Summary of the invention
Based on this, be necessary to provide a kind of detection method for trace protein that can keep antiserum activity the long period.
A kind of detection method for trace protein, is characterized in that, comprising:
Configuration damping fluid: configuration antiserum treating fluid, configuration reaction buffer;
Antiserum process: adopt antiserum treating fluid that antiserum is diluted to 1-2mg/ml, room temperature is placed 2-8 DEG C of temperature environment and preserved after placing;
Operation: add reaction buffer in reaction vessels, then adds detection sample, and adds the antiserum forming reactions solution after antiserum disposal methods;
Response analysis: tested reaction solution by protein analyzer, reads the range of reaction detecting sample;
Standard curve fit: utilize the calibration solution of variable concentrations to make typical curve, line nonlinearity matching of going forward side by side.
In a preferred embodiment, in described antiserum processing procedure, after adopting antiserum treating fluid that antiserum is diluted to 1-2mg/ml, room temperature is placed after 12-24 hour, then places 4 DEG C of refrigerator environment and preserves.
In a preferred embodiment, in operation, be further: in reaction vessels, add reaction buffer, then add detection sample, the reaction vessels adding detection sample is placed into the correspondence position of the reacting hole of protein analyzer, and adds the antiserum after process.
In a preferred embodiment, in described operation, the sero-fast volume ratio after the reaction buffer added, detection sample, process is 400:20:40; Response analysis is further: after the antiserum after protein analyzer senses process joins reaction vessels from reacting hole, automatically test, the range of reaction of read test sample.
In a preferred embodiment, in described operation, blank time is 6-8 second, and the reaction time is 122-124 second.
In a preferred embodiment, described protein analyzer adopts determined wavelength to be 650-690nm, and protein analyzer reads blank value according to blank time, reads range of reaction according to the reaction time.
In a preferred embodiment, described standard curve fit comprises further: the mALB calibration solution utilizing the RANDOX of variable concentrations, makes typical curve, to typical curve carry out non-linear, repeatedly, piecewise fitting.
In a preferred embodiment, described standard curve fit comprises further: detect steep lifting line section or curve, smoothing curve amendment.
In a preferred embodiment, described antiserum treating fluid: the Gly of the NaCl solution+40mM of the phosphate buffer+150mM of 20mM, PH7.0-7.8.
In a preferred embodiment, the reaction buffer of described configuration: the isinglass of the NaCl solution+0.01-0.5 mass percent of the phosphate buffer+30-100mM of 20mM, PH7.0-7.8.
The detection method of above-mentioned trace of albumin, adopt the antiserum after the process of antiserum treating fluid, adopt to place and process at room temperature, various component in antiserum treating fluid and the antibody in antiserum are fully acted on, because the condition of room temperature is relatively gentle for condition antibody, for antiserum treating fluid various components temperature be unlikely to too low, thus be conducive to the abundant effect of various component in antibody and antiserum treating fluid; Due to the abundant effect of various component in antibody and antiserum treating fluid, to make antiserum have good stability, place 1 year under 2-8 DEG C of condition of storage, activity can not reduce.
Embodiment
The detection method for trace protein of one embodiment of the invention, comprises the steps:
Configuration damping fluid: configuration antiserum treating fluid, configuration reaction buffer;
Antiserum process: adopt antiserum treating fluid that antiserum is diluted to 1-2mg/ml, room temperature is placed after 12-24 hour, to make the various component in antiserum treating fluid and the antibody in antiserum fully act on, then places 2 DEG C of-8 DEG C of temperature environments and preserves.Preferred further, Conservation environment is the temperature environment of 4 DEG C.
Operation: add reaction buffer in reaction vessels, then adds detection sample, and adds the antiserum after antiserum processing reaction;
Response analysis: carry out test reading by protein analyzer and get range of reaction;
Confirmed standard curve: the calibration solution confirmed standard curve utilizing variable concentrations, and nonlinear fitting is carried out to typical curve.
Preferably, adopt antiserum treating fluid that antiserum is diluted to 1-2mg/ml, room temperature was placed after 12-24 hour, placed in the refrigerator of 4 DEG C and preserved.Antiserum is diluted to 1-2mg/ml by antiserum treating fluid, room temperature is adopted to place process to make the various component in antiserum treating fluid and the antibody in antiserum fully act on, because the condition of room temperature is relatively gentle for condition antibody, for antiserum treating fluid various components temperature be unlikely to too low, thus be conducive to the abundant effect of various component in antibody and antiserum treating fluid.
Preferably, in the present embodiment, antiserum treating fluid: the Gly of the NaCl solution+40mM of the phosphate buffer+150mM of 20mM, PH7.0-7.8.
Preferably, in the present embodiment, reaction buffer: the isinglass of the NaCl solution+0.01-0.5 mass percent of the phosphate buffer+30-100mM of 20mM, PH7.0-7.8.
Further, in operation, the sero-fast volume ratio after reaction buffer, detection sample, antiserum processing reaction is 400:20:40.
The concrete operations of the operation in one embodiment of the present invention are as follows: add 400 microlitre reaction buffers at reaction vessels (as in reaction cup); The detection sample (as urine specimen) of 20 microlitres is added in the reaction cup that damping fluid is housed; Detect the reaction cup of sample be put into adding in the reacting hole of protein analyzer, and the antiserum after the antiserum added after the process of 40 microlitres and the process of antiserum treatment step.
Further, in operation, blank time preferably 6-8 second, preferably 122-124 second in reaction time.
In response analysis, when reaction cup is placed in the reacting hole of protein analyzer, after the antiserum after protein analyzer detects 40 microlitre process adds from reacting hole, protein analyzer is tested automatically, reads the range of reaction of test sample book.
In the present embodiment, antiserum preferably, ADVY company of India produce antiserum.
Further, the determined wavelength that protein analyzer adopts, preferably with antigen in reaction solution, that antibody response combines the particle size diameter formed is identical or close.Preferably, protein analyzer adopts determined wavelength to be 650-690nm.
Further, protein analyzer is according to the blank time reading blank value of setting, the range of reaction according to the reaction time read test sample arranged.
Further, the standard curve fit of the present embodiment comprises: the mALB calibration solution utilizing the RANDOX of variable concentrations, makes typical curve.Due to W-response trend, be non-linear, in the present embodiment, to typical curve adopt fitting software carry out non-linear, repeatedly, piecewise fitting.The range of linearity according to the detection of the typical curve after matching can reach 5-330mg/L.
The ideal response cohesive process of antigen and antibody is a level and smooth curve, but due to the impact of various factors, in the course of reaction of reality, a certain section of phenomenon suddenly raised appears in the curve occasional of antigen and antibody response, thus can affect testing result.Preferably, in the present embodiment to typical curve carry out non-linear, repeatedly, piecewise fitting process is further: detect steep lifting line section or curve, to this line segment or the smoothing transient curve correction of curve.Thus eliminate this impact to greatest extent, make the curve of matching more accurate, testing result is also more reliable.
Preferred embodiment 1:
The detection method for trace protein of the preferred embodiment of the present invention 1, comprises the steps:
Configuration damping fluid: configuration antiserum treating fluid: the Gly of the NaCl solution+40mM of the phosphate buffer+150mM of 20mM, PH7.0-7.8.
Configuration reaction buffer: the isinglass of the NaCl solution+0.01-0.5 mass percent of the phosphate buffer+30-100mM of 20mM, PH7.0-7.8.
Antiserum process: adopt antiserum treating fluid that antiserum is diluted to 1mg/ml, after room temperature places 12 hours, to make the various component in antiserum treating fluid and the antibody in antiserum fully act on, then places in the refrigerator of 4 DEG C of temperature and preserves.In the present embodiment, antiserum preferably, ADVY company of India produce antiserum.
Reaction time is arranged: arranging blank time is 6 seconds, and the reaction time is 122 seconds.
Operation: add 400 microlitre reaction buffers in reaction cup, then the urine specimen of 20 microlitres is added, the reaction cup of the urine specimen added is put in the reacting hole of protein analyzer, and antiserum after adding 40 microlitre reactions after the process of antiserum processing procedure.
Response analysis: after the antiserum after protein analyzer senses process adds, automatically test, reads blank value according to the blank time arranged, and reads range of reaction according to the reaction time arranged.
In the present embodiment, the protein analyzer that protein analyzer preferably selects Shenzhen Genius Electronics Co., Ltd. to manufacture.Preferably, protein analyzer selects wavelength to be the detection ripple of 650nm.
Confirmed standard curve: utilize the mALB calibration solution of variable concentrations RANDOX to make typical curve, adopt standard curve fitting software and to typical curve adopt non-linear, repeatedly, piecewise fitting, can 5-330mg/L be reached to make the detection range of linearity.
Preferred embodiment 2:
The detection method for trace protein of the preferred embodiment of the present invention 2, comprises the steps:
Configuration damping fluid: configuration antiserum treating fluid: the Gly of the NaCl solution+40mM of the phosphate buffer+150mM of 20mM, PH7.0-7.8.
Configuration reaction buffer: the isinglass of the NaCl solution+0.01-0.5 mass percent of the phosphate buffer+30-100mM of 20mM, PH7.0-7.8.
Antiserum process: adopt antiserum treating fluid that antiserum is diluted to 2mg/ml, after room temperature places 24 hours, to make the various component in antiserum treating fluid and the antibody in antiserum fully act on, then places in the refrigerator of 4 DEG C of temperature and preserves.In the present embodiment, antiserum preferably, ADVY company of India produce antiserum.
Reaction time is arranged: arranging blank time is 8 seconds, and the reaction time is 124 seconds.
Operation: add 400 microlitre reaction buffers in reaction cup, then the urine specimen of 20 microlitres is added, the reaction cup of the urine specimen added is put in the reacting hole of protein analyzer, and antiserum after adding 40 microlitre reactions after the process of antiserum processing procedure.
Response analysis: after the antiserum after protein analyzer senses process adds, automatically test, reads blank value according to the blank time arranged, and reads range of reaction according to the reaction time arranged.
In the present embodiment, the protein analyzer that protein analyzer preferably selects Shenzhen Genius Electronics Co., Ltd. to manufacture.Preferably, protein analyzer selects wavelength to be the detection ripple of 690nm.
Confirmed standard curve: utilize the mALB calibration solution of variable concentrations RANDOX to make typical curve, adopt fitting software or program and to typical curve adopt non-linear, repeatedly, piecewise fitting, can 5-330mg/L be reached to make the detection range of linearity.
Further, adopt fitting software or program and to typical curve adopt non-linear, repeatedly, piecewise fitting is specially the section of skyrocketing in detectable antigens and antibody response process, curve occurred, adopt to seamlessly transit curve and carry out auto modification.
Table 1: the range of linearity (antiserum preferably India ADVY company is produced) of detection
In the present embodiment, experiment is compared to the sero-fast range of reaction of different dilute concentration:
Table 2: the range of reaction of different antiserum dilute concentration compares
In antagonistic Serum processing procedure, the range of reaction after the antiserum after dilution adopts different disposal time (room temperature standing time) process is compared:
Table 3: the comparison of antiserum different disposal time response amplitude
Stability is tested:
By the antiserum after process 2 DEG C-8 DEG C, in the light protected environment of non-corrosiveness gas storage after 12 months, get the pattern detection of effect after date, analyze the relative deviation situation with target value.Test the antiserum after process with the mALB calibration solution of RANDOX, duplicate detection 10 times, takes the mean, and then calculates the relative deviation with target value.
Form 4-1: stability test
Form 4-2: stability
Accuracy measures: under repeated condition, test the reaction solution after operation process with the mALB calibration solution of RANDOX, replication ten times, then calculates inaccuracy and inaccuracy.
Form 5: inaccuracy and inaccuracy tables of data
Sensitivity behaviour is tested: be the reference material of 5mg/L with the target value of RANDOX, tests, replication ten times to the reaction solution after operation process, calculating variance.Then calculate the scope of the variance of target value plus-minus twice, and all values measured for ten times is all in scope.
Form 6: sensitivity
By table 1,2,3,4-2,4-2,5,6 and Fig. 1 and Fig. 2 can draw:
The detection method of trace of albumin of the present invention, the detection of particularly suitable microdose urine protein, by improving the processing mode of antibody, reaction time, the component of reaction buffer, the fit approach of typical curve, effectively alleviate prozone phenomenon, make that mALB sensing range is wider can reach 5-330mg/L, the accuracy detected improves greatly, thus is more conducive to clinical diagnosis.
Meanwhile, the antiserum after the present embodiment antiserum treating fluid and disposal methods has good stability, and place 1 year under 4 DEG C of conditions of storage, activity can not reduce.
Antibody property indices is given prominence to: accuracy: inaccuracy≤5% in batch; Inaccuracy≤6% between batch; Inaccuracy≤3%; Sensitivity: the mALB concentration that can detect 5mg/L, and the concentration value measured is all in ± 2SD.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.