CN107828855B - Seminal plasma neutral alpha-glucosidase detection kit and application thereof - Google Patents
Seminal plasma neutral alpha-glucosidase detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses a seminal plasma neutral alpha-glucosidase detection kit and application thereof, wherein the kit comprises an R1 reagent, an R2 reagent and a non-constant value quality control product; the R1 reagent is a phosphate buffer solution containing SDS; the R2 reagent is phosphate buffer solution containing urea, sodium salicylate and PNPG; the non-constant value quality control product is seminal plasma freeze-dried powder. The kit disclosed by the invention is matched with a full-automatic biochemical analyzer for detecting neutral alpha-glucosidase in seminal plasma, the kit is simple in components, no stop solution exists, and the production and the use are relatively simple; when the method is used for detecting the seminal plasma neutral alpha-glucosidase, the reaction time is about 5min, the required reagent amount and the sample amount are small, the linear range is wide, the accuracy and the precision of the result are high, and simultaneously, the method can be used for joint detection with other items of seminal plasma biochemistry. The kit is suitable for detecting large-batch samples clinically; the entire course of the reaction can be monitored by changes in the reaction curve.
Description
Technical Field
The invention relates to a second type of in-vitro diagnostic reagent, in particular to a seminal plasma neutral alpha-glucosidase detection kit and application thereof.
Background
The epididymis is an important organ for sperm maturation, and the neutral alpha-glucosidase in the seminal plasma is derived from the epididymis and is a specific enzyme and a marker enzyme of the epididymis. When the epididymis itself is diseased, such as epididymis, epididymis nodule, etc., the secretion of neutral alpha-glucosidase is reduced, and the activity of the enzyme in seminal plasma is reduced, so that the determination of the activity of the neutral alpha-glucosidase in the seminal plasma has important significance for early diagnosis, treatment and follow-up of epididymis and male infertility. In addition, the obstruction of epididymis and vas deferens can cause the activity of seminal plasma neutral alpha-glucosidase to be obviously reduced, so that the detection of the activity of the seminal plasma neutral alpha-glucosidase provides a new method for accurately and non-invasively diagnosing obstructive azoospermia.
The currently clinically adopted method for detecting seminal plasma neutral alpha-glucosidase is an improved Cooper method, the shortest reaction time reported in the literature is about 25min, and all the operations are manual methods, so that the method cannot meet the detection of clinical large-batch samples and cannot monitor the detection process in real time.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a seminal plasma neutral alpha-glucosidase detection kit which has simple components and no stop solution, and has the advantages of high detection speed, short time, wide linear range, and high accuracy and precision of results when the seminal plasma neutral alpha-glucosidase is detected.
The invention also provides application of the seminal plasma neutral alpha-glucosidase detection kit, and the kit can be applied to a full-automatic biochemical analyzer.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a seminal plasma neutral alpha-glucosidase assay kit, comprising an R1 reagent, an R2 reagent, and a non-constant quality control substance; the R1 reagent is a phosphate buffer comprising Sodium Dodecyl Sulfate (SDS); the R2 reagent is a phosphate buffer solution containing urea, sodium salicylate and 4-nitrobenzene-alpha-D-glucopyranoside (PNPG); the non-constant value quality control product is seminal plasma freeze-dried powder.
Wherein the concentration of SDS in the R1 reagent is 0.2-0.5% g/mL.
The concentration of PNPG in the R2 reagent is 0.25-0.6% g/mL, the concentration of urea is 0.1-1% g/mL, and the concentration of sodium salicylate is 0.8-3.0% g/mL.
Preferably, the phosphate buffer solutions of the R1 reagent and the R2 reagent are obtained by 0.1-0.4mol/L disodium hydrogen phosphate dodecahydrate and 0.1-0.4mol/L sodium dihydrogen phosphate dihydrate, and the intermodulation pH value is 6.0-7.0.
The reagent R1 and the reagent R2 contain one or more of preservatives penicillin, streptomycin, penicillin streptomycin, gentamicin sulfate and sodium azide, and the content is 0.01-0.05% g/ml or 0.01-0.05% ml/ml.
Further, the non-constant value quality control product is freeze-dried powder formed by freeze-drying seminal plasma, wherein the seminal plasma contains one or more of preservatives penicillin, streptomycin, gentamicin sulfate and sodium azide. The seminal plasma has been tested negative for HBsAg, HCV antibodies, HIV1+2 antibodies, and syphilis.
Wherein the concentration of the preservative in the mixed seminal plasma is 0.01-0.05% g/ml or 0.01-0.05% ml/ml.
Further, the seminal plasma neutral alpha-glucosidase detection kit comprises an R1 reagent, an R2 reagent, a non-fixed value quality control product, an instruction book and a lining.
The seminal plasma neutral alpha-glucosidase detection kit provided by the invention is applied to a full-automatic biochemical analyzer.
Wherein the detection step of the application is
(1) Sample preparation: semen forbidden for 2-7 days is collected by masturbation method and put in a sampling cup, and then liquefied in a 37 ℃ water bath box, centrifuged at 3000-12000 rpm for 5-10 min to separate seminal plasma, and immediately used for detection; preparation of a kit: taking out the kit from 2-8 ℃, balancing for 30-35 min at room temperature, then placing R1 at 35-37 ℃ for 5-10 min to clarify the R1 solution, and then performing on-machine detection;
(2) setting primary and secondary wavelengths of a full-automatic biochemical analyzer to be 400-420 nm and 600-700 nm respectively, wherein the volume ratio of R1/sample/R2 sample adding amount is 15: 1: 15, and the range is (75-151 mu l): 5-10.1 mu l): 75-151 mu l.
(3) The detection method comprises the following steps: a speed method, adding R1 and a sample, uniformly mixing, and incubating at 37 ℃ for 4-6 min; adding R2, mixing uniformly, and incubating for 2-5 min at 37 ℃; continuously measuring the absorbance within 2-5 min, and calculating the absorbance change rate delta A/min by an instrument, wherein the specific process is shown in table 1;
TABLE 1 seminal plasma neutral alpha-glucosidase assay protocol
(4) Calculating neutral alpha-glucosidase activity in the sample;
(5) taking purified water to redissolve the non-fixed value quality control product, generally taking 2-5 mL of purified water to redissolve the non-fixed value quality control product in a bottle of 4mL before freeze-drying, uniformly mixing, and subpackaging into 300 mu L/piece, and storing at-25 to-20 ℃; taking out one redissolution every day, detecting the redissolved non-constant value quality control product (the setting of detection method and apparatus is the same as that of the above sample) with kit, repeating the detection for 5-20 days, repeating the detection for 5 days every day-10 times, the test results are statistically analyzed and averaged
And standard deviation(s) of
Drawing a quality control graph for the quality control line; and then, detecting the quality control product together with the sample (the detection method is the same as the above), and judging whether the detection result of the sample is controlled according to whether the detection result of the quality control product is in the quality control line.
Wherein the neutral α -glucosidase activity in the step (4) is calculated as (U/L) ═ Δ a/min × F, where
F: theoretical factors on a corresponding full-automatic biochemical analyzer;
VT: the sum of the volumes of the R1 reagent, R2 reagent and sample aspirated by the reagent needle and sample needle, in units: mu.l;
: molar absorptivity, unit: l/(mol. cm);
l: optical path, unit: cm;
Vs: sample volume, unit: mu.l;
the delta A/min is obtained by automatically measuring a sample by a full-automatic biochemical analyzer.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the invention provides a kit for detecting neutral alpha-glucosidase in seminal plasma, which is matched with a full-automatic biochemical analyzer for use, the kit has simple components, no stop solution and relatively simple production and use; when the neutral alpha-glucosidase in seminal plasma is detected, the reaction time is about 5min, the detection time of a single sample needs 10-15min, the required reagent amount and the sample amount are small, the linear range is wide, the accuracy and the precision of the result are high, and simultaneously, the method can be used for joint detection with other items of seminal plasma biochemistry.
(2) The kit is matched with a full-automatic biochemical analyzer for use, and the whole reaction process is monitored through the change of a reaction curve; the activity of the enzyme is calculated by monitoring the change rate of the absorbance by using a rate method, and the detection result of a sample is directly given by an instrument without making a standard curve; the stability of the detection system and the accuracy of the detection result can be reflected by the detection of the quality control product, and simultaneously, the quality control product can be jointly detected with other biochemical items of seminal plasma.
(3) The reagent kit of the invention contains urea and sodium salicylate, which can effectively improve the accuracy and repeatability of the whole reagent kit.
Drawings
FIG. 1 is a graph showing the linear relationship of the linear parameter confirmation of the kit 1 of the present invention;
FIG. 2 is a graph showing the relationship between the accuracy of the kit of example 1 of the present invention and the accuracy of the comparative example;
FIG. 3 is a graph showing the relationship between the accuracy of the kit of example 2 of the present invention and the accuracy of the comparative example;
FIG. 4 is a graph showing the relationship between the accuracy of the kit of example 3 of the present invention and the accuracy of the comparative example.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
The seminal plasma neutral alpha-glucosidase detection kit comprises the following components:
r1 reagent: phosphate buffer containing SDS and a preservative; the phosphate buffer solution was adjusted to pH 6.5 by 0.25mol/L disodium phosphate dihydrate to 0.25mol/L disodium phosphate dihydrate, and 3.5g SDS and 0.5ml penicillin streptomycin were contained per liter of the phosphate buffer solution (Sigma; 10000 units penicillin and 10mg streptomycin per ml).
R2 reagent: a phosphate solution containing PNPG and a preservative; the phosphate buffer solution was adjusted to pH 6.5 with 0.25mol/L disodium phosphate dihydrate to 0.25mol/L disodium phosphate dihydrate, and contained 4.25g PNPG, 0.5ml streptomycin, 5g urea, and 20g sodium salicylate per liter of the phosphate buffer solution.
The quality control product is prepared from seminal plasma which is detected to be negative by HBsAg, HCV antibody, HIV1+2 antibody and syphilis, and 0.05% (mL/mL) of preservative streptomycin by mixing, subpackaging with 4 mL/bottle, and lyophilizing to obtain lyophilized powder.
Example 2
The seminal plasma neutral alpha-glucosidase detection kit comprises the following components:
r1 reagent: phosphate buffer containing SDS and a preservative; the phosphate buffer solution was adjusted to pH 7.0 with 0.4mol/L disodium phosphate dihydrate by 0.4mol/L sodium dihydrogen phosphate dihydrate, and contained 5g SDS and 0.1g gentamicin sulfate per liter of the phosphate buffer solution.
R2 reagent: a phosphate solution containing PNPG and a preservative; the phosphate buffer solution was adjusted to pH 7.0 with 0.4mol/L disodium phosphate dihydrate to 0.4mol/L disodium phosphate dodecahydrate, and each liter of the phosphate buffer solution contained 6g of PNPG, 0.1g of gentamicin sulfate, 10g of urea, and 30g of sodium salicylate.
The quality control product is prepared from seminal plasma which is detected to be negative by HBsAg, HCV antibody, HIV1+2 antibody and syphilis, 0.01% (g/mL) preservative gentamicin sulfate, mixing, subpackaging with 4 mL/bottle, and lyophilizing to obtain lyophilized powder.
Example 3
The seminal plasma neutral alpha-glucosidase detection kit comprises the following components:
r1 reagent: phosphate buffer containing SDS and a preservative; the phosphate buffer solution was adjusted to pH 6.0 by 0.1mol/L sodium dihydrogen phosphate dihydrate to 0.1mol/L disodium hydrogen phosphate dodecahydrate, and contained 2g SDS and 0.1g sodium azide per liter of the phosphate buffer solution.
R2 reagent: a phosphate solution containing PNPG and a preservative; the phosphate buffer solution was adjusted to pH 6.0 with 0.1mol/L disodium hydrogenphosphate dihydrate to 0.1mol/L disodium hydrogenphosphate dodecahydrate, and contained 2.5g of PNPG, 0.1g of sodium azide, 1g of urea, and 8g of sodium salicylate per liter of the phosphate buffer solution.
The quality control product is prepared from seminal plasma which is detected to be negative by HBsAg, HCV antibody, HIV1+2 antibody and syphilis, adding 0.01% (g/mL) preservative sodium azide, mixing, subpackaging with 4 mL/bottle, and lyophilizing to obtain lyophilized powder.
Example 4
The components of the seminal plasma neutral alpha-glucosidase assay kit of examples 1-3 were prepared using the following procedure, wherein the mass or volume of each material of the different examples was calculated separately from the final concentration:
(1) preparation process of phosphate buffer solution
Weighing sodium dihydrogen phosphate dihydrate to dissolve in purified water to obtain 0.1-0.4mol/L sodium dihydrogen phosphate solution, weighing disodium hydrogen phosphate dodecahydrate to dissolve in purified water to obtain 0.1-0.4mol/L disodium hydrogen phosphate solution, adjusting the disodium hydrogen phosphate solution with the same concentration of the sodium dihydrogen phosphate solution to ensure that the pH value is 6.0-7.0, and storing at 2-8 ℃ for later use.
(2) Preparation process of R1 reagent
Weighing a certain amount of SDS into a beaker filled with phosphate buffer solution, adding preservative, stirring until the mixture is uniformly mixed, fixing the volume by using the phosphate buffer solution, uniformly mixing, subpackaging, labeling, checking and storing at the temperature of 2-8 ℃.
(3) Preparation process of R2 reagent
Weighing a certain amount of urea, sodium salicylate and PNPG in a beaker filled with phosphate buffer solution, adding a preservative, stirring to mix uniformly, fixing the volume by using the phosphate buffer solution, mixing uniformly, subpackaging, labeling, checking and storing at 2-8 ℃.
(4) Preparation process of quality control product
Collecting completely liquefied male fresh semen with negative HCV antibody, HIV1+2 antibody, HBsAg and syphilis detection, liquefying, centrifuging, collecting supernatant, mixing, adding antiseptic, stirring, dissolving, mixing, packaging into 4 mL/bottle, and lyophilizing.
The freeze-drying step of the vacuum freeze-drying machine: the lyophilization procedure was set up as in table 2. Starting the vacuum freeze dryer, pre-freezing for 6h at-30 ℃, and starting the freeze-drying program. And (4) after the freeze-drying is finished, deflating and taking out, and sealing the cover to obtain the dry powder-shaped quality control product.
TABLE 2 non-constant quality control Freeze drying procedure
| Temperature (. degree.C.) | Time (min) |
| -20 | 900 |
| -15 | 360 |
| -10 | 360 |
| -5 | 180 |
| 0 | 180 |
| 10 | 180 |
| 15 | 180 |
| 20 | 240 |
Example 5
The method for detecting the activity of the neutral alpha-glucosidase in the seminal plasma by using the seminal plasma neutral alpha-glucosidase detection kit comprises the following specific steps:
first, sample preparation and request
Semen of abstinence for 2 days is collected by masturbation method, liquefied in a water bath box at 37 ℃, centrifuged at 3000r/min for 10min, and the upper layer is separated to obtain seminal plasma which is immediately used for detection. The amount of the seminal plasma is 2.5mL, the liquefaction is good, and the seminal plasma sample which cannot be detected immediately can be stored for 1 month at the temperature of minus 20 ℃, so that repeated freeze thawing is avoided.
Second, reagent preparation
Any one of the kits of examples 1-3 was selected.
And taking the R1 reagent, the R2 reagent and the quality control product out of a refrigerator at the temperature of 2-8 ℃, balancing for 30min at room temperature, and then placing the R1 reagent at the temperature of 37 ℃ for 5min to clarify the R1 solution.
Third, detection method
(1) Basic parameters:
the full-automatic biochemical analyzer of Merrill BS330E model is adopted, and the main/auxiliary wavelength: 420nm/600 nm; temperature: 37 ℃; the method comprises the following steps: a rate method; the reaction direction is as follows: and (4) rising.
(2) The method comprises the following operation steps:
(3) calculating the formula:
neutral alpha-glucosidase activity (U/mL) ═ Fx Δ A/min in the samples
In the formula:
VT：312.1μl；
4-nitrophenol is prepared into solutions with the concentrations of 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 16 mu mol/L, 32 mu mol/L, 64 mu mol/L and 100 mu mol/L by using a phosphate buffer solution, the absorbance of the solutions is detected at 420nm by using a spectrophotometer, and the molar absorption coefficient of the 4-nitrophenol is calculated to be 7.3 × 10 according to the Lambert-beer law3L/(mol·cm)；
l：1cm；
Vs：10.1μl；
The delta A/min is obtained by automatically measuring a sample by a Mirey BS330E full-automatic biochemical analyzer.
Definition of enzyme units: the amount of enzyme catalyzing the formation of 1. mu. mol of 4-nitrophenol per minute at 37 ℃ is defined as one activity unit;
f: under these conditions, F is 4247.
(4) And (3) assignment and detection of quality control products: taking 2mL of non-fixed value quality control product 4 mL/bottle before redissolving and freeze-drying, mixing uniformly, subpackaging into 300 mu L/bottle, and storing at-20 ℃; taking one subpackaged non-fixed value quality control product out of minus 20 ℃ every day, redissolving, detecting the redissolved non-fixed value quality control product by using the kit of the embodiment, setting the detection method and the instrument to be the same as the detection of the sample of the embodiment, repeatedly detecting 20 times every day for 5 days, statistically analyzing 100 data for 5 days, calculating the average value
And standard deviation(s) of
Drawing a quality control graph for the quality control line; and then the quality control product is detected together with the sample, the detection method is the same, and whether the detection result of the sample is controlled is judged according to whether the detection result of the quality control product is in the quality control line.
Fourthly, main performance indexes of the kit
(1) The change rate of blank absorbance (delta A/min) of the reagent is less than or equal to 0.06;
(2) linear range: [5-65] U/L, r is more than or equal to 0.9900;
(3) repeatability: CV is less than or equal to 7 percent;
(4) inter-batch difference: r is less than or equal to 10 percent.
Fifth, report the results
And after the sample detection is finished, outputting the sample in a summary report form.
Sixth, reference interval
The normal reference value range is determined as the 5 th percentile according to the fact that the detection result of seminal plasma neutral alpha-glucosidase of at least 200 normal male breeding cases is biased distribution and the clinical significance of seminal plasma neutral alpha-glucosidase, and the normal reference value range of seminal plasma neutral alpha-glucosidase is more than or equal to 10.12U/L.
Seven clinically applicable symptoms
The kit is used for detecting the activity of the neutral alpha-glucosidase in seminal plasma in vitro, and has important significance for early diagnosis, treatment and follow-up of epididymitis and male infertility. The neutral alpha-glucosidase is an epididymis marker used clinically and can be used as a functional index of epididymis.
Example 6
The method for detecting the activity of the seminal plasma neutral alpha-glucosidase by using the seminal plasma neutral alpha-glucosidase detection kit comprises the following specific steps:
first, sample preparation and request
Semen forbidden for 7 days is collected by masturbation method, liquefied in a water bath box at 37 deg.C, centrifuged at 10000r/min for 5min, and the upper layer is separated to obtain seminal plasma which is immediately used for detection. The amount of the concentrate was 0.5mL, and the liquefaction was good. Seminal plasma samples which could not be detected immediately could be stored for 1 month at-20 deg.C, avoiding repeated freeze thawing.
Second, reagent preparation
Any one of the kits of examples 1-3 was selected.
Taking the R1 reagent, the R2 reagent and the quality control product out of a refrigerator at the temperature of 2-8 ℃, balancing for 35min at room temperature, and then placing R1 at the temperature of 35 ℃ for 10min to clarify the R1 solution for later use.
Third, detection method
(1) Basic parameters:
adopting an Olympus full-automatic biochemical analyzer, wherein the main/auxiliary wavelength is as follows: 400nm/700 nm; temperature: 37 ℃; the method comprises the following steps: a rate method; the reaction direction is as follows: and (4) rising.
(2) Procedure for the preparation of the
(3) Calculating the formula:
neutral alpha-glucosidase activity (U/L) ═ F x delta A/min in the sample
Wherein, as described in example 5, F-4247;
the delta A/min is obtained by automatically measuring a sample by an olympus full-automatic biochemical analyzer.
Definition of enzyme units: the amount of enzyme catalyzing the formation of 1. mu. mol of 4-nitrophenol per minute at 37 ℃ is defined as one activity unit.
(4) Quality controlAnd (3) assignment and detection of the product: taking 5mL of purified water to redissolve and freeze-dry 4 mL/bottle of non-fixed value quality control product, mixing uniformly, subpackaging into 300 mu L/bottle, and storing at-25 ℃; taking out one subpackaged lyophilized seminal plasma quality control material from-25 ℃ every day, redissolving, detecting the redissolved non-fixed value quality control material by using the kit of the embodiment, setting the detection method and the instrument as the detection of the sample of the embodiment, repeating the detection for 20 days, repeating the detection for 5 times every day, counting and analyzing 100 data in total for 20 days, calculating the average value
And standard deviation(s) of
Drawing a quality control graph for the quality control line; and detecting the quality control product with the sample in the same way as above, and judging whether the detection result of the sample is controlled according to whether the detection result of the quality control product is in the quality control line.
Test example 1
The linear range of the kit was confirmed using the detection method of example 5 and the kit prepared in example 1. Linear range confirmation: diluting the high-value neutral alpha-glucosidase sample of the seminal plasma into 9 neutral alpha-glucosidase seminal plasma samples with different theoretical activities of 0, 5, 10, 20, 30, 40, 50, 60 and 65U/L by using physiological saline, then detecting on a machine, testing each theoretical activity sample for 3 times, and respectively calculating the mean value of each theoretical activity detection result. And (3) taking the theoretical activity of the diluted sample as an independent variable, taking the mean value of each theoretical activity detection result as a dependent variable to calculate a linear regression equation, and calculating a correlation coefficient of linear regression. The theoretical activity in the above method was substituted into the linear regression equation to calculate the estimated value of the theoretical activity and the relative deviation of the measured mean from the estimated value, and the results are shown in Table 3.
TABLE 3 kit Linear Range parameters confirmation of test results
As can be seen from Table 3, the test examples show that the detection activity of the diluted sample of the kit in [0-65] U/L and the theoretical activity show good linear relationship, but when the detection theoretical activity is 0U/L, the relative deviation is large, so the linear range of the kit is defined as [5-65] U/L. In view of the reference interval of the kit: the sample is more than or equal to 10.12U/L, which indicates that the kit is reliable in detecting the results of normal and abnormal samples. The results were similar to those described above using the kits of examples 2 and 3 or the detection method of example 6 together.
Test example 2
The detection method of example 5 was used, and the kits prepared in examples 1 to 3 were used to perform repetitive detection of the kit. The repeatability detection method comprises the following steps: collecting sample, testing, measuring the same seminal plasma with the kit, repeating the test for 10 times, and averaging
The Coefficient of Variation (CV) in repeatability was calculated according to the following formula,
in the formula (I), the compound is shown in the specification,
the average value of the results obtained by repeating the detection 10 times; s: the standard deviation of the results obtained by repeating the detection 10 times; CV: the coefficient of variation. The results are shown in Table 4.
TABLE 4 repeatability test results of the kit
Test example 3
The accuracy of the kit was determined by using the detection method of example 5 and the kits prepared in examples 1 to 3, respectively, and the accuracy of the kit was determined by using 40 seminal plasma samples having different activities within the detection activity range and the kits produced by the marketed company (seminal plasma neutral α -glucosidase quantitative detection kit (modified Cooper method) manufactured by Shenzhen Boruide Biotechnology Co., Ltd., lot number: 20140401; product registration number: Yuejian monitor (Standard) word 2010 No. 2400800), each sample was detected by using the kit of examples 1 to 3 and the comparison method, respectively, and the detection result (xi) of the comparison method was used as an independent variable and the detection result (yi) of the kits of examples 1 to 3 was used as a dependent variable to determine a linear regression equation and calculate a correlation coefficient (r) of the linear regression.
And substituting the detection result (xi) of the comparison method into a linear regression equation to calculate the estimated value of yi and the relative deviation between xi and xi.
Relative deviation (estimated value of yi-xi)/xi × 100%
The results are shown in fig. 2, fig. 3, fig. 4 and table 5.
TABLE 5 accuracy test results of the kit
Example 1 kit assay results (fig. 2): correlation coefficient r is 0.9900, linear equation: y 0.9618x + 0.6885;
example 2 kit assay results (fig. 3): correlation coefficient r is 0.9887, linear equation: y 0.9412x + 1.1623;
example 3 kit assay results (fig. 4): correlation coefficient r is 0.9917, linear equation: y 0.9359x + 1.2425.
The results of fig. 2, fig. 3, fig. 4 and table 5 are combined to show that the test accuracy of the kit is good, and the results are similar to those described above by using the detection method of example 6.
And (4) conclusion: the test results show that when the kit is used for detecting the activity of the seminal plasma neutral alpha-glucosidase, the linear range is wide, the accuracy and precision of the result are high, the detection time is short, and the kit is suitable for detecting a large number of samples clinically; meanwhile, the detection process of the sample can be tracked in real time.
Test example 4
Joint inspection with other items
Detecting the activity of plasma neutral alpha-glucosidase (NAG for short) in a plasma sample by the detection method of example 5 and using the kit of example 1;
four fully-automatic kits which are marketed by Nanjing Xindi biological pharmaceutical engineering Limited liability company are used at the same time: a seminal plasma fructose detection kit (hexose kinase method) (registration number: Su mechanical Standard 20172400748) (FHK for short), a seminal plasma zinc quantitative detection kit (PAN method) (registration number: Su mechanical Standard 2014 No. 2400711) (ZN for short), a seminal plasma superoxide dismutase detection kit (rate method) (registration number: Su mechanical Standard 20172400749) (SOD for short) and a seminal plasma gamma-L-glutamyl transpeptidase quantitative detection kit (rate method) (registration number: Su mechanical Standard 2014 No. 2400710) (GGT for short) are used for detecting seminal plasma fructose, seminal plasma zinc, seminal plasma superoxide dismutase and seminal plasma gamma-L-glutamyl transpeptidase in the seminal plasma sample on a full-automatic biochemical analyzer, and the detection method refers to the use specifications of the four kits.
On a full-automatic biochemical analyzer, the same seminal plasma sample is applied and detected simultaneously by a seminal plasma neutral alpha-glucosidase detection project, seminal plasma fructose, seminal plasma zinc, seminal plasma superoxide dismutase and seminal plasma gamma-L-glutamyl transpeptidase projects, the detection is repeated for 10 times, and the detection results are shown in table 6.
Table 6 results of item Joint examination
As can be seen from the results in Table 6, the kit and other kits can simultaneously detect the same sample, and the repeatability of the kit meets the requirements in performance indexes; the results were similar to those described above using the kits of examples 2 and 3 or the detection method of example 6 together.
Test example 5
The influence of urea and salicylic acid in the kit on the whole kit is detected.
Wherein comparative example 2 is the same as the reagent of the kit of example 1 except that urea is not contained;
comparative example 3 the same reagents as the kit of example 1 except that sodium salicylate was not included;
comparative example 4 the same reagents as the kit of example 1, except that urea and sodium salicylate were absent;
the linear range, reproducibility and accuracy of the kits of comparative examples 1, 2 and 3 were respectively measured using the detection method of example 5, and compared with the kit of example 1 of the present invention, and the results are shown in table 7.
TABLE 7 influence of Urea and salicylic acid on the test results of the kit
As shown in Table 7, compared with comparative examples 2-4, the linear range, repeatability and accuracy of the kit disclosed by the embodiment 1 of the invention are obviously superior to those of the comparative examples, and the urea and the sodium salicylate in the R2 reagent play an important role in the aspects of accuracy and repeatability.
Claims (4)
1. The seminal plasma neutral alpha-glucosidase detection kit is characterized by comprising an R1 reagent, an R2 reagent and a non-fixed value quality control product; the R1 reagent is a phosphate buffer solution containing SDS; the R2 reagent is a phosphate buffer solution containing urea, sodium salicylate and PNPG; the non-constant value quality control product is seminal plasma freeze-dried powder; the concentration of SDS in the R1 reagent is 0.2% -0.5% g/mL; the concentration of PNPG in the R2 reagent is 0.25-0.6% g/mL, the concentration of urea is 0.1-1% g/mL, and the concentration of sodium salicylate is 0.8-3.0% g/mL; the phosphate buffer solutions of the R1 reagent and the R2 reagent are respectively obtained by 0.1-0.4mol/L disodium hydrogen phosphate dodecahydrate and 0.1-0.4mol/L sodium dihydrogen phosphate dihydrate, wherein the intermodulation pH value is 6.0-7.0.
2. The seminal plasma neutral alpha-glucosidase detection kit of claim 1 wherein the R1 reagent and R2 reagent contains one or more of preservatives penicillin, streptomycin, penicillin, gentamicin sulfate and sodium azide.
3. The seminal plasma neutral alpha-glucosidase test kit of claim 1 wherein the non-constant quality control is lyophilized powder formed by lyophilizing seminal plasma containing one or more of the preservatives penicillin, streptomycin, gentamicin sulfate and sodium azide.
4. The seminal plasma neutral alpha-glucosidase assay kit of claim 1 comprising R1 reagent, R2 reagent, non-constant quality control, instructions and lining.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060141542A1 (en) * | 2002-08-29 | 2006-06-29 | Cohen Barb A | Alpha-amylase assay and uses thereof |
| CN103048282A (en) * | 2012-10-26 | 2013-04-17 | 潍坊鑫泽生物科技有限公司 | Detection method of bilirubin and detection kit |
| CN103760331A (en) * | 2014-02-11 | 2014-04-30 | 郑州安图生物工程股份有限公司 | Combined detection kit for seminal plasma |
| EP2766476A1 (en) * | 2011-10-11 | 2014-08-20 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| CN105021543A (en) * | 2015-06-30 | 2015-11-04 | 广州金域医学检验中心有限公司 | Alpha-amylase detection reagent and application thereof |
| CN105264384A (en) * | 2013-05-06 | 2016-01-20 | 生物辐射实验室股份有限公司 | Stabilization of labile analytes in reference materials |
| CN105543198A (en) * | 2016-01-21 | 2016-05-04 | 河南仰韶生化工程有限公司 | Application of sodium salicylate in preparation of pullulanase by fermenting |
-
2017
- 2017-10-31 CN CN201711044672.2A patent/CN107828855B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060141542A1 (en) * | 2002-08-29 | 2006-06-29 | Cohen Barb A | Alpha-amylase assay and uses thereof |
| EP2766476A1 (en) * | 2011-10-11 | 2014-08-20 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| CN103048282A (en) * | 2012-10-26 | 2013-04-17 | 潍坊鑫泽生物科技有限公司 | Detection method of bilirubin and detection kit |
| CN105264384A (en) * | 2013-05-06 | 2016-01-20 | 生物辐射实验室股份有限公司 | Stabilization of labile analytes in reference materials |
| CN103760331A (en) * | 2014-02-11 | 2014-04-30 | 郑州安图生物工程股份有限公司 | Combined detection kit for seminal plasma |
| CN105021543A (en) * | 2015-06-30 | 2015-11-04 | 广州金域医学检验中心有限公司 | Alpha-amylase detection reagent and application thereof |
| CN105543198A (en) * | 2016-01-21 | 2016-05-04 | 河南仰韶生化工程有限公司 | Application of sodium salicylate in preparation of pullulanase by fermenting |
Non-Patent Citations (1)
| Title |
|---|
| 24种精浆生化指标与精液常规参数的相关性研究;张红烨等;《中华男科学杂志》;20151220;第21卷(第12期);第1087-1092页 * |
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