A kind of latex enhancing immune of Troponin I is than turbid detection kitTechnical field
This Gong Kai Wataru and medicine, immune and in-vitro diagnosis field, Ju Ti Wataru and a kind of Immunity transmission turbidity detection fleshThe kit of calcium protein I.
Background technology
Cardiac muscle troponin I (cTn I) is one of three kinds of subunits of cardiac troponin (cTn), in addition two kinds of subunits pointIt Wei not serum cardiac troponin T (cTnT) and troponin C (TnC).CTnI is about made of 209 amino acid residues, molecular weight23.9kD, isoelectric point 9.87 are a kind of protein rich in α spirals.CTnI only 3-6% are present in cytoplasm in a free formIn, the overwhelming majority is present in the form of compound on actin filament.After myocardial damage, cardiac troponin complex is discharged intoIn blood, after 4-6 hours, start to increase in blood, raised Troponin I can in blood be kept 6-10 days.
In these protein related with Muscle cell contract, Troponin I has height Cardiac-specific and sensitivity,It is unique Troponin I different from being found in skeletal muscle.It is thus extensively studied as the sensitive and special of myocardial damageDifferent marker.
The detection method of cardiac muscle troponin I is more at present, such as colloidal gold method, immunofluorescence technique, enzyme-linked immunization, chemistryLuminescence method, Electrochemiluminescince, latex enhancing immune turbidimetry etc..The shortcomings that colloidal gold method, is accurately to be quantified,And differences between batches are larger, can not continue the level for monitoring patient's Troponin I；The immunofluorescence technique reaction time is longer, to environmentIt is more demanding, be easy to be influenced by dirt in air, and mostly 96 person-portions/box, using the shortcomings of rare earth marker but also this is immuneMethod can not large-scale use；Enzyme-linked immunization is easy to operate but sensitivity is low；Chemoluminescence method is to instrument requirements height and reagentOf high cost, detection speed is relatively slow, and popularity rate is low；The application of latex enhancing immune turbidimetry reagent is convenient, at low cost, automationBy force, facilitate clinical application, but this reagent mostly uses how anti-preparation, sensitivity is low, and zero negative value and differences between batches are big etc. asksTopic, also brings prodigious puzzlement to clinical application.
Rheumatoid factor existing for patient's body (RF) can interfere many immunological detection methods.IgM type RF and IgG types RFIt is combined with the Fc of capture antibody and label secondary antibody in detection reagent, causes to detect false positive results.RF is to different detection methodsAnnoyance level it is different, influence degree is not directly proportional to RF concentration.Currently, the detection reagent used in clinic does not have mostlyMeasure (progress that rheumatoid factor interferes immunoassays, the Medical review 2015 for preventing RF from interfering are taken wellVol 21,19：3495).Currently, prevent RF interfere strategy include：For capturing antibody, with F (ab ')2Segment substitutes completeIgG；Sample to be tested is pre-processed (Euroimmune companies) with the solid-phase adsorbent for being connected with thermal denaturation IgG, and 4 DEG C overnightIt is detected again after centrifugation；It when detecting antigen, can be added to 2 mercapto ethanol in Sample dilution or sample, IgM types RF is made to dropSolution.
For example, disclosing a kind of method and its detection kit of detection cardiac muscle troponin I in CN102539784A.The kit includes reagent 1, reagent 2, and wherein reagent 1 is buffer solution, reagent 2 by coupled antibody composition polystyrene latexParticle, buffer solution composition.The buffer solution is PBS buffer solution, Tris buffer solutions, glycine buffer, borate buffer solution, vinegarPhthalate buffer, citrate phosphate buffer, carbonate bicarbonate buffer, 2-morpholine ethane sulfonic acid buffer solution, ammonium chlorideOne or more of buffer solution.The composition that at least three kinds of above antibody are used in the kit, meets high-precision as much as possibleSpend condition needed for cTnI detection antibody, balanced sensibility of the antibody to various determination influences factors, can be minimized it is negative orPositive interference, increases the accuracy of assay method；Meanwhile also cTnI antibody being coated with to latex particle using orientation coupling methodOn, antibody coupling position is Fc segments, and antigen binding site is made to be directed toward mobile phase, and the feelings of antigen binding capacity loss will not occurCondition；In addition to this, due to during chemical coupling Fc segments the change in structure has occurred, reduce RF and heterophil antibodyInterference.
There is still a need for a kind of high sensitivity, the cardiac muscle troponin I kits of good in anti-interference performance for this field.
Invention content
According to some embodiments, a kind of detection kit of Troponin I is provided, it includes the first reagent and secondReagent.
In some embodiments, the first reagent includes：
In some embodiments, second reagent includes：
In some embodiments, buffer solution is selected from：Glycine buffer, Tris buffer solutions, MOPS buffer solutions, MES are slowFliud flushing, Hepes buffer solutions.In some embodiments, the buffer solution in first reagent and second reagent is identicalOr it is different.In some embodiments, the buffer solution in first reagent and second reagent is 50mM to 100mMGlycine buffer.
In some embodiments, the electrolyte is selected from：Sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, zinc chloride, chlorineChange calcium.
In some embodiments, the stabilizer is selected from the following a kind of or combines：Mannose, glucose, chitosan,Sorbierite, bovine serum albumin(BSA), trehalose, fructose, sucrose.
In some embodiments, the surfactant is selected from：Triton、Tween20、Tween80、NP40、thesit、BrijL2；Preferably Tween80.
In some embodiments, it is selected from for described discrete dose：Potassium rhodanide, choline chloride.
In some embodiments, the preservative is selected from：Sodium azide, phenol, P-hydroxybenzoic acid, PC300.
In some embodiments, the monoclonal antibody is originated from：Mouse, rabbit or fowl.
In some embodiments, the blocking agent is selected from：Mouse IgG, Mouse IgM, mice serum.
In some specific embodiments, the blocking agent is Mouse IgM.
In some specific embodiments, the blocking agent is mouse anti human IgM monoclonal antibody.
Without wishing to be held to specific theoretical limitation, it is however to be understood that present inventor it was unexpectedly found thatNon-specific antibody (such as mouse IgG, Mouse IgM, mice serum) is affinity to RF, to block RF, to avoid false sunProperty.
In some specific embodiments, the surfactant is 10g/L to 20g/LTween 80.
In some specific embodiments, the latex particle is polystyrene latex particles, and surface modification group isCarboxyl.
In some specific embodiments, the concentration of the blocking agent is 12g/L to 18g/L, preferably 15g/L.
In other specific embodiments, anti-human Troponin I monoclonal antibody is anti-comprising two different mouse sourcesHuman troponin I monoclonal antibody identifies different epitopes (epitope aa 60-90 and the aa 20- of human troponin I respectively40).In some specific embodiments, the ratio of the amount of the substance of two different anti-human Troponin I monoclonal antibodies is2：1.
In some specific embodiments, the detection kit of the application also includes calibration object or quality-control product.Calibration objectOr it can also be commercially available that quality-control product, which can be prepared voluntarily,.
According to some embodiments, a kind of detection kit of Troponin I is provided, it includes the first reagent and secondReagent；
Wherein, first reagent includes：
Second reagent includes：
The blocking agent is the mouse anti human IgM monoclonal antibody of purifying.
According to some embodiments, purposes of the blocking agent in anti-interference is additionally provided.The blocking agent is selected from the followingA kind of or combination：Mouse IgG, Mouse IgM, mice serum.
In some embodiments, providing the mouse anti human IgM monoclonal antibody of purifying is improving Troponin I detectionThe purposes of middle anti-interference ability.Preferably, interference refers to the interference of rheumatoid factor；More specifically, interference refers to due to class windTroponin I testing result false positive caused by the interference of the wet factor.
According to some embodiments, additionally providing the mouse anti human IgM monoclonal antibody of purifying is improving Troponin I inspectionPurposes in the false positive results of survey.
According to some embodiments, a kind of method improving anti-interference ability in Troponin I detection is additionally provided, includingStep：Blocking agent is introduced in Troponin I detection reagent, the blocking agent is selected from the following a kind of or combines：Mouse IgG,Mouse IgM, mice serum.
According to some embodiments, a kind of method improving false positive in Troponin I detection, including step are additionally providedSuddenly：Blocking agent is introduced in Troponin I detection reagent, the blocking agent is selected from the following a kind of or combines：It is mouse IgG, smallMouse IgM, mice serum.
In some embodiments, Troponin I detection reagent refers to latex enhancing immune than turbid detection reagent.
According to some embodiments, additionally providing the combination of blocking agent and surfactant is improving Troponin I detectionThe purposes of middle anti-interference ability.The blocking agent is selected from the following a kind of or combines：Mouse IgG, Mouse IgM, mice serum；AndThe surfactant is Tween 80.In some embodiments, anti-interference ability is selected from the following a kind of or combines：Anti- RFInterference, the interference of anti-binding bilirubin, antivitamin C interference, the interference of anti-Intralipid fat emulsions, antiheparin sodium interfere, are anti-Hemoglobin interferes.In some embodiments, the mass ratio of blocking agent and surfactant in same reagent is 3：2.
Description of the drawings
Fig. 1：Disclosure Troponin I kit standard curve.
Specific implementation mode
Embodiment 1：The preparation of the kit of the application
1. the first reagent preparation process is as follows：
First reagent includes
Under room temperature, according to the preparation of above-mentioned the first reagent of carry out, the blocking agent is that the mouse anti human IgM of purifying is mono-It is anti-(to be purchased from California, USA Scantibodies Laboratory Inc.9336Abraham Way, Santee, 92071, article No.3KC542；95%) purity is more than.
2. the second reagent preparation process is as follows：
4.5ml 0.05M MES are added in the polystyrene colloidal milk solution (concentration 10%) of 0.5ml diameters 300nm(PH6.0) and 100 μ l EDC (carbodiimides, concentration 5mg/mL), 1 hour is stood in 37 DEG C；
Troponin I monoclonal antibody is added to above-mentioned immediately after being diluted with 5ml 0.05M Tris buffer solutions (PH7.5)In buffer solution, reacted 2 hours at 37 DEG C；
1ml 0.1M glycine buffers (PH7.0) are added and stir to terminate for 1 hour and react；
It is washed with the glycine buffer (PH7.0) of 20ml 100mM, supernatant is removed in centrifugation, washs 3 times；
It is allowed to be dispersed into white latex suspension with buffer solution resuspension latex microsphere is preserved；
A concentration of 0.25% (% tables of the coated polystyrene latex particles of Troponin I monoclonal antibody in second reagentShow g/100ml)；Concentration of the antibody in the second reagent is 0.08g/L.
Wherein, the composition for preserving buffer solution：
The example more preferred as one：Monoclonal antibody in second reagent can with more than one monoclonal antibody,Such as two or more monoclonal antibodies, the different loci (coating of different monoclonal antibodies of Troponin I is identified respectivelyMethod is also pressed above-mentioned steps and is carried out).In an example, the mass ratio (or amount ratio of substance) of two kinds of monoclonal antibodies is 2:1, know respectivelyEpitope aa 60-90 and the aa 20-40 of other Troponin I.
Embodiment 2：The preparation of contrast agents box
The method that the preparation method is the same as that of Example 1, difference is only that is substituted for polyclonal antibody by monoclonal antibody.
The comparison of 3. surfactant of embodiment
According to the method reagent preparation box of embodiment 1, difference is only that：Tween 80 in first reagent is substituted for downEach surfactant of various various concentrations in table；And the standard items of various concentration are tested respectively, it is as a result as follows.
The comparison of 1. surfactant of table
The data of S1-S6 are calibration reaction absorbances, and reaction gradient (i.e. slope) is bigger, and blank reacts absorbance (i.e. S1React absorbance) it is more lower better.From the results, it was seen that effect is best when Tween 80 is 10g/L, when 20g/L Tween80Take second place.
Influence of 4. surfactant of embodiment to anti-interference ability
According to the method reagent preparation box of embodiment 1, step difference is only that following：Tween80 is substituted for following table instituteThe different surfaces activating agent of the various various concentrations shown.
Contained with each kit test of preparation or ((in conjunction with bilirubin, also referred to as direct courage is red by DB without chaff interferentElement), Vc, Intralipid fat emulsion, heparin sodium, hemoglobin) sample.As a result as follows：
Influence of 2. surfactant of table to anti-interference ability
From the results, it was seen that when reagent addition 10g/L 80 anti-jamming effectiveness of Tween are more preferable；The Tween 80 of 8g/LTake second place.
The comparison of the different blocking agents of embodiment 5.
According to the method reagent preparation box of embodiment 1, step difference is only that without blocking agent.
The sample contained or without RF is tested with each kit of preparation.As a result as follows：
The comparison of the different blocking agents of table 3.
From data as can be seen that obviously having anti-RF interference effects when containing blocking agent, and specific absorbance is influencedIt is relatively small.
Embodiment 6. determines the concentration of blocking agent
According to the method reagent preparation box of embodiment 1, step difference, which is only that, blocks agent concentration different (see the table below).With systemStandby each kit test contains or the sample without RF.As a result as follows：
The different comparisons for blocking agent concentration of table 4.
From the results, it was seen that the optimal dosage of blocking agent is 15g/L, 18g/L takes second place.
Influence of 7. blocking agent of embodiment to calibration
According to the method reagent preparation box of embodiment 1, step difference is only that uninterrupted dose.With each kit of preparationTest the standard items of various concentration.As a result as follows：
Whether there is or not the comparisons of blocking agent for table 5.
The degree of fitting of calibration curve is smaller, and illustrative graph fitting is better.
8. blocking agent of embodiment improves false positive
It prepares the kit for containing or not contain blocking agent respectively according to the method for embodiment 7, each kit is used in combination to distinguishTest false positive sample, and conventional sample (i.e. true positives and true negative sample).
Siemens's measured value is the negative or lower sample of measured value, if the application reagent does not add blocking agent, a other styleOriginally it will appear extremely high value, as shown in the table, false positive is significantly improved after blocking agent is added.
Whether there is or not the comparisons of blocking agent for table 6.
Whether there is or not the comparisons of blocking agent for table 7.
The result shows that blocking agent, which is added, corrects for false positive issue, to the measured values of true positives and true negative sample without statisticsSignificantly affect.
9. difference between batch of embodiment compares
Experimental method：According to the form below experiment parameter table is tested, and embodiment 1 takes 3 batches of reagents (when being related to two kinds of monoclonal antibodies), realIt applies example 2 and takes 2 batches of reagents (only 2 batches of antibody at present), calibrate and detect 10 samples.
8. experiment parameter table of table
| R1:R2:S | 150:50:15 | Pair/dominant wavelength | Without/600 |
| Read point | 19-34 | Calibrate type | spline |
| The Direction of Reaction | It is incremented by | Point (point)/skip point (span point) | 6/3 |
10. embodiment of table, 1 kit the calibration results
11. embodiment of table, 2 kit the calibration results
The comparison of 12.10 pattern detection results of table
It can be, it is evident that embodiment 1 (disclosure) can improve how anti-flesh calcium from the calibration results and pattern detection resultProtein I latex enhancing immune is than difference between batch problem existing for turbid reagent, and 2 the calibration results of embodiment and pattern detection result are all depositedIn notable difference.
10. repeatability of embodiment
Experimental method：With kit (when being related to using two kinds of monoclonal antibodies) detection low value sample and height of the embodiment of the present application 1It is worth sample, retest 10 times calculates mean value and the coefficient of variation.
13. repeated experiment result of table
| Sequencing | Low concentration sample | High concentration sample |
| 1 | 1.147 | 4.794 |
| 2 | 1.149 | 4.71 |
| 3 | 1.193 | 4.722 |
| 4 | 1.147 | 4.811 |
| 5 | 1.186 | 4.789 |
| 6 | 1.201 | 4.784 |
| 7 | 1.134 | 4.461 |
| 8 | 1.128 | 4.756 |
| 9 | 1.195 | 4.732 |
| 10 | 1.095 | 4.747 |
| Mean value | 1.1575 | 4.7306 |
| SD | 0.035 | 0.100 |
| CV | 3.02% | 2.12% |
The CV of upper table show lower concentration sample and high concentration sample is respectively 3.02% and 2.12%, the results showed that the disclosureMeasured value repeatability is good.
11. accelerated stability of embodiment
Experimental method：The reagent of the embodiment of the present application 1 is put into 37 DEG C of incubators to carry out accelerating processing 7 days, before comparing accelerationThe variation of reaction signal and sample measured value is calibrated afterwards.
Reaction signal is calibrated in the acceleration of table 14. before and after the processing
Table 15. accelerates the variation of sample measured value before and after the processing
| Catalogue number(Cat.No.) | Before acceleration | After acceleration |
| 1 | 1.65 | 1.65 |
| 2 | 2.17 | 2.19 |
| 3 | 2.22 | 2.17 |
| 4 | 1.61 | 1.60 |
| 5 | 1.55 | 1.59 |
| 6 | 1.97 | 2.06 |
| 7 | 0.85 | 1.09 |
| 8 | 10.36 | 10.58 |
| 9 | 11.37 | 11.05 |
| 10 | 5.00 | 4.47 |