC label is mixed with the CRP monoclonal antibody solution marked with ALP respectively
It closes and incubates, each sample should replace pipettor head and avoid cross contamination.With multitube vortex mixer oscillation mix 30 seconds (2000 turns/
Point), set 37 ± 0.5 DEG C of water-bath 15min.Then the 50 coated magnetic particle solution of μ l anti-FITC polyclonal antibody are added to each examination
Guan Zhong is vibrated with multitube vortex mixer and mixes 30 seconds (2000 revs/min), sets 37 ± 0.5 DEG C of water-baths 5 minutes, test tube frame linking is put to magnetic
On separator, it is ensured that test tube is contacted with magnetic sheet, is precipitated 2 minutes.Separator is reversed with a big and slow circular motion to pour out
Clear liquid is placed on the test tube of reversing on filter paper together with separator, slap, to remove the drop being stained on tube wall.Add 300 μ l
Cleaning solution after dilution sets the oscillation of multitube vortex mixer and mixes 30 seconds (2000 revs/min) into each test tube.Repeat above-mentioned cleaning behaviour
Make, cleans 3 times altogether.Add the Chemoluminescent substrate of 100 μ l alkaline phosphatase catalytic luminescences into each test tube, mixes 3 seconds,
It is detected in 5 minutes with luminometer, obtains the recurrence side of calibration object dose-response curve using four parameter logistic fits
Journey, referring to figs. 2 and 3.
According to it is conventional in the art manufacture and vertification regulation kit of the invention is examined and determine, to its accuracy,
Accuracy and stability are tested.
(1) accuracy and precision
The standard items of high, medium and low three concentration are selected, are respectively 200ng/ml and 300ng/ containing Lp-PLA2 and CRP concentration
Ml, 100ng/ml and 150ng/ml, 50ng/ml and 50ng/ml.With the chemical luminescence reagent kit of the invention designed and in the market
Conventional detection kit (detection Lp-PLA2 ELISA method, detect CRP latex enhancing immune turbidimetry) is carried out while being detected,
Each Concentration Testing 10 times, acquired results are compared, and determine the accuracy and precision of this kit, obtained detection knot
Fruit is as follows:
1 Lp-PLA2 testing result of table compares
| Methodology | Mean value 1 (height) | S1 | Mean value 2 (in) | S2 | Mean value 3 (low) | S3 |
| ELISA | 202.23 | 43.67 | 101.33 | 13.76 | 49.55 | 4.42 |
| Board-like luminescence method | 201.21 | 42.65 | 98.36 | 12.67 | 50.05 | 3.76 |
The T for doing paired sample to this group of data is examined, and is obtained P > 0.05, is obtained the difference between two groups of results without statistics
Meaning, illustrate two methods no matter it is high, in or the Lp-PLA2 detection of low concentration in, have preferable consistency, this patent
The accuracy of the detection method of design is similar to commercially available detection kit.
2 CRP testing result of table compares
| Methodology | Mean value 1 (height) | S1 | Mean value 2 (in) | S2 | Mean value 3 (low) | S3 |
| Latex-enhanced turbidimetry | 305.01 | 72.03 | 144.33 | 23.23 | 48.53 | 7.53 |
| Chemoluminescence method | 302.32 | 63.75 | 155.36 | 28.75 | 48.43 | 6.84 |
The T for doing paired sample to this group of data is examined, and is obtained P > 0.05, is obtained the difference between two groups of results without statistics
Meaning, illustrate two methods no matter it is high, in or the CRP detection of low concentration in, have preferable consistency, this patent design
Detection method accuracy it is similar to commercially available detection kit.
Meanwhile this method coefficient of variation of the repetition testing result of two indexes under more concentration is respectively less than 10%, have compared with
Good precision.
(2) stabilization of kit is tested
Kit preservation condition is 2-8 DEG C, and after saving 6 months, the indices of assay kit are found in normal model
Within enclosing.Consider in transport and use process, has improper preservation condition and occur, the condition that kit is saved at 37 DEG C
It is lower to place 6 days, carry out accelerated aging tests, the results showed that the kit indices comply fully with requirement.In view of kit
Freezing happens, and kit is put into -20 DEG C of refrigerator freezings 5 days, and measurement result also indicates that kit indices completely just
Often.It can show that kit can at least save 6 months or more at 2~8 DEG C from result above.
The testing principle of kit provided by the invention is as shown in Figure 1, wherein luminous marker 1 is isosulfocyanic acid fluorescence
Element, luminous marker 2 are alkaline phosphatase, and sample to be tested, FITC the Lp-PLA2 monoclonal antibody solution marked and ALP are marked
The CRP monoclonal antibody solution that the Lp-PLA2 monoclonal antibody solution of note, which mixes, to be incubated, while sample to be tested, FITC being marked
Incubation is mixed with the CRP monoclonal antibody solution marked with ALP respectively.Then the magnetic for being coated with anti-FITC polyclonal antibody is added
Particle, is then added the magnetic particle for being coated with anti-FITC polyclonal antibody, and immune complex is adsorbed to magnetic particle surface.Washing
Luminous substrate is added after removal unbonded antibody and impurity, ALP catalysis substrate shines, and measures relative luminous intensity (RLU).?
RLU and Lp-PLA2 and CRP antigen concentration is proportional in a certain range, by interpolation method can be read from standard curve to
Lp-PLA2 and CRP content in test sample sheet.
A kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content provided in the above-described embodiments is adopted
With the reaction pattern of double antibody sandwich method, efficiently utilizes chemiluminescence detection technology combination magnetic particle and isolation technics original is immunized
It manages, Lp-PLA2 the and CRP content in quantitatively determining human blood sample, it is ensured that the sensitivity of detection, and indices reach
The analytic approach of similar import reagent box is horizontal, while low to the pre-treatment of sample requirement, and sample pretreatment process is simple and reliable, can
Quickly, high-throughput detection gross sample, convenient for operation and production, reagent cartridge configuration provided in this embodiment is easy, easy to use,
High sensitivity, high specificity, detection range is wide, easy to operate, and kit is at low cost, and "dead" pollution, clinical applicability is strong,
It is more suitable for China's clinical detection, situation of all-level hospitals at home is suitble to promote the use of.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content, which is characterized in that the kit includes
The coated magnetic particle of anti-fluorescein isothiocynate polyclonal antibody, marked by fluorescein isothiocyanate Lp-PLA2 monoclonal antibody,
CRP monoclonal antibody, the Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark, alkaline phosphatase of marked by fluorescein isothiocyanate
The CRP monoclonal antibody of enzyme label and Chemoluminescent substrate, dilution, cleaning solution, the Lp- of alkaline phosphatase catalytic luminescence
PLA2 series of calibration product and CRP series of calibration product.
2. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody is resisted different by the magnetic particle surface covalent bond of carboxyl modified
It is 0.7~1 μm that magnetic particle partial size, which is prepared, in thiocyanic acid fluorescein polyclonal antibody, and kernel is coated with for ferroso-ferric oxide
Carboxylic group.
3. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the anti-coated magnetic particle of fluorescein isothiocynate polyclonal antibody is suspended in the dilution, is configured to 0.5~2 μ g/
The magnetic particle solution of mL；The Lp-PLA2 monoclonal antibody and marked by fluorescein isothiocyanate of the marked by fluorescein isothiocyanate
CRP monoclonal antibody be dissolved in the dilution, be configured to 0.25~0.5 μ g/mL marked by fluorescein isothiocyanate respectively
Lp-PLA2 monoclonal antibody solution and 0.25~0.5 μ g/mL marked by fluorescein isothiocyanate CRP monoclonal antibody solution；
The Lp-PLA2 monoclonal antibody of the alkali phosphatase enzyme mark and the CRP monoclonal antibody of alkali phosphatase enzyme mark are dissolved in
In the dilution, be configured to respectively 0.25~0.5 μ g/mL alkali phosphatase enzyme mark Lp-PLA2 monoclonal antibody solution and
The CRP monoclonal antibody solution of alkali phosphatase enzyme mark.
4. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the Lp-PLA2 monoclonal antibody solution of marked by fluorescein isothiocyanate and the CRP monoclonal of marked by fluorescein isothiocyanate
Antibody-solutions are prepared by dialysis.
5. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the Lp-PLA2 monoclonal antibody of alkali phosphatase enzyme mark and the CRP monoclonal antibody of alkali phosphatase enzyme mark pass through respectively
By the Lp-PLA2 Dan Ke after activation after activation Lp-PLA2 monoclonal antibody, CRP monoclonal antibody and alkaline phosphatase enzyme solutions
Grand antibody and CRP monoclonal antibody obtain after mixing with the alkaline phosphatase enzyme solutions after activation.
6. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the Tris-HCl buffer that Chemoluminescent substrate is 0.1~0.3M, pH value is 8~10, Chemoluminescent substrate contains
The dioxane of 0.2~0.4mg/ml.
7. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
Be, the Tris-HCl buffer and the dilution that dilution is 0.05~0.5M, pH value is 7.0~8.0 containing 0.4~
The sodium azide of the bovine serum albumin(BSA) BSA and 0.05~0.3wt% of 0.6wt%；The cleaning solution is 0.1~0.2M, pH
The Tris-HCl buffer that value is 8~9, Tris-HCl buffer contain the polysorbas20 and 15~20wt% of 0.01~0.04wt%
Sodium chloride.
8. a kind of kit based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1, feature
It is, the concentration range of Lp-PLA2 series of calibration product and CRP series of calibration product is respectively 0~50ng/ml and 0~100ng/ml.
9. a kind of method of the kit detection Lp-PLA2 and CRP content as described in claim 1-8 any one, feature
It is, includes the following steps:
Step 1: room temperature is equilibrated to after all reagents in kit are taken out, using preceding that anti-FITC polyclonal antibody is coated
Magnetic particle solution thoroughly mixes, it is ensured that magnetic particle suspends uniformly, with deionized water by cleaning solution dilution, mixes, sets 37 DEG C
Water-bath keeps chemiluminescence detector in a state to be tested；
Step 2: the Lp-PLA2 of sample to be tested or calibration object, the Lp-PLA2 monoclonal antibody solution of FITC label and ALP label
Monoclonal antibody solution mixing incubates, while the CRP monoclonal antibody solution of sample to be tested or calibration object, FITC label being distinguished
Incubation is mixed with the CRP monoclonal antibody solution marked with ALP, is vibrated and is mixed with multitube vortex mixer, set 37 ± 0.5 DEG C of water-baths,
Then the coated magnetic particle solution of anti-fluorescein isothiocynate polyclonal antibody is added into each test tube, is shaken with multitube vortex mixer
Mixing is swung, 37 ± 0.5 DEG C of water-baths are set, test tube frame linking is put to magnetic separator, it is ensured that test tube is contacted with magnetic sheet, and precipitating is poured out
Clear liquid, then plus the cleaning solution after dilution is into each test tube, sets the oscillation of multitube vortex mixer and mixes, repeats above-mentioned cleaning operation,
It cleans 3 times altogether；
Step 3: adding the Chemoluminescent substrate of alkaline phosphatase catalytic luminescence into each test tube, mix, in 5 minutes with hair
Optical detector is detected, and obtains the regression equation of calibration object dose-response curve using four parameter logistic fits, further according to
The relative luminous intensity of test sample sheet can be from the concentration for returning determinand in calculating sample on regression curve.
10. a kind of examination based on chemoluminescence method detection Lp-PLA2 and CRP content as described in claim 1-8 any one
The application of agent box, which is characterized in that be used to prepare nervous function damage degree and Infarction volume evaluation after occurring for cerebral apoplexy
Reagent on.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811624633.4A CN109490555A (en) | 2018-12-28 | 2018-12-28 | A kind of kit, method and application based on chemoluminescence method detection Lp-PLA2 and CRP content |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811624633.4A CN109490555A (en) | 2018-12-28 | 2018-12-28 | A kind of kit, method and application based on chemoluminescence method detection Lp-PLA2 and CRP content |
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| CN109490555A true CN109490555A (en) | 2019-03-19 |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462075A (en) * | 2020-11-13 | 2021-03-09 | 三诺生物传感股份有限公司 | Alkaline phosphatase-labeled AMH antibody conjugate and preparation method and application thereof |
| CN113777326A (en) * | 2021-09-13 | 2021-12-10 | 北京森美希克玛生物科技有限公司 | Kit for high-specificity detection of heparin binding protein and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104820102A (en) * | 2015-05-05 | 2015-08-05 | 南京格耀生物科技有限公司 | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof |
-
2018
- 2018-12-28 CN CN201811624633.4A patent/CN109490555A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104820102A (en) * | 2015-05-05 | 2015-08-05 | 南京格耀生物科技有限公司 | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462075A (en) * | 2020-11-13 | 2021-03-09 | 三诺生物传感股份有限公司 | Alkaline phosphatase-labeled AMH antibody conjugate and preparation method and application thereof |
| CN113777326A (en) * | 2021-09-13 | 2021-12-10 | 北京森美希克玛生物科技有限公司 | Kit for high-specificity detection of heparin binding protein and application thereof |
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Application publication date: 20190319 |