CN109813908A - Eliminate the method and kit of hook effect in cystatin C detection - Google Patents
Eliminate the method and kit of hook effect in cystatin C detection Download PDFInfo
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Abstract
The invention discloses a kind of methods of hook effect in elimination cystatin C detection, reagent used includes reagent R1, reagent R2, and calibration object, the method for eliminating hook effect in cystatin C detection is reacted using competitive immunization, it include: that anti-cystatin C antibody is added in reagent R1, cystatin C sensitizing latex particle is added in reagent R2, calibration object uses cystatin C calibration object；When detection, by combining the cystatin C antibody of the known concentration of cystatin C corresponding thereto, then with cystatin C sensitizing latex particle reaction, by measuring the variable quantity of absorbance, to determine the concentration of measurand in sample.Advantage is: accuracy in detection is high, detection range is wide.The invention also discloses a kind of kits of hook effect in elimination cystatin C detection.
Description
Technical field
The present invention relates to biochemical reagents technical fields, more particularly, to hook effect in a kind of elimination cystatin C detection
Method.The invention further relates to a kind of kits of hook effect in elimination cystatin C detection.
Background technique
Preceding band and rear band are just proposed by Heidelberger early in nineteen twenty-nine.He adds not same amount antigen in constant basis antibody
In immune complex (IC) the precipitating quantifier elimination formed afterwards, relational graph shown in FIG. 1 is obtained.He (turns vertex of a parabola
Point) at left and right sides of, various trait shown by same reaction is known as two-phase response (biphasic response), and the song
Conversion zone included by line is roughly divided into antibody excess region with the relative scale of Ag-Ab, also known as preceding band (prozone),
The roughly equal equivalent area of the two concentration is also known as equivalence zone, the also known as rear band (postzone) of antibody excess region.In immunological test
In, when with quantitative antigen measuring antibody, if measured antibody concentration is lower than antibody equivalent concentration, the IC amount of formation is proportional to anti-
Bulk concentration；Reduced instead greater than the IC amount formed after the equivalent concentration, antibody excess is more, and the IC amount of formation is fewer, occur with
The similar response curve of Fig. 1.This phenomenon was called prozone phenomenon in the past.If otherwise when with quantitative antibody determination antigen, when
Tested antigen concentration is greater than the case where IC amount occurred after the equivalent concentration of antibody is reduced, referred to as Postzone phenomenon.These name sources
In classical Heidelberger two-phase response curve, and adopt for a long time until now.After the advent of monoclonal antibody,
Miles etc. has founded the double site two step method of radiommunoassay, is developed again as double site one-step method soon thereafter.At him
Measure in the research of serum ferritin and find, in determining solid phase antibody concentration and labelled antibody concentration, when tested ferritin
After to a certain degree, reaction signal is no longer directly proportional to analyte concentration and turns to inverse relation.Such case is one
It is particularly acute in footwork, that is, the detection range measured is narrower compared with two step method.Again and again by many researcher institutes after this phenomenon
It confirms, it is more common in ELISA reaction.Green in 1977 etc. proposes hook effect (hook according to response curve shape
Effect title).Therefore strictly speaking, prozone phenomenon is meant makes reaction signal weaken and make signal-instead when antibody excess
Dosage (concentration) curve is in hook-shaped phenomenon；Postzone phenomenon makes reaction signal-dose curve in hook-shaped when referring to antigen excess
Phenomenon.Therefore hook effect summarises forward and backward zoning, more definite in name.Because habit makes so, prozone phenomenon is existing still to exist
It uses, one word of Postzone phenomenon is not mostly used, often mixed with prozone phenomenon in former document.Hook effect refers to antigen mistake more at present
For surplus, therefore there is high dose hook effect (high dose hook effect) word again, with low dose not in competition law
Measure hook effect.The present prozone phenomenon situation mixed with hook effect is still more common, not yet unified on glossology.
The reason of hook effect, once there are many kinds of explain.Tend to approval viewpoint be, when Ag-Ab ratio where appropriate,
More complete IC network structure is formed, makes the hydrophobic structure sufficiently exposure of protein molecule and is also easy to produce precipitation reaction.Conversely, working as
When the two is out of proportion, the structural intergrity of IC will be influenced to some extent, with certain solubility or occurred reversible
Dissolution, i.e. IC are more imperfect, more soluble or invertible dissolution.Therefore for immune precipitation, hook effect is
Since the sufficiently large and small IC of solubility cannot be generated.For agglutinating reaction, then because incomplete IC network structure is through shaking
And reversible dissociation occurs.For various label immunoassays, then because forming the IC of solubility IC or formation easily from solid phase carrier
Elution.Excessive measured object falls over each other to combine with Solid phase protein and labelled protein in a step sandwich method, it is difficult to or cannot be effectively
Form sandwich conjugate.It is weakly positive so that false negative result that high dose hook effect, which surveys strong positive sample accidentally,.In competition law
Low dosage hook effect mean and generate stronger reaction signal when analyte concentration is lower instead, its reason is still being studied
In.For exceptional value and the less big Pharmaceutical Analysis etc. of Upper Limit of Normal Value spacing, low dosage hook effect can produce error result,
In general its severity is not so good as high dose hook effect.
The detection method that the country applies at present mainly has dry chemical method of inspection, immunofluorescence technique, enzyme immunoassay, radio-immunity
Method and immunoturbidimetry.Dry chemical method of inspection can only provide sxemiquantitative or qualitatively as a result, dry chemical scrip detects simultaneously
As a result by compared with multifactor impact, it inevitably will appear false negative and false positive results in clinical examination.Immunofluorescence technique
And enzyme immunoassay is complicated for operation and time-consuming, inconvenient extensive clinical application.Radioimmunology high sensitivity, high specificity, but have
There is the disadvantages of radioactive pollution, reagent storage life is short.Immunoturbidimetry has easy to operate, the measurement period is short, precision is high etc.
Advantage, using relatively broad on Biochemical Analyzer.But since the testing principle of immunoturbidimetry is that reaction generation is insoluble
Antigen-antibody complex, and certain turbidity is generated, the content of antigen, leads to existing exempt from the height reflection sample of turbidity
Inevitably there is the lower deficiency of sensitivity in epidemic disease reaction detection kit.
Summary of the invention
The object of the present invention is to provide a kind of methods of hook effect in elimination cystatin C detection, it has and can directly answer
For automatic clinical chemistry analyzer, as a result detection range is wide, is able to solve the influence, easy to use of hook effect, and is convenient for
The characteristics of large-scale promotion.The invention also discloses a kind of kits of hook effect in elimination cystatin C detection.
First technical solution of the present invention is:
The method for eliminating hook effect in cystatin C detection, reagent used include reagent R1, reagent R2, and calibration
Product, it is characterised in that: the method for eliminating hook effect in cystatin C detection is reacted using competitive immunization, comprising:
(1) anti-cystatin C antibody is added in reagent R1, cystatin C sensitizing latex particle, calibration are added in reagent R2
Product use cystatin C calibration object；
(2) when detecting, press down by combining the cystatin C antibody of the known concentration of cystatin C corresponding thereto, then with Guang
Plain C sensitizing latex particle reaction, by measuring the variable quantity of absorbance, to determine the concentration of measurand in sample.
The component of the reagent R1 are as follows: buffer 10-300mmol/L, surfactant 0.1-10.0g/L, preservative
0.1-10.0g/L, anti-cystatin C antibody 20-50mg/L, wherein the pH value of buffer is 5.0-9.0；
The component of the reagent R2 are as follows: buffer 10-300mmol/L, preservative 0.1-10.0g/L, suspending agent 0.1-
10.0g/L, cystatin C sensitizing latex particle 50-800ml/L, wherein the pH value of buffer is 5.0-9.0.
Second technical solution of the present invention is:
The kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object are eliminated,
The component of the reagent R1 are as follows: buffer 10-300mmol/L, surfactant 0.1-10.0g/L, preservative
0.1-10.0g/L, anti-cystatin C antibody 20-50mg/L, wherein the pH value of buffer is 5.0-9.0；
The component of the reagent R2 are as follows: buffer 10-300mmol/L, preservative 0.1-10.0g/L, suspending agent 0.1-
10.0g/L, cystatin C sensitizing latex particle 50-800ml/L, wherein the pH value of buffer is 5.0-9.0.
The buffer be Tris buffer, MES buffer, MOPS buffer, TEA buffer, HEPES buffer solution,
One of PIPES buffer, PBS buffer solution.
The surfactant is one of polyoxyethylene deriv, polyoxyethylene alkyl ether sulfate salt, polidocanol
Or it is a variety of, the suspending agent is polyoxyethylene deriv, polyoxyethylene alkyl ether sulfate salt, arlacels, lauryl
One of betaines are a variety of.
The HLB value of the polyoxyethylene deriv is 12-18.8, and the HLB value of arlacels is 1.8-8.6.
The HLB value of the polyoxyethylene deriv is 13.3-15, and the HLB value of arlacels is 4.3-6.7.
The preservative be CAA, 5-Bromo-5-notrp-1,3-dioxane, ProClin 300,
One of Imidazolidinylurea (IZUH2O), Sodium azide, potassium sorbate, gentamicin.
The partial size of cystatin C sensitizing latex particle is 60-300nm in the reagent R2.
The invention has the advantages that compared with the prior art:
1, accuracy in detection is high.From principle, increases cystatin C antibody in reagent R1, used in reagent R2
The latex particle of cystatin C sensitization can be effectively prevented from false negative.It prescribes a time limit when the antigen in sample exceeds detection of the invention,
Measurement result is not less than linear value, not will cause erroneous judgement.Also that is, compared to current immune detection reagent (to detect antigen
For), calibration object basic indifference does not add antibody in reagent R1, and antibody is added in reagent R2 and (or is coated with the glue of antibody
Newborn antibody), and testing result is calculated according to the increase of turbidity, for the reaction that absorbance rises, testing result depends on being added
Amount of antibody.When amount of antigen is more than amount of antibody, there is hook effect, it may appear that the result of false negative.Such as normal, abnormal
Cut off value is 5mg/L, it is assumed that setting-out line is 30mg/L, then the result that 55mg/L is shown is exactly 5mg/L, and 100mg/L may
It is exactly negative value.In the present invention, the antibody of known concentration is added in reagent R1, and the latex of antigen coat is added in reagent R2
Grain, calculates testing result according to the reduction of turbidity.When antigen concentration is more than the concentration of known antibodies, excessive antigen and sample
Latex particle in this does not react, and with antigen excess, turbidity is no longer reduced, and absorbance tends towards stability, the additional amount of value and antibody
It is consistent, the amount of antibody of addition is more than exceptional value.Such as normal, abnormal cut off value is 5mg/L, it is assumed that setting-out line
Property is 30mg/L, then the result that 55mg/L is shown is exactly 30mg/L, and 100mg/L is also 30mg/L, and false negative will not occur.Such as
This, eliminates hook effect, improves detection accuracy.In addition, passing through the restriction of Surfactant and suspending agent HLB value, rise
The effect of linear degree of fitting in improvement method is arrived.
2, detection range is wide.Improve the detection range that antibody content significantly improves clinical diagnosis.With detection method before
It compares, same to add the antibody that potency is 20mg/L, detection method before is 8.5mg/L in the sample value of measurement 30mg/L,
And when using reagent of the invention, measured value 20.5mg/L.Detection method before use, when the D-dimer in sample is super
After crossing 20mg/L, redundance D-dimer so that turbidity reduction, causes measurement result relatively low, and can use this in conjunction with antibody
The reagent of invention, theoretically, the sample more than 20mg/L can react without remaining antibody with latex, and turbidity is not
Reduce again, therefore result always show 20.5mg/L, it can be seen that, when addition antibody be more than clinical diagnosis range, would not
Generate erroneous judgement.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples:
Fig. 1 is hook effect schematic diagram；
Fig. 2 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 1 is eliminating hook
Measurement result figure after shape effect；
Fig. 3 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 2 is eliminating hook
Measurement result figure after shape effect；
Fig. 4 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 3 is eliminating hook
Measurement result figure after shape effect；
Fig. 5 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 4 is eliminating hook
Measurement result figure after shape effect；
Fig. 6 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 5 is eliminating hook
Measurement result figure after shape effect；
Fig. 7 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 6 is eliminating hook
Measurement result figure after shape effect；
Fig. 8 is that the kit of hook effect in elimination cystatin C detection prepared by the embodiment of the present invention 7 is eliminating hook
Measurement result figure after shape effect；
Fig. 9 is commercially available cystatin C kit measurement result figure.
Specific embodiment
Embodiment 1
Eliminate the kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object.This implementation
In example, the cystatin C calibration object of Ningbo Puri cypress Biotechnology Ltd. is used using commercially available calibration object, in use,
Cystatin C concentration is respectively adopted in calibration object: 0.0,0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, 8.0mg/L, calibration
Also contain buffer 10mmol/L in product.
Specifically:
The component of reagent R1 are as follows: buffer 150mmol/L, surfactant 5g/L, preservative 10.0g/L, the suppression of anti-Guang
Plain C antibody 10mg/L, wherein the pH value of buffer is 9.0；
The component of reagent R2 are as follows: buffer 10mmol/L, preservative 0.1g/L, suspending agent 0.1g/L, cystatin C sensitization
Latex 50ml/L, wherein the pH value of buffer is 6.0.
Wherein:
Triton X-405 (the isooctyl phenyl polyoxyethylene that buffer is Tris buffer, surfactant is 2:1
Ether) and KAO B66 (polyoxyethylene radical derivative, HLB13.2), preservative be Imidazolidinylurea (IZUH2O),
Antibody is sheep anti-human polyclonal antibody, preservative is Imidazolidinylurea (IZUH2O), suspending agent is KAO 20AB,
The partial size of cystatin C sensitizing latex is 60nm.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 2, as a result as can be seen that when sample final concentration result is super
When crossing 10mg/L, measurement result no longer occurs significantly to change, and detection range is up to 15mg/L.
Embodiment 2
Eliminate the kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object.This implementation
In example, the cystatin C calibration object of Ningbo Puri cypress Biotechnology Ltd. is used using commercially available calibration object, in use,
Cystatin C concentration is respectively adopted in calibration object: 0.0,0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, 8.0mg/L, calibration
Also contain buffer 50mmol/L in product.
Specifically:
The component of reagent R1 are as follows: buffer 300mmol/L, surfactant 10g/L, preservative 0.1g/L, the suppression of anti-Guang
Plain C antibody 15mg/L, wherein the pH value of buffer is 7.0；
The component of reagent R2 are as follows: buffer 10mmol/L, preservative 0.3g/L, suspending agent 3g/L, cystatin C sensitization glue
Newborn 800ml/L, wherein the pH value of buffer is 8.0.
Wherein: buffer is MES buffer, surfactant is Triton X-405 (isooctyl phenyl polyoxyethylene
Ether), Span40 (the anhydrous sorbitol list that preservative is potassium sorbate, antibody is rabbit anti-human polyclonal antibody, suspending agent is 9:4
Palmitate, HLB value 6.7) and KAO 24B (lauryl betaine), the partial size of cystatin C sensitizing latex is 220nm.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 3, as a result as can be seen that when sample final concentration result is super
When crossing 15mg/L, measurement result no longer occurs significantly to change, and detection range is up to 30mg/L.
Embodiment 3
Eliminate the kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object.This implementation
In example, the cystatin C calibration object of Ningbo Puri cypress Biotechnology Ltd. is used using commercially available calibration object, in use,
Cystatin C concentration is respectively adopted in calibration object: 0.0,0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, 8.0mg/L, calibration
Also contain buffer 30mmol/L in product.
Specifically:
The component of reagent R1 are as follows: buffer 10mmol/L, surfactant 0.8g/L, preservative 5g/L, anti-cystatin C
Antibody 20mg/L, wherein the pH value of buffer is 5.0；
The component of reagent R2 are as follows: buffer 300mmol/L, preservative 9g/L, suspending agent 2g/L, cystatin C sensitization glue
Newborn 500ml/L, wherein the pH value of buffer is 7.0.
Wherein: buffer is PIPES buffer, surfactant is Thesit (polidocanol), preservative CAA, is resisted
Body is mouse anti-human monoclonal's antibody, suspending agent is Tween 80 (polyoxyethylene (20EO) sorbitan monooleate, HLB value
15), the partial size of cystatin C sensitizing latex is 300nm.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 4, as a result as can be seen that when sample final concentration result is super
When crossing 20mg/L, measurement result no longer occurs significantly to change, and detection range is up to 28mg/L.
Embodiment 4
Eliminate the kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object.This implementation
In example, the cystatin C calibration object of Ningbo Puri cypress Biotechnology Ltd. is used using commercially available calibration object, in use,
Cystatin C concentration is respectively adopted in calibration object: 0.0,0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, 8.0mg/L, calibration
Also contain buffer 100mmol/L in product.
Specifically:
The component of reagent R1 are as follows: buffer 50mmol/L, surfactant 0.5g/L, preservative 2g/L, anti-cystatin C
Antibody 30mg/L, wherein the pH value of buffer is 9.0；
The component of reagent R2 are as follows: buffer 150mmol/L, preservative 10g/L, suspending agent 5g/L, cystatin C sensitization glue
Newborn 100ml/L, wherein the pH value of buffer is 7.0.
Wherein: the Thesit (polidocanol) and Atlas G- that buffer is TEA buffer, surfactant is 10:1
1794 (Emulsifier EL-60, HLB13.3), preservative are Imidazolidinylurea (IZUH2O), antibody is rabbit-anti
People's polyclonal antibody, suspending agent be KAO 20AB, the partial size of cystatin C sensitizing latex is 160nm.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 5, as a result as can be seen that when sample final concentration result is super
When crossing 30mg/L, measurement result no longer occurs significantly to change, and detection range is up to 45mg/L.
Embodiment 5
Difference with embodiment 1 is only that: buffer is Tris buffer, preservative is Proclin 300, suspending agent is
The Span40 (sorbitan monopalmitate, HLB value 6.7) plus KAO 24B (lauryl betaine) of 3:7.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 6, as a result as can be seen that when sample final concentration result is super
When crossing 10mg/L, measurement result no longer occurs significantly to change, and detection range is up to 20mg/L.
Embodiment 6
Difference with embodiment 2 is only that: the Brij 35 that buffer is HEPES buffer solution, surfactant is 3:1 is (poly-
Ethylene oxide (20EO) sorbitan monooleate, HLB16.9) and EMAL 20C (sodium laureth sulfate),
Preservative is KATHONLX150.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 7, as a result as can be seen that when sample final concentration result is super
When crossing 15mg/L, measurement result no longer occurs significantly to change, and detection range is up to 25mg/L.
Embodiment 7
Difference with embodiment 3 is only that: the KAO20AB and Span that buffer is PBS buffer solution, suspending agent is 2:7
20, preservative is potassium sorbate.
The reagent being prepared is liquid reagent, can directly be used on full automatic biochemical apparatus.According to following ratios, in life
Change and set on instrument, can start to measure sample.Parameter is by taking 7180 automatic biochemistry analyzer of Hitachi as an example:
Measuring method: two point method；
Detection wavelength: 546nm；
The Direction of Reaction: reaction downwards；
V sample: VR1:VR2=3 μ l:240 μ l:60 μ l；
Calibration and calibrating mode: water is carried out curve fitting after calibrating with calibration object using Spline.
Operating procedure:
In 37 DEG C of incubation 3-5min after sample and reagent R1 mixing, the 1st reading point absorbance A 1 is read, reagent is added immediately
R2,37 DEG C of incubation 5min after mixing read the 2nd reading point absorbance A 2.
As a result it calculates:
It is calibrated using calibration object and water (zero point), by absorbance change value (A calibration-A blank) to its respective concentration
Figure is done, calibration curve is drawn.Containing for cystatin C in sample is acquired on calibration curve according to (A sample-A blank) value of sample
Amount.
Cystatin C sample is detected, acquired results are detailed in Fig. 8, as a result as can be seen that when sample final concentration result is super
When crossing 20mg/L, measurement result no longer occurs significantly to change, and detection range is up to 30mg/L.
Effect example 3
Prepared by certain commercially available cystatin C kit (hereinafter referred to as commercial reagent box 1) and the embodiment of the present invention 1 to 7
Cystatin C kit (kit 1 to 7 hereinafter referred to as of the present invention) be compared, as shown in connection with fig. 9:
The detection range of commercial reagent box 1 are as follows: 8mg/L；
The detection range of kit 1 of the present invention are as follows: 15mg/L；
The detection range of kit 2 of the present invention are as follows: 30mg/L；
The detection range of kit 3 of the present invention are as follows: 28mg/L；
The detection range of kit 4 of the present invention are as follows: 45mg/L；
The detection range of kit 5 of the present invention are as follows: 20mg/L；
The detection range of kit 6 of the present invention are as follows: 25mg/L；
The detection range of kit 7 of the present invention are as follows: 30mg/L；
Illustrate: kit accuracy in detection of the invention is higher, detection range is wider.Meanwhile it can be directly full-automatic
Biochemical instruments carry out using.
Meanwhile by Fig. 2 to Fig. 8 it can be found that being more than the antibody concentration of addition, value will not reduce, illustrate of the invention
Kit does not have hook effect.
In conclusion the present invention is in reagent R1 by increasing the corresponding antigen of measurand or antibody, in reagent R2
The middle latex particle for increasing the corresponding antibody of measurand or antigen sensibilization, can be effectively prevented from false negative, when anti-in sample
Original is prescribed a time limit beyond detection of the invention, and measurement result is not less than linear value, not will cause erroneous judgement, and antibody content can be improved can
Improve detection range.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
Equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content is applied directly or indirectly in other correlations
Technical field, be included within the scope of the present invention.
Claims (9)
1. the method for eliminating hook effect in cystatin C detection, reagent used includes reagent R1, reagent R2 and calibration object,
It is characterized by: the method for eliminating hook effect in cystatin C detection is reacted using competitive immunization, comprising:
(1) anti-cystatin C antibody is added in reagent R1, cystatin C sensitizing latex particle is added in reagent R2, calibration object is adopted
With cystatin C calibration object；
(2) when detecting, by combining the cystatin C antibody of the known concentration of cystatin C corresponding thereto, then with cystatin C
Sensitizing latex particle reaction, by measuring the variable quantity of absorbance, to determine the concentration of measurand in sample.
2. the method according to claim 1 for eliminating hook effect in cystatin C detection, it is characterised in that:
The component of the reagent R1 are as follows: buffer 10-300mmol/L, surfactant 0.1-10.0g/L, preservative 0.1-
10.0g/L, anti-cystatin C antibody 20-50mg/L, wherein the pH value of buffer is 5.0-9.0；
The component of the reagent R2 are as follows: buffer 10-300mmol/L, preservative 0.1-10.0g/L, suspending agent 0.1-10.0g/
L, cystatin C sensitizing latex particle 50-800ml/L, wherein the pH value of buffer is 5.0-9.0.
3. eliminating the kit of hook effect in cystatin C detection, including reagent R1, reagent R2 and calibration object, feature exist
In:
The component of the reagent R1 are as follows: buffer 10-300mmol/L, surfactant 0.1-10.0g/L, preservative 0.1-
10.0g/L, anti-cystatin C antibody 20-50mg/L, wherein the pH value of buffer is 5.0-9.0；
The component of the reagent R2 are as follows: buffer 10-300mmol/L, preservative 0.1-10.0g/L, suspending agent 0.1-10.0g/
L, cystatin C sensitizing latex particle 50-800ml/L, wherein the pH value of buffer is 5.0-9.0.
4. the kit according to claim 3 for eliminating hook effect in cystatin C detection, it is characterised in that: described slow
Fliud flushing is Tris buffer, MES buffer, MOPS buffer, TEA buffer, HEPES buffer solution, PIPES buffer, PBS are slow
One of fliud flushing.
5. the kit according to claim 3 for eliminating hook effect in cystatin C detection, it is characterised in that: the table
Face activating agent is one of polyoxyethylene deriv, polyoxyethylene alkyl ether sulfate salt, polidocanol or a variety of, the suspending
Agent is one of polyoxyethylene deriv, polyoxyethylene alkyl ether sulfate salt, arlacels, lauryl betaine class
Or it is a variety of.
6. the kit according to claim 5 for eliminating hook effect in cystatin C detection, it is characterised in that: described poly-
The HLB value of ethylene oxide derivative is 12-18.8, and the HLB value of arlacels is 1.8-8.6.
7. the kit according to claim 5 for eliminating hook effect in cystatin C detection, it is characterised in that: described poly-
The HLB value of ethylene oxide derivative is 13.3-15, and the HLB value of arlacels is 4.3-6.7.
8. the kit according to claim 3 for eliminating hook effect in cystatin C detection, it is characterised in that: described anti-
Rotten agent is CAA, 5-Bromo-5-notrp-1,3-dioxane, ProClin 300, Imidazolidinylurea (IZU
H2O), one of Sodium azide, potassium sorbate, gentamicin.
9. the kit according to claim 3 for eliminating hook effect in cystatin C detection, it is characterised in that: the examination
The partial size of cystatin C sensitizing latex particle in agent R2 is 60-300nm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112946254A (en) * | 2021-01-18 | 2021-06-11 | 上海云泽生物科技有限公司 | Latex-enhanced competitive immunoturbidimetric assay method and kit |
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