CN112176026A - Detection kit for alpha-galactosidase activity - Google Patents
Detection kit for alpha-galactosidase activity Download PDFInfo
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Abstract
The invention discloses a detection kit for alpha-galactosidase activity, which belongs to the field of biochemical detection and comprises reaction liquid, a precipitator, a stop solution, a linear standard substance and a reference substance, wherein the linear standard substance is 4-methylumbelliferone solution, and the reaction liquid is 4-methylumbelliferone acyl-alpha-D-galactoside solution; the kit can rapidly and accurately carry out in-vitro detection on the activity of the alpha-galactosidase in a human blood sample, can realize the purpose of rapidly, conveniently and accurately diagnosing Fabry disease patients in batches by detecting collected dry blood sheet samples or venous blood samples of filter paper, and has the advantages of high detection sensitivity, good selectivity, strong anti-interference capability, good reproducibility, simplicity and convenience in operation, good product stability and easiness in clinical use.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a detection kit for alpha-galactosidase activity.
Background
Fabry disease is an X-chromosomal inherited multisystem lysosomal storage disease caused by a genetic mutation in alpha-galactosidase a (alpha-Gal a). The incidence rate of the newborn male is 1/110000-1/40000. Men and women are affected and usually develop diseases from children to teenagers, and female symptoms are usually lighter than male symptoms. Fabry disease is due to mutation of the gene encoding alpha-galactosidase a, resulting in partial or complete loss of the function of the encoded alpha-galactosidase a, normal degradation of hexosylceramide is hindered, and undegraded substrates accumulate in the lysosomes of cells in various tissues, causing dysfunction of the relevant tissues. Fabry disease is divided into classic and atypical: the onset of typical patients occurs in early stage in children and adolescents, and the onset of atypical patients occurs in adulthood and the onset of the patients occurs in single organ.
The alpha-galactosidase is widely present in animals, plants, microorganisms and cultured cells, can specifically catalyze the hydrolysis of alpha-galactoside bonds, and is mainly involved in the degradation of galactoside. Fabry disease is caused by the reduction or deletion of alpha-galactosidase activity, is X-linked recessive inherited sphingolipid storage disease, and the severity of the disease is often more or less closely related to the proportion of enzyme activity residues.
At present, there is no fast and convenient diagnostic reagent product for diagnosing Fabry disease in domestic clinic, and diagnosis is usually performed by combining typical clinical symptoms such as face change, rough skin, joint deformation, organ involvement and the like through means such as gene mutation detection, CT, X-ray and the like. However, for patients lacking typical clinical symptoms, none of the above approaches can effectively achieve the purpose of early diagnosis, and the methods have the disadvantages of complex operation, high cost, and secondary damage to patients repeatedly, and cannot be used as a more direct diagnosis standard to diagnose the disease.
Disclosure of Invention
The invention aims to provide a detection kit for alpha-galactosidase activity, which solves the problems in the prior art, and the kit can be used for assisting in diagnosing Fabry disease patients by measuring the alpha-galactosidase activity in a dry blood sample or a venous blood sample of filter paper, has the characteristics of trace amount, rapidness and convenience, and solves the problem that no similar mature detection kit product is used at present.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a detection kit for alpha-galactosidase activity, which comprises a reaction solution, a precipitating agent, a stop solution, a linear standard substance and a reference substance, wherein the linear standard substance is a 4-methylumbelliferone solution, and the reaction solution is a 4-methylumbelliferyl-alpha-D-galactoside solution.
Further, the 4-methylumbelliferyl-alpha-D-galactoside solution is an acidic solution, and the pH is adjusted by a buffer.
Further, the pH value of the 4-methylumbelliferyl-alpha-D-galactoside solution is 4.0-6.0;
the buffer solution is at least one selected from acetic acid-sodium acetate buffer solution, PBS buffer solution, Goods buffer solution, tris buffer solution, sodium citrate-dipotassium phosphate buffer solution, mops buffer solution, potassium dihydrogen phosphate-dipotassium phosphate buffer solution, pips buffer solution and hepes buffer solution.
Further, the preparation method of the reaction solution comprises the following steps: adding 20mmol of 4-methylumbelliferyl-alpha-D-galactoside into purified water, stirring and fully dissolving, adding a buffer solution to adjust the pH of the solution to 4.0-6.0, and fixing the volume to 1000 mL.
Further, the concentrations of the linear standard include 40nmol/L,100nmol/L,200nmol/L,400nmol/L, and 500 nmol/L.
Further, the precipitator is one or a mixed solution of several of barium hydroxide, sodium carbonate, zinc sulfate, sodium hydroxide and sodium bicarbonate, and the ion concentration of the precipitator is 0.25 mol/L.
Further, the terminating solution is a mixed solution of one or more of sodium carbonate, sodium bicarbonate, sodium hydroxide and barium hydroxide, and the ion concentration of the terminating solution is 0.5-1.0 mol/L.
Further, the reference substance is a dried blood slice prepared from a whole blood sample of a healthy human or a pure enzyme sample.
Further, the dry blood slice reference substance is divided into a negative reference substance, a weak positive reference substance and a positive reference substance, and is a dry blood slice sample prepared from blood cells and a normal human whole blood sample, wherein the hematocrit is 50-55%, and the preparation method of each level of reference substance comprises the following steps:
| enterprise reference product | Composition of |
| Negative reference substance | Normal human whole blood, which has a hematocrit of about 50% |
| Weak positive reference substance | 20% normal human whole blood, wherein the hematocrit is about 50% |
| Positive reference | 5% normal human whole blood, with a hematocrit of about 50% |
Further, the pure enzyme reference substance is an alpha-galactosidase solution, and the preparation method of each level reference substance is as follows:
alpha-galactosidase initial concentration of 0.484U/ml, diluted 2 times with 0.2ug/ul BSA solution to 3.88X 10-5The concentration of U/ml is recorded as a quality control product 1; dilution of the quality control 1 to 1.94X 10 with 0.2ug/ul BSA solution-5The concentration of U/ml is recorded as a quality control product 2; diluting the quality control product 2 with 0.2ug/ul BSA solution according to the following table to obtain quality control products 3-8
The invention discloses the following technical effects:
the detection and analysis principle of the detection kit of the invention is as follows:
alpha-galactosidase is a lysosomal enzyme widely present in human blood, which is capable of hydrolyzing the artificially synthesized substrate 4-methylumbelliferyl-alpha-D-galactosidase (4-methylumbelliferyl-alpha-D-galactosidase) under acidic conditions to produce the fluorescent products 4-methylumbelliferone and galactose; the 4-methyl umbelliferone can detect specific fluorescence under the conditions that the excitation wavelength is 360nm and the emission wavelength is 460nm, and the alpha-galactosidase activity can be calculated according to the detected fluorescence value.
The invention develops an in-vitro diagnosis kit capable of quickly and accurately diagnosing alpha-galactosidase in a human blood sample based on the principle, and can realize the purpose of quickly, conveniently and accurately diagnosing Fabry disease patients in batches by detecting the collected filter paper dry blood sample or venous blood sample. The kit has the advantages of high detection sensitivity, good selectivity, strong anti-interference capability, good reproducibility, simple operation and good stability, and can be used for the activity detection of alpha-galactosidase.
The blood sample of the filter paper dried blood sheet is a blood sample collection mode which is generally adopted clinically at present, has the advantages of simple and easy operation, small blood demand (about 20 mu L/blood spot) and easy storage and transportation, and is particularly suitable for infant groups. The rapid detection technology of the filter paper dried blood sample by adopting the alpha-galactosidase fluorogenic substrate method is not interfered by blood cells contained in the dried blood sample.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
In the embodiment of the invention, the filter paper dried blood slices are prepared, transported and used according to the expert consensus of neonatal disease screening filter paper blood slice collection, delivery and preservation.
EXAMPLE 1 composition of a kit for detecting alpha-galactosidase Activity
The detection kit comprises the following parts:
1) reaction solution: weighing 20mmol of 4-methylumbelliferyl-alpha-D-galactoside, adding into purified water, stirring and fully dissolving, adjusting the pH value to 4.0-6.0 by using acetic acid-sodium acetate buffer solution, and fixing the volume to 1000 mL.
2) A precipitant: 0.125mol of barium hydroxide and 0.125mol of sodium carbonate are weighed and added into 1000mL of purified water to be fully dissolved by stirring.
3) Stopping liquid: 0.5mol of sodium carbonate is weighed and added into 1000mL of purified water to be fully dissolved by stirring, and the pH value of the solution is adjusted to be more than 10.0.
4) Linear standards: weighing 500nmol of 4-methyl umbelliferone, adding into 1000mL of purified water, and stirring to dissolve completely; diluting with purified water to 40nmol/L,100nmol/L,200nmol/L, and 400nmol/L to obtain the final product.
5) Comparison products: at least 3 parts of whole blood samples are taken within 24 hours of health examination people who have got rid of hepatitis B, hepatitis C, AIDS, syphilis and normal liver function. Negative reference substance: after mixing, 20 mu L/mm2Preparing a negative reference substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; weak positive reference substance: diluting 20% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20 μ L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; positive reference substance: diluting 5% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20. mu.L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use.
EXAMPLE 2 composition of the detection kit for alpha-galactosidase Activity
The detection kit comprises the following parts:
1) reaction solution: weighing 20mmol of 4-methylumbelliferyl-alpha-D-galactoside, adding into purified water, stirring to dissolve completely, adjusting pH to 4.0-6.0 with PBS buffer solution, and diluting to 1000 mL.
2) A precipitant: 0.1mol of barium hydroxide and 0.15mol of sodium carbonate are weighed and added into 1000mL of purified water to be fully dissolved by stirring.
3) Stopping liquid: 0.8mol of sodium hydroxide is weighed and added into 1000mL of purified water to be fully dissolved by stirring, and the pH value of the solution is adjusted to be more than 10.0.
4) Linear standards: weighing 500nmol of 4-methyl umbelliferone, adding into 1000mL of purified water, and stirring to dissolve completely; diluting with purified water to 40nmol/L,100nmol/L,200nmol/L, and 400nmol/L to obtain the final product.
5) Comparison products: removing hepatitis B, hepatitis C, AIDS, and mume fructusAt least 3 whole blood samples within 24 hours of a healthy physical population with normal toxicity and liver function. Negative reference substance: after mixing, 20 mu L/mm2Preparing a negative reference substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; weak positive reference substance: diluting 20% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20 μ L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; positive reference substance: diluting 5% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20. mu.L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use.
EXAMPLE 3 composition of the detection kit for alpha-galactosidase Activity
The detection kit comprises the following parts:
1) reaction solution: weighing 20mmol of 4-methylumbelliferyl-alpha-D-galactoside, adding into purified water, stirring to dissolve completely, adjusting pH to 4.0-6.0 with sodium citrate-dipotassium hydrogen phosphate buffer solution, and diluting to 1000 mL.
2) A precipitant: 0.15mol of barium hydroxide and 0.1mol of zinc sulfate are weighed and added into 1000mL of purified water to be stirred and fully dissolved.
3) Stopping liquid: 0.5mol of sodium bicarbonate and 1mol of sodium hydroxide are weighed and added into 1000mL of purified water to be fully dissolved by stirring, and the pH value of the solution is adjusted to be more than 10.0.
4) Linear standards: weighing 500nmol of 4-methyl umbelliferone, adding into 1000mL of purified water, and stirring to dissolve completely; diluting with purified water to 40nmol/L,100nmol/L,200nmol/L, and 400nmol/L to obtain the final product.
5) Comparison products: at least 3 parts of whole blood samples are taken within 24 hours of health examination people who have got rid of hepatitis B, hepatitis C, AIDS, syphilis and normal liver function. Negative reference substance: after mixing, 20 mu L/mm2Preparing a negative reference substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; weak positive reference substance: diluting 20% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20 μ L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; positive reference substance: diluting 5% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20. mu.L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use.
EXAMPLE 4 composition of the detection kit for alpha-galactosidase Activity
The detection kit comprises the following parts:
1) reaction solution: 20mmol of 4-methylumbelliferyl-alpha-D-galactoside is weighed and added into purified water to be stirred and dissolved sufficiently, pH is adjusted to 4.0-6.0 by Goods buffer solution, and the volume is adjusted to 1000 mL.
2) A precipitant: 0.125mol of sodium carbonate and 0.125mol of zinc sulfate are weighed and added into 1000mL of purified water to be stirred and fully dissolved.
3) Stopping liquid: 1mol of sodium bicarbonate and 0.5mol of sodium carbonate are weighed and added into 1000mL of purified water to be fully dissolved by stirring, and the pH value of the solution is adjusted to be more than 10.0.
4) Linear standards: weighing 500nmol of 4-methyl umbelliferone, adding into 1000mL of purified water, and stirring to dissolve completely; diluting with purified water to 40nmol/L,100nmol/L,200nmol/L, and 400nmol/L to obtain the final product.
5) Comparison products: at least 3 parts of whole blood samples are taken within 24 hours of health examination people who have got rid of hepatitis B, hepatitis C, AIDS, syphilis and normal liver function. Negative reference substance: after mixing, 20 mu L/mm2Preparing a negative reference substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; weak positive reference substance: diluting 20% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20 μ L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use; positive reference substance: diluting 5% of the mixed blood with inactivated blood cells and inactivated serum, wherein the hematocrit is about 50%, 20. mu.L/mm2Preparing a positive control substance by using filter paper, drying for 24 hours, sealing by using a self-sealing bag, and storing at 2-8 ℃ for later use.
Example 5 Using procedure and result judgment of the detection kit for alpha-galactosidase Activity
1) Preparation of reagents: the kit components of example 1 were removed from 2-8 ℃ and allowed to equilibrate at room temperature for more than 30 minutes.
2) Sample preparation and loading: taking a dry blood sheet of filter paper to be detected, taking one sample (the diameter is approximately equal to 3mm) of a central blood spot of the dry blood sheet by using a special puncher, adding the sample into a U-shaped deep pore plate, simultaneously arranging 1 pore for each of a blank control pore, a negative control pore and a positive control pore, and respectively adding 1 piece of the blank filter paper sheet, the negative control dry blood sheet and the positive control dry blood sheet.
3) Liquid adding: the reaction solution was added at 200. mu.L/well.
4) And (3) incubation: shaking at 37 deg.C and 200rpm/min, and reacting for 24h in dark.
5) And (3) precipitation: adding 100 μ L of precipitant, shaking, mixing, and standing in dark for 10 min.
6) Terminating the centrifugation: adding 760 μ L of stop solution, shaking, mixing, and centrifuging at 2500rpm for 15min at 4 deg.C.
7) Color comparison: accurately taking 200 mu L of the solution to be detected on an enzyme label plate, and recording the absorbance value of each detection hole by using the excitation wavelength/emission wavelength of 360/460 nm.
8) Calculating the biological enzyme activity:
drawing a standard curve, drawing a standard curve y as ax according to the detection result of the standard product, and calculating the slope a.
4MU standard curve formula: y is ax
Wherein:
y: detected fluorescence value
a: slope of standard curve
x: 4MU concentration, unit: nmol/L;
② calculating the enzyme Activity of the sample
Definition of enzyme activity: the nanomolar number (nmol) of substrate 4-methylumbelliferyl-alpha-D-galactoside converted to product 4-methylumbelliferone per minute by alpha-galactosidase per liter of blood at 37 ℃ is one unit (nmol/L/min).
Enzyme activity calculation formula:
wherein: y: detected fluorescence value
a: the slope of the standard curve;
note: each blood spot was 3mm in diameter and contained a blood volume of about 5. mu.L.
The alpha-galactosidase activity was measured as 1050. mu.L reaction.
The reaction time was 24h × 60 min-1440 min.
Example 6 Using procedure and result judgment of the detection kit for alpha-galactosidase Activity
First, sample pretreatment
Sample treatment is carried out by adopting special sample releasing agents 1, 2 and 3 of Nodaokouke (Beijing) Biotechnology limited company, and the method comprises the following steps:
1. the blood collection tube containing anticoagulation is inverted and mixed evenly to avoid blood stratification;
2. centrifuging the specimen: 3mL of whole blood, 1500g and 6min of centrifugation; centrifuging above 3mL of whole blood at 1500g for 10 min;
3. discarding the supernatant (blood plasma), uniformly blowing and sucking the subnatant, and transferring to a 10mL centrifuge tube;
4. adding sample releasing agent 1 until the total volume in the tube reaches 10mL, reversing and mixing uniformly, centrifuging 1500g for 6min after 10min, and discarding the supernatant;
5. adding 1mL of sample release agent 1, uniformly blowing and sucking, and repeating the previous step;
6. adding 1mL of sample releasing agent 2, uniformly mixing by blowing and sucking, and transferring to a 1.5mL centrifuge tube; 1200g, centrifuging at 4 ℃ for 3min, and removing supernatant;
7. adding 1mL of sample release agent 3, and uniformly mixing by blowing and sucking; 2000g, centrifuging for 3min at 4 ℃, and discarding the supernatant;
8. resuspending with appropriate amount of purified water, packaging, and storing in-80 deg.C refrigerator if not immediately detected.
Second, calibrating the protein concentration of the sample
The protein concentration of the sample is calibrated by using a commercially available reagent, and a Coomassie brilliant blue G-250 reagent can be selected, and the concentration of the sample to be detected is diluted to about 0.2 +/-0.02 ug/uL by using purified water for later use.
The concentration of the calibration protein was recorded as Cug/uL.
Third, the enzyme activity of the sample is detected
The reagents of each component are added in sequence and reacted according to the following contents:
fourthly, calculating a result:
drawing a standard curve, drawing a standard curve y as ax according to the detection result of the standard products 1-6, and calculating the slope a.
4MU standard curve formula: y is ax
Wherein:
y: detected fluorescence value
a: slope of standard curve
x: 4MU concentration, unit: nmoL/L
② calculating the enzyme Activity of the sample
Definition of enzyme activity: the nanomolar number (nmoL) of substrate 4-methylumbelliferyl-alpha-D-galactoside converted to product 4-methylumbelliferone per gram of alpha-galactosidase per minute (min) in leukocytes (g) at 37 ℃ is one unit (nmoL/g/min).
Enzyme activity calculation formula:
wherein y: detected fluorescence value
a: slope of standard curve
C: calibration of protein concentration
Note: reaction system with alpha-galactosidase activity detected as 1000uL
The reaction time is 2 hX 60 min-120 min
Fifth, quality control
The quality control product of the company is used, and proper quality control intervals and limit values are adopted according to the specific requirements of each laboratory, and the detection value is in a set range.
EXAMPLE 7 evaluation of technical standards of the detection kit for alpha-galactosidase Activity (Dry blood sample)
1) Absorbance of blank
And determining that the light absorption value of a blank filter paper sheet of the kit is less than or equal to 200 by detecting at least three batches of products.
2) Accuracy of
The accuracy of the kit in three continuous batches is verified by adopting special 10 negative (the enzyme activity is approximately equal to 100% of normal people) dry blood sheets, 10 weak positive (the enzyme activity is approximately equal to 15% of normal people) dry blood sheets and 10 strong positive (the enzyme activity is approximately equal to 5% of normal people) dry blood sheet quality control products, and the negative and positive results are judged according to the determined cut off value.
The result shows that the negative quality control products are all negative, the coincidence rate is 100 percent, and the positive and weak positive quality control products are all positive and weak positive, and the coincidence rate is 100 percent.
3) Precision degree
And taking one part of each of the negative, positive and weak positive quality control substances, verifying with the precision of the kit of three consecutive batches, repeatedly detecting for 8 times every day for 5 days, and judging the negative and positive results with the determined cut off value.
The result shows that the negative quality control substances of the three continuous batches of the kit have no positive result, and the positive and weak positive quality control substances are all positive and have no negative result.
4) Sensitivity of the probe
And detecting three weak positive quality control products by using three continuous batches of kits to obtain no negative result.
5) Detection of actual clinical samples
The kit provided by the invention summarizes the results of 624 clinical detection samples within one year, as shown in the following table, 2 positive results are found, and the clinical diagnosis is confirmed to be a patient with Fabry disease; the blood samples of 3 clinically confirmed Fabry disease patients are detected, and the detection results are positive. The blood samples of 152 clinically collected physical examination people were tested and all were negative.
Test results of clinical specimens
| Total number of samples | Number of positive cases | Consistency with clinical diagnosis | |
| Clinical sample data within one year | 624 | 5 | ■ yes at □ no |
| Has confirmed diagnosis of clinical sample data | 3 | 3 | ■ yes at □ no |
| Sample data of normal physical examination population | 152 | 0 | ■ yes at □ no |
EXAMPLE 8 evaluation of technical standards of detection kit for alpha-galactosidase Activity (venous blood sample)
1. Reagent blank
The blank light absorption value is less than or equal to 15.
2. Linear range
In the range of the sample enzyme activity of 2.2 nmoL/g/min-88.1 nmoL/g/min: the correlation coefficient r is more than or equal to 0.975, and the relative deviation is within the range of +/-15%.
3. Accuracy of
And (3) determining samples with the labeled enzyme activity of 53.2-58.8 nmoL/g/min, 20.9-23.1 nmoL/g/min and 6.27-6.93 nmoL/g/min, wherein the determination result is not more than +/-10% of the labeled value.
4. Repeatability of
Samples with the enzyme activity of 22.0 +/-1.1 nmoL/g/min and 6.6 +/-0.33 nmoL/g/min are determined by the same batch of reagents, each sample is tested repeatedly for 10 times, and the detected CV value is less than or equal to 15 percent.
5. Difference between batches
Three batches of reagents are taken to determine samples with the enzyme activity of 22.0 +/-1.1 nmoL/g/min and 6.6 +/-0.33 nmoL/g/min, each sample is tested repeatedly for 3 times, and the CV value detected is less than or equal to 15 percent.
5) Detection of actual clinical samples
The results of 241 clinical detection samples in one year are summarized by using the kit, as shown in Table 1, 4 positive results are found, and the clinical diagnosis of the results is confirmed to be a Fabry disease type I patient; the blood samples of 2 clinically confirmed Fabry disease patients are detected, and the detection results are positive. The test on 88 clinical blood samples collected from the physical examination population shows negative results.
TABLE 1 test results of clinical specimens
| Total number of samples | Number of positive cases | Consistency with clinical diagnosis | |
| Clinical sample data within one year | 241 | 8 | ■ yes at □ no |
| Has confirmed diagnosis of clinical sample data | 2 | 2 | ■ yes at □ no |
| Sample data of normal physical examination population | 88 | 0 | ■ yes at □ no |
The results show that the alpha-galactosidase activity detection kit has higher accuracy and specificity in alpha-galactosidase activity detection, can meet the clinical requirement for Fabry disease auxiliary diagnosis, solves the problem that similar products are not available at present, and takes a filter paper dry blood sheet sample as a detection object, and the usage amount of the blood sample is about 5 mu L (3 mm)2) The pain of the patient is relieved every time; the activity of the alpha-galactosidase is detected to be 5% of that of normal people at the lowest, the sensitivity is high, and the Fabry disease type 1 disease can be accurately diagnosed.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The detection kit for the activity of the alpha-galactosidase is characterized by comprising a reaction solution, a precipitating agent, a stop solution, a linear standard substance and a reference substance, wherein the linear standard substance is a 4-methylumbelliferone solution, and the reaction solution is a 4-methylumbelliferyl-alpha-D-galactoside solution.
2. The kit for detecting alpha-galactosidase activity of claim 1, wherein the 4-methylumbelliferyl-alpha-D-galactoside solution is an acidic solution and the pH is adjusted by a buffer.
3. The detection kit for alpha-galactosidase activity according to claim 2, wherein the pH of the 4-methylumbelliferyl-alpha-D-galactoside solution is 4.0-6.0;
the buffer solution is at least one selected from acetic acid-sodium acetate buffer solution, PBS buffer solution, Goods buffer solution, tris buffer solution, sodium citrate-dipotassium phosphate buffer solution, mops buffer solution, potassium dihydrogen phosphate-dipotassium phosphate buffer solution, pips buffer solution and hepes buffer solution.
4. The kit for detecting alpha-galactosidase activity of claim 1, wherein the reaction solution is prepared by a method comprising: adding 20mmol of 4-methylumbelliferyl-alpha-D-galactoside into purified water, stirring and fully dissolving, adding a buffer solution to adjust the pH of the solution to 4.0-6.0, and fixing the volume to 1000 mL.
5. The detection kit for alpha-galactosidase activity according to claim 1, wherein the concentration of the linear standard comprises 40nmol/L,100nmol/L,200nmol/L,400nmol/L and 500 nmol/L.
6. The kit for detecting the activity of alpha-galactosidase according to claim 1, wherein the precipitating agent is one or a mixture of barium hydroxide, sodium carbonate, zinc sulfate, sodium hydroxide and sodium bicarbonate, and the ion concentration of the precipitating agent is 0.25 mol/L.
7. The alpha-galactosidase activity detection kit according to claim 1, wherein the stop solution is a mixed solution of one or more of sodium carbonate, sodium bicarbonate, sodium hydroxide and barium hydroxide, and the ion concentration of the stop solution is 0.5mol/L to 1.0 mol/L.
8. The detection kit for alpha-galactosidase activity according to claim 1, wherein the control is a dried blood sheet prepared from a whole blood sample of a healthy human or a pure enzyme sample.
9. The kit for detecting alpha-galactosidase activity according to claim 8, wherein the dry blood control sample is divided into a negative control, a weak positive control and a positive control, and is a dry blood sample prepared from blood cells and a normal human whole blood sample, wherein the hematocrit is 50-55%.
10. The detection kit for alpha-galactosidase activity according to claim 8, wherein the pure enzyme control is an alpha-galactosidase solution, and the preparation method of each level of reference is as follows:
alpha-galactosidase initial concentration of 0.484U/ml, diluted 2 times with 0.2ug/ul BSA solution to 3.88X 10-5The concentration of U/ml is recorded as a quality control product 1; dilution of the quality control 1 to 1.94X 10 with 0.2ug/ul BSA solution-5The concentration of U/ml is recorded as a quality control product 2; the quality control 2 was diluted with 0.2ug/ul BSA solution according to the following table to obtain quality control 3-8, respectively.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112557381A (en) * | 2021-01-25 | 2021-03-26 | 河南省科学院生物研究所有限责任公司 | Detection test strip for alpha-galactosidase and detection method thereof |
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