CN102384893A - Seminal plasma gamma-L-glutamyl transpeptidase activity quantitative detecting reagent kit and application thereof - Google Patents
Seminal plasma gamma-L-glutamyl transpeptidase activity quantitative detecting reagent kit and application thereof Download PDFInfo
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Abstract
The invention discloses a seminal plasma gamma-L-glutamyl transpeptidase activity quantitative detecting reagent kit, which comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following ingredients: 100 to 200 mmol/L trihydroxymethyl aminomethane buffer with pH value of 8.0 to 8.3, 90 to 110mmol/L glycylglycine and 0.01%to 0.1% antiseptic; and the reagent R2 comprises the following ingredients: 100 to 200mmol/L trihydroxymethyl aminomethane buffer with pH value of 8.0 to 8.3, 0.5 to 3.0mmol/L substrate and 0.01% to 0.1% antiseptic. The two liquid reagents can be directly used without being frozen at the temperature of minus 20 DEG C, the type and the consumption of the reagent can be remarkably reduced, and the sample is free from being diluted for more than 200 times; the substrate has good specificity and good appetency to the enzyme, so the reaction speed is fast; and the reagent kit is matched with an automatic biochemical instrument to use, so the procedures can be implified, the reagent can be saved, the manual error can be reduced, automatic and large-scale detection can be realized, the detection result is more accurate and more reliable, and the detection efficiency can be greatly improved.
Description
Technical field
The invention belongs to biomedical check determination techniques field, relate to a kind of in-vitro diagnosis with kit and application thereof, specifically a kind of refining γ-L-glutamyl transpeptidase active level detection kit and application thereof.
Background technology
Refining γ-L-glutamyl transpeptidase (being called for short γ-L-glutamyl transpeptidase) is mainly derived from prostate, and its content is 200-500 times in the serum approximately, and its active detection can be used for estimating prostatic function; In addition, refining γ-L-glutamyl transpeptidase is the key enzyme of glutathione metabolism, avoids also playing important effect aspect the oxygen radical infringement at the protection sperm.In prostate hypofunction patient, its γ-L-glutamyl transpeptidase is active obviously to descend; In prostate cancer and benign prostatic hyperplasia patient, its activity significantly increases.
At present, the method that detects refining γ-L-glutamyl transpeptidase mainly is a chemical colorimetry, and this fado is craft or semi-automatic operation, because its hyperactivity; Sample needs dilution in advance, and the application of sample program is many, and operation steps is complicated, reagent and after the sample mixing regular hour; Through spectrophotometer colorimetric estimation one by one, calculate the result according to formula again, consumptive material consumption power not only consuming time; Do not meet scale and benefit, and personal error is also bigger, consistance is not fine as a result.
Now, the clinical biochemical check has all realized automated analysis basically.Full automatic biochemical apparatus is exactly with the sampling in the original process hand-manipulated, mixing, incubation, detection, calculating as a result, judgement, demonstration and all or part of automatic operation of steps such as print result and cleaning; Because its measuring speed is fast, accuracy is high, the consumption amount of reagent is little; Obtained in epidemic prevention station, hospital at different levels, family planning service station being widely used at present, be used the income and the efficient that can improve the routine biochemistry check greatly.Regrettably; By the end of at present, no matter be abroad or domestic, for the detection of refining γ-L-glutamyl transpeptidase project; Except early stage scientific payoffs; Still do not have a kind of detection kit that can match with full automatic biochemical apparatus, commercial to come out, therefore, set up a kind of and full automatic biochemical apparatus refining γ-L-glutamyl transpeptidase active level detection kit and the detection method of using that match and just seem particularly urgent and important.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, fill up the vacancy on the market, a kind of refining γ-L-glutamyl transpeptidase active level detection kit that matches and use with full automatic biochemical apparatus is provided.
Another object of the present invention is to provide the application of said refining γ-L-glutamyl transpeptidase active level detection kit in detecting refining γ-L-glutamyl transpeptidase activity.
For solving the problems of the technologies described above, the present invention has adopted following technical scheme:
A kind of refining γ-L-glutamyl transpeptidase active level detection kit comprises R1 reagent and R2 reagent; Wherein, R1 reagent comprises the component of content: 100-200mmol/L, pH8.0-8.3 TRIS buffer, 90-110mmol/L glycylglycine and 0.01-0.1% antiseptic; R2 reagent comprises the component of following content: 100-200mmol/L, pH8.0-8.3 TRIS buffer, 0.5-3.0mmol/L substrate and 0.01-0.1% antiseptic; Wherein, said substrate is L-γ-Gu Anxianji-p-nitroaniline or L-γ-Gu Anxianji-3-carboxyl-4-nitroaniline; Described antiseptic is any in Sodium azide, thimerosal, gentamicin or the ProClin series antiseptic.
The application of described refining γ-L-glutamyl transpeptidase active level detection kit in measuring refining γ-L-glutamyl transpeptidase activity.
The application of described refining γ-L-glutamyl transpeptidase active level detection kit in measuring refining γ-L-glutamyl transpeptidase activity comprises the steps:
1) specimen preparation: fresh semen liquefaction back is through the centrifugal 10～20min of 3000～5000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is following in the function software of full automatic biochemical apparatus:
Test item: γ-GT;
Sample reagent ratio: 1: 100;
Detection method: rate method;
Calibrating method: factorization method;
Predominant wavelength: 405-410nm;
The Direction of Reaction: rise;
Reaction time: 36-54 second;
Detection time: 90-120 second;
3) scale operation: adopt the factorization method calibration, directly input hypothesis factor F value gets final product;
4) pattern detection:
The refining sample for preparing and R1 reagent, R2 reagent are placed on the relevant position of full automatic biochemical apparatus, and full automatic biochemical apparatus goes out the activity of γ in the sample-L-glutamyl transpeptidase according to theoretical factor calculation:
The U of unit of enzyme activity definition: under above-mentioned reaction conditions, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol p-nitroaniline;
Active (the U/L)=Δ A/min * F of γ in the refining-L-glutamyl transpeptidase;
Wherein, Δ A/min is the rate of change of per minute absorbance, and F is known.
Said R1 reagent is liquid solution, and said buffer solution is the Tris damping fluid of PH8.0-8.3, is used to reaction suitable reaction conditions is provided; In the R1 reagent, glycylglycine is the effective binding capacity that decomposes the L-γ-Gu Anxianji that produces with γ-L-glutamyl transpeptidase catalysis capacity substrate, as the acceptor of L-γ-Gu Anxianji.
Said R2 reagent is liquid solution, and said buffer solution is trishydroxymethylaminomethane (Tris) damping fluid of PH8.0-8.3, is used to reaction suitable reaction conditions is provided; Substrate in the R2 reagent is L-γ-Gu Anxianji-p-nitroaniline (L-γ-glutamyl-p-nitroanilide); Can also be L-γ-Gu Anxianji-3-carboxyl-4-nitroaniline (L-γ-glutamyl-3-carboxy-4-nitroanilide), be used for the substrate of γ-L-glutamyl transpeptidase effect; In the said R2 reagent, the content of substrate is 0.5-3.0mmol/L, when being lower than the lower limit 0.5mmol/L of substrate; Can cause the range of linearity of kit to reduce, also can shorten the linear phase of reagent reacting, when being higher than the upper limit 3.0mmol/L of substrate; The cost of kit is increased, and absorbance is not in the sensing range of instrument, in the 0.5-3.0mmol/L scope; The concentration of substrate is high more, and the rate of change of per minute absorbance is fast more.
Said antiseptic is a Sodium azide, and content is 0.01-0.1%, and this scope can be to not reacted any spinoff, the term of validity that can prolong kit in effective sterilization; Can also be any etc. in other thimerosal, gentamicin or the ProClin series antiseptic, in buffer solution, play the corrosion-resistant effect with antisepsis.
In the method for above-mentioned application, in the said step 1), prepared refining sample can detect in separating the same day, also can preserve more than 2 weeks at least, but should avoid multigelation in frozen subsequent use below-20 ℃; Said step 2) in, sample reagent should be taken into account the minimum application of sample amount of full automatic biochemical apparatus than deciding according to the parameter of full automatic biochemical apparatus, the reactant liquor volume scope of cuvette etc.; In the said step 3), theoretical factor F value can be different on different full automatic biochemical apparatus, and the computing formula of theoretical factor F value is:
Wherein, 10
3Transformation ratio for mmol to μ mol; Vt is the reactant liquor cumulative volume; ε is the theoretical molar extinction coefficient of p-nitroaniline; L is cuvette optical path (1cm); Vs is a sample volume; In the said step 4), the hyperactivity of γ in sample-L-glutamyl transpeptidase when exceeding the range of linearity of kit, is used and is resurveyed after physiological saline dilutes, and the result multiply by extension rate and gets final product.
Detection method of the present invention is that enzyme process detects, and described R1 reagent, R2 reagent are the buffer solution that contains L-γ-Gu Anxianji-p-nitroaniline, glycylglycine, Tris, antiseptic, detect principle to be,
With L-γ-Gu Anxianji-p-nitroaniline is substrate; Glycylglycine is the acceptor of glutamyl, and under the catalysis of γ-L-glutamyl transpeptidase, glutamyl is transferred on the glycylglycine molecule; Discharge coloured product p-nitroaniline simultaneously; The p-nitroaniline has maximum absorption band in the 405-410nm wavelength, through the per minute absorbance rate of change of monitoring 405-410nm wavelength p-nitroaniline, and then detects the activity of γ in the sample-L-glutamyl transpeptidase.
Compared with prior art, outstanding technique effect is:
1, this kit adopts rate method to detect the activity of refining γ-L-glutamyl transpeptidase; Only adopt 2 kinds of liquid reagents; Can be subsequent use in 4 ℃ of preservations, to compare with the end-point method of manual method, rate method is more suitable for the active detection of γ-L-glutamyl transpeptidase; And reagent do not need in-20 ℃ frozen, the kind and the consumption of reagent all significantly reduce;
2, adopt this kit and detection method, do not need after sample prepares to dilute more than 200 times again, testing process does not need to do separately blank pipe, standard pipe yet again, has simplified operation steps, has reduced personal error; Selected substrate specificity is good, and strong with the affinity of enzyme, reaction velocity is fast, and testing result is more reliable and more stable;
3, the present invention has realized from manual transformation to full-automatic detection method; Filled up the vacancy on the market; Adopt full automatic biochemical apparatus to carry out the detection of robotization, scale, discharged great amount of manpower, practiced thrift a large amount of consumptive materials; To the accurate control and the monitoring of reaction time, course of reaction, significantly improved the accuracy and the reliability of testing result;
4, employing the present invention can be when detecting same this γ of increment-L-glutamyl transpeptidase activity; Kits such as the seminal plasma fructose that can also match with full automatic biochemical apparatus, alpha-glucosidase, zinc are united use; And detect these projects simultaneously, detect finish after, can print all results of this institute of same increment survey project with the form of report; Can satisfy the needs of mechanisms such as the relevant section office of hospital's male sterility, human sperm bank, family planning research institute to semen quality, sperm screening; And for the auxiliary diagnosis of male sterility etc., improved efficient and income that the seminal fluid routine biochemistry is analyzed greatly, be very suitable for conventional sense and popularization.
Embodiment:
According to following embodiment, can better understand the present invention.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1
A kind of refining γ in the present embodiment-L-glutamyl transpeptidase active level detection kit is a double reagent, comprises R1 reagent and R2 reagent, respectively by following composition and consumption preparation:
1) R1 reagent
PH8.2Tris damping fluid 180mmol/L;
Glycylglycine 100mmol/L;
Sodium azide 0.05%;
2) R2 reagent
PH8.2Tris damping fluid 180mmol/L;
L-γ-Gu Anxianji-p-nitroaniline 1.25mmol/L;
Thimerosal 0.02%;
Mentioned reagent is divided the bottle of packing into after all dissolving fully, processes liquid double reagent, and is subsequent use in 2-8 ℃ of preservation.
Embodiment 2
A kind of refining γ in the present embodiment-L-glutamyl transpeptidase active level detection kit is a double reagent, comprises R1 reagent and R2 reagent, respectively by following composition and consumption preparation:
1) R1 reagent
PH8.3Tris damping fluid 150mmol/L;
Glycylglycine 90mmol/L;
ProClin300 0.02％；
2) R2 reagent
PH8.3Tris damping fluid 150mmol/L;
L-γ-Gu Anxianji-3-carboxyl-4-nitroaniline 0.6mmol/L;
Thimerosal 0.01%;
Mentioned reagent is divided the bottle of packing into after all dissolving fully, processes liquid double reagent, and is subsequent use in 2-8 ℃ of preservation.
Embodiment 3
A kind of refining γ in the present embodiment-L-glutamyl transpeptidase active level detection kit is a double reagent, comprises R1 reagent and R2 reagent, respectively by following composition and consumption preparation:
1) R1 reagent
PH8.1Tris damping fluid 100mmol/L;
Glycylglycine 100mmol/L;
ProClin150 0.1％；
2) R2 reagent
PH8.1Tris damping fluid 100mmol/L;
L-γ-Gu Anxianji-p-nitroaniline 3.0mmol/L;
ProClin300 0.05％；
Mentioned reagent is divided the bottle of packing into after all dissolving fully, processes liquid double reagent, and is subsequent use in 2-8 ℃ of preservation.
Embodiment 4
Utilize the kit among the embodiment 1 to detect the active method of γ in the refining-L-glutamyl transpeptidase, the concrete operations step is following:
1) specimen preparation: fresh semen liquefaction back is through the centrifugal 10min of 3000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is following in the function software of Olympus AU400 full automatic biochemical apparatus:
Test item: γ-GT;
Sample size: 3 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1, the R1 amount of reagent: 240 μ l; R2 amount of reagent: 60 μ l;
Detection method: rate method;
Calibration pattern: MB (factorization method);
Predominant wavelength: 410nm;
The Direction of Reaction: rise;
Main reading point 1:13;
Main reading point 2:17;
3) scale operation:
The calibration of employing factorization method, directly input hypothesis factor F value 10202 gets final product;
The computing formula of F value is following:
The theoretical factor
Wherein, 10
3Transformation ratio for mmol to μ mol; Vt is 303 μ l for the reactant liquor cumulative volume; ε is that the theoretical molar extinction coefficient of p-nitroaniline is 9.9 * 10
3L is cuvette optical path (1cm); Vs is the i.e. 3 μ l of sample volume.
4) pattern detection:
With 1) in the refining sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.Behind the EOT, full automatic biochemical apparatus provides the activity of γ in the sample-L-glutamyl transpeptidase automatically according to the theoretical factor.
The U of unit of enzyme activity definition: under above-mentioned reaction conditions, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol p-nitroaniline;
Active (the U/L)=Δ A/min * F of γ in the refining-L-glutamyl transpeptidase, wherein, Δ A/min is the rate of change of per minute absorbance, on full automatic biochemical apparatus, measures automatically to provide, the theoretical factor is known.
For example, the Δ A/min that full automatic biochemical apparatus records in certain part of refining is 0.1560, then γ in this part refining-L-glutamyl transpeptidase activity=0.1560 * 10202=1591.51U/L
Embodiment 5
Utilize the kit among the embodiment 2 to detect the active method of γ in the refining-L-glutamyl transpeptidase, the concrete operations step is following:
1) specimen preparation: fresh semen liquefaction back is through the centrifugal 20min of 4000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is following in the function software of Laura Faith-1000 full automatic biochemical apparatus:
Test item: γ-GT;
Sample size: 4 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1, the R1 amount of reagent: 320 μ l; R2 amount of reagent: 80 μ l;
Detection method: rate method;
Calibration pattern: factorization method;
Predominant wavelength: 405nm;
The Direction of Reaction: rise;
Main reading point 1:17;
Main reading point 2:22;
3) scale operation:
The calibration of employing factorization method, directly input hypothesis factor F value 10202 gets final product;
The computing formula of F value is following:
The theoretical factor
Wherein, 10
3Transformation ratio for mmol to μ mol; Vt is 404 μ l for the reactant liquor cumulative volume; ε is that the theoretical molar extinction coefficient of p-nitroaniline is 9.9 * 10
3L is cuvette optical path (1cm); Vs is the i.e. 4 μ l of sample volume.
4) pattern detection:
With 1) in the refining sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.Behind the EOT, full automatic biochemical apparatus calculates the activity of γ in the sample-L-glutamyl transpeptidase automatically according to the theoretical factor.
The U of unit of enzyme activity definition: under above-mentioned reaction conditions, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol p-nitroaniline;
Active (the U/L)=Δ A/min * F of γ in the refining-L-glutamyl transpeptidase, wherein, Δ A/min is that rate of change automatic mensuration on full automatic biochemical apparatus of per minute absorbance provides, the theoretical factor is known.
For example, the Δ A/min that full automatic biochemical apparatus records in certain part of refining is 0.1826, then γ in this part refining-L-glutamyl transpeptidase activity=0.1826 * 10202=1862.89U/L
Embodiment 6
Utilize the kit among the embodiment 3 to detect the active method of γ in the refining-L-glutamyl transpeptidase, the concrete operations step is following:
1) specimen preparation: fresh semen liquefaction back is through the centrifugal 10min of 5000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is following in the function software of Olympus AU640 full automatic biochemical apparatus:
Test item: γ-GT;
Sample size: 2 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1, the R1 amount of reagent: 160 μ l; R2 amount of reagent: 40 μ l;
Detection method: rate method;
Calibration pattern: MB (factorization method);
Predominant wavelength: 410nm;
The Direction of Reaction: rise;
Main reading point 1:12;
Main reading point 2:17;
3) scale operation:
The calibration of employing factorization method, directly input hypothesis factor F value 10202 gets final product;
The computing formula of F value is following:
The theoretical factor
Wherein, 10
3Transformation ratio for mmol to μ mol; Vt is 202 μ l for the reactant liquor cumulative volume; ε is that the theoretical molar extinction coefficient of p-nitroaniline is 9.9 * 10
3L is cuvette optical path (1cm); Vs is the i.e. 2 μ l of sample volume.
4) pattern detection:
With 1) in the refining sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.Behind the EOT, full automatic biochemical apparatus provides the activity of γ in the sample-L-glutamyl transpeptidase automatically according to the theoretical factor;
The U of unit of enzyme activity definition: under above-mentioned reaction conditions, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol p-nitroaniline;
Active (the U/L)=Δ A/min * F of γ in the refining-L-glutamyl transpeptidase, wherein, Δ A/min is that rate of change automatic mensuration on full automatic biochemical apparatus of per minute absorbance provides, the theoretical factor is known;
For example, after certain part of refining diluted 2 times with physiological saline, the Δ A/min that full automatic biochemical apparatus records was 0.1060, then γ in this part refining-L-glutamyl transpeptidase activity=0.1060 * 10202 * 2=2162.82U/L.
Embodiment 7
In on the basis of embodiment 1 and embodiment 4, criticize and the detection (being the reappearance of the testing result of kit of the present invention) of betweenrun precision
Select each 1 part in low, the high mixing refining sample that is worth of γ-L-glutamyl transpeptidase activity; Prepare 5 batches detection kit; Each batch kit is surveyed same concentration refining sample 8 times, has 40 data, and the result of each sample replication 8 times calculates withinrun precision from 5 batches; Calculate betweenrun precision from all 40 results, the result is that CV% representes with the percentage of the coefficient of variation all.Wherein, CV%=standard deviation/mean value * 100%, this coefficient is more little, shows that same this duplicate detection of increment result's dispersion degree is more little, and promptly the reappearance of testing result is good more.Table 1 is to adopt the precision result of said kit and detection method and manual or semi-automatic detection to compare, and shows the activity that adopts the present invention to detect refining γ-L-glutamyl transpeptidase, as a result favorable reproducibility.
Manual, the semi-automatic and full automatic precision result of table 1 relatively
| Manual inspection | Semi-automatic detection | Full-automatic detection of the present invention | |
| Withinrun precision | CV％≤13％ | CV％≤10％ | CV％≤5％ |
| Betweenrun precision | CV％≤15％ | CV％≤12％ | CV％≤5％ |
Embodiment 8
Detection (being the field of activity that kit of the present invention can accurately be measured) in the basic enterprising line linearity scope of embodiment 1 and embodiment 4.
Choose each 1 part in the mixing refining sample of low, the high value of γ-L-glutamyl transpeptidase activity, the low value sample is No. 1 sample, and high value sample is No. 5, and the two is mixed into No. 2 at 3: 1; Five equilibrium is mixed into No. 3, is mixed into No. 4 at 1: 3, is mixed into No. 5 at 1: 4; Be prepared into the sample of 5 isoconcentration gradients, each concentration samples duplicate detection equal absorbance of making even for 4 times is according to experimental result; With the catalogue number(Cat.No.) is the X axle, is the Y axle with the mean light absorbency, carries out straight-line regression; Find coefficient R >=0.990, multiply by scaling factor 10202 according to absorbance, drawing the active range of linearity of γ-L-glutamyl transpeptidase is 334-2832U/L; Be illustrated in this scope, the activity and the correlativity between the absorbance of γ-L-glutamyl transpeptidase are good, are the field of activities that kit of the present invention can accurately be measured.
Embodiment 9
On the basis of embodiment 1 and embodiment 4, verify accuracy through recovery test.
Choose each 1 part of active high value of γ-L-glutamyl transpeptidase and low value mixing sample; With the low value mixing sample high value mixing sample is carried out the different proportion dilution; The low value sample accounts for 75%, 50%, 25% respectively; Every this replication of increment 4 times calculates average recovery rate respectively, and its recovery is expressed as the recovery 1, the recovery 2 and the recovery 3 respectively.Wherein, the recovery=measured result/notional result * 100%, the recovery is more near 100%, and the accuracy of expression testing result is high more.Table 2 is to adopt the recovery result of said kit and detection method and manual or semi-automatic detection to compare, and shows the activity that adopts the present invention to detect refining γ-L-glutamyl transpeptidase, and result precision is good.
Manual, the semi-automatic and full automatic recovery result of table 2 relatively
| Manual inspection | Semi-automatic detection | Full-automatic detection of the present invention | |
| The recovery 1 | 82％ | 84％ | 116.1％ |
| The recovery 2 | 75％ | 79％ | 112.8％ |
| The recovery 3 | 78％ | 80％ | 108.4％ |
| Average recovery rate | 79％ | 81％ | 112.4％ |
Embodiment 10
On the basis of embodiment 1 and embodiment 4, carry out the detection of reagent blank absorbance rate of change.
Change the sample in γ-L-glutamyl transpeptidase reaction system into distilled water and detect, other testing conditions is constant, and the result that surveys is reagent blank absorbance rate of change (Δ A/min), is one of quality index of this kit.Repeated detection result shows, Δ A/min≤0.0015, and when exceeding this scope, the prompting possible operation is improper or reagent is out of date.
Embodiment 11
On the basis of embodiment 1 and embodiment 4, carry out confirming of normal reference value.
Collect the equal normal healthy examinee's refining of 195 routine seminal fluid conventional analysis parameters sample; 19～45 years old age; On full automatic biochemical apparatus, detect; Testing result if confirm as normal distribution, is then calculated term of reference according to x (mean value) ± 1.96S (standard deviation) through test of normality, calculates reference interval otherwise press method of percentiles.Find that with the analysis of SPSS17.0 statistical analysis software its distribution of results is normal distribution, normal reference range is 914.49-2206.13U/L.
Embodiment 2 according to the invention and embodiment 5, embodiment 3 and embodiment 6 and interior kit and the detection method of said other concentration range; The detection method of its precision, accuracy, the range of linearity and reagent blank absorbance rate of change and step and embodiment 1 and embodiment's 4 is similar, repeats no more at this.
Claims (3)
1. refining γ-L-glutamyl transpeptidase active level detection kit is characterized in that, comprises R1 reagent and R2 reagent; Wherein, R1 reagent comprises the component of following content: 100-200mmol/L, pH8.0-8.3 TRIS buffer, 90-110mmol/L glycylglycine and 0.01-0.1% antiseptic; R2 reagent comprises the component of following content: 100-200mmol/L, pH8.0-8.3 TRIS buffer, 0.5-3.0mmol/L substrate and 0.01-0.1% antiseptic;
Wherein, said substrate is L-γ-Gu Anxianji-p-nitroaniline or L-γ-Gu Anxianji-3-carboxyl-4-nitroaniline; Said antiseptic is any in Sodium azide, thimerosal, gentamicin or the ProClin series antiseptic.
2. the application of the described refining γ of claim 1-L-glutamyl transpeptidase active level detection kit in measuring refining γ-L-glutamyl transpeptidase activity.
3. application according to claim 2 is characterized in that comprising the steps:
1) specimen preparation: fresh semen liquefaction back is through the centrifugal 10～20min of 3000～5000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is following on full automatic biochemical apparatus:
Test item: γ-GT;
Sample reagent ratio: 1: 100;
Detection method: rate method;
Calibrating method: factorization method;
Predominant wavelength: 405-410nm;
The Direction of Reaction: rise;
Reaction time: 36-54 second;
Detection time: 90-120 second;
3) scale operation:
The calibration of employing factorization method, directly input hypothesis factor F value gets final product;
4) pattern detection:
The refining sample for preparing and R1 reagent, R2 reagent are placed on the relevant position of full automatic biochemical apparatus, and full automatic biochemical apparatus goes out the activity of γ in the sample-L-glutamyl transpeptidase according to theoretical factor calculation;
The U of unit of enzyme activity definition: under above-mentioned reaction conditions, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol p-nitroaniline;
Active (the U/L)=Δ A/min * F of γ in the refining-L-glutamyl transpeptidase;
Wherein, Δ A/min is the rate of change of per minute absorbance, and the F value is known.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215631A (en) * | 2014-08-14 | 2014-12-17 | 苏州康铭诚业医用科技有限公司 | Buffer for determining human serum glutamyltransferase |
| CN112608977A (en) * | 2020-12-10 | 2021-04-06 | 武汉伊莱瑞特生物科技股份有限公司 | Gamma-glutamyl transpeptidase detection kit and detection method thereof |
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| CN112608977A (en) * | 2020-12-10 | 2021-04-06 | 武汉伊莱瑞特生物科技股份有限公司 | Gamma-glutamyl transpeptidase detection kit and detection method thereof |
| CN112608977B (en) * | 2020-12-10 | 2022-05-10 | 武汉伊莱瑞特生物科技股份有限公司 | Gamma-glutamyl transpeptidase detection kit and detection method thereof |
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