CN108866153A - A kind of Application of Sialic Acid Measurement kit and its measuring method - Google Patents
A kind of Application of Sialic Acid Measurement kit and its measuring method Download PDFInfo
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Abstract
The present invention provides a kind of Application of Sialic Acid Measurement kit, including reagent R1 and reagent R2, reagent R1 include following component and its concentration：8~20g/L of trishydroxymethylaminomethane, 5~15ml/L of HCL, 0.5~5KU/L of neuraminidase, 0.5~5KU/L of lactic dehydrogenase, 0.1~2mmol/L of NADH；Reagent R2 includes following component and its concentration：8~20g/L of trishydroxymethylaminomethane, 5~15ml/L of HCL, 0.5~5KU/L of Neu 5 Ac aldolase, 25~100g/L of sucrose, 0.6~1.2ml/L of triethanolamine.The invention belongs to technical field of biological, Application of Sialic Acid Measurement kit provided by the invention significantly enhances anti-interference ability while improving stability, the interference that bilirubin, hemoglobin and triglycerides etc. can be significantly reduced, the measurement accuracy for sialic acid are good.
Description
Technical field
The invention belongs to technical field of biological more particularly to a kind of Application of Sialic Acid Measurement kit and its measuring methods.
Background technique
Sialic acid (Sialic acid, SA) is also known as N-acetyl-neuraminate, be a variety of natural neuraminic acid derivatives it
One, it is the general name of a compounds of group that precursor structure, which is the interior cyclic amino acids of nine carbon atoms composition,.Sialic acid is cell membrane sugar
The important component of albumen, is distributed widely in animal body, and the sialic acid in human serum includes free sialic acid and combination
The content of sialic acid, free sialic acid is lower, is sialic acid in serum with the bound sialic acid of lipoprotein and Glycoprotein binding
It is primarily present form.
Sialic acid is related with many biological functions of organism, and with malignant change of cell, metastasis of cancer, infiltrate, lose and contact
Property inhibit, cell adhesion reduce and tumor antigenicity it is closely related.A large number of studies show that the one kind of SA as malignant tumour
Marker has diagnostic significance to Several Kinds of Malignancy.When eubolism, the sialic acid content of serum is stablized, and works as histocyte
When canceration, cell surface glycoprotein is structurally and functionally being substantially change, and is incorporated in the sialic acid on glycoprotein with different
The glycoprotein often expressed falls off into blood, is the raised possible cause of sialic acid in blood.The abnormal of sialic acid content increases and disease
The severity of feelings is proportional to, and is measured serum sialic acid concentration, be can be used as cancerous swelling diagnostic assistance index and observation of curative effect
Index.
The detection method of sialic acid content mainly includes HPLC method, enzyme process, chemical colorimetry.HPLC method has sensitivity
Advantage high, specificity is good, but expensive equipment and complicated for operation, unsuitable clinical batches detection.Although chemical colorimetry valence
Lattice are lower, but specificity is low, complicated for operation cumbersome, need high-temperature extraction, are not suitable for automated analysis.Enzyme process has specificity
Height, it is simple and quick, it is accurate safety, can automated analysis the advantages of.
Chinese patent application CN101469344 discloses a kind of enzymatic assays sialic acid liquid double reagent, by reagent 1 and examination
Agent 2 forms, and reagent 1 includes neuraminidase, lactic dehydrogenase, buffer；Reagent 2 contracts including N-acetyl-neuraminate aldehyde
Enzyme, NADH and buffer etc.；Reagent 1 and reagent 2 can also include stabilizer, surfactant, preservative, and stabilizer is selected from Mg
Ion, Nacl, Kcl, seralbumin, antibiotic, EDTA class, polyethylene glycol, disaccharides, polyalcohol etc..
Chinese patent application CN 102399851 discloses a kind of Sialidase detection kit, by following two reagent
It constitutes, reagent 1 includes buffer, neuraminidase, lactic dehydrogenase, surfactant, stabilizer and preservative；Reagent 2
Including buffer, reducibility coenzyme, neuraminic acid aldolase, stabilizer and preservative；Stabilizer be polyethylene glycol, glycerine,
One of propylene glycol, sucrose, trehalose, sorbierite, BSA are several.
Chinese patent application CN 106092942 discloses a kind of kit for measuring sialic acid, including reagent R1 and reagent
R2 biliquid component, reagent R1 include：AMP buffer, neuraminidase, lactic dehydrogenase, Tween-20 and sodium benzoate；
Reagent R1 includes：AMP buffer, NADH, neuraminic acid aldolase, polyethylene glycol 2000 and sodium benzoate；By adding poly- second
Glycol -2000 accelerates the reaction speed of reaction, improves the stability of reagent as preservative by addition sodium benzoate.
The stabilizer type that existing kit uses is more, although stability can be improved, anti-interference ability is often
It is not ideal enough.Therefore it provides the Application of Sialic Acid Measurement kit that a kind of stability is good and anti-interference ability is good is of great significance.
Summary of the invention
(anti-interference ability is not ideal enough) to solve problems of the prior art, the present invention provide a kind of sialic acid survey
Determine kit, by adding certain density sucrose and triethanolamine, is significantly enhanced while improving stabilization of kit
Anti-interference ability can significantly reduce the interference of bilirubin, hemoglobin and triglycerides etc., the measurement accuracy for sialic acid
It is good.
The purpose of the present invention will further illustrate by the following detailed description.
The present invention provides a kind of Application of Sialic Acid Measurement kit, including reagent R1 and reagent R2, the reagent R1 include as follows
Component and its concentration：8~20g/L of trishydroxymethylaminomethane, 5~15ml/L of HCL, 0.5~5KU/L of neuraminidase, cream
0.5~5KU/L of acidohydrogenase, 0.1~2mmol/L of NADH；The reagent R2 includes following component and its concentration：Trihydroxy methyl
8~20g/L of aminomethane, 5~15ml/L of HCL, 0.5~5KU/L of Neu 5 Ac aldolase, 25~100g/L of sucrose,
0.6~1.2ml/L of triethanolamine.
Preferably, the reagent R1 includes following component and its concentration：10~15g/L of trishydroxymethylaminomethane, HCL 6
~10ml/L, 0.5~2KU/L of neuraminidase, 1~3KU/L of lactic dehydrogenase, 0.2~1mmol/L of NADH；The reagent
R2 includes following component and its concentration：10~15g/L of trishydroxymethylaminomethane, 6~10ml/L of HCL, N-acetyl-neuraminate
1~3KU/L of aldolase, 50~70g/L of sucrose, 0.8~1.2ml/L of triethanolamine.
It is highly preferred that the reagent R1 includes following component and its concentration：Trishydroxymethylaminomethane 12g/L, HCL
8ml/L, neuraminidase 1KU/L, lactic dehydrogenase 2KU/L, NADH 0.5mmol/L；The reagent R2 includes following component
And its concentration：Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, Neu 5 Ac aldolase 2KU/L, sucrose 50g/L,
Triethanolamine 1ml/L.
Preferably, the reagent R1 is also comprised the following components and its concentration：0.8~1.2g/L of potassium sorbate；The reagent
R2 is also comprised the following components and its concentration：0.8~1.2g/L of potassium sorbate.
It is highly preferred that the reagent R1 is also comprised the following components and its concentration：Potassium sorbate 1g/L；The reagent R2 is also wrapped
Include following component and its concentration：Potassium sorbate 1g/L.
Preferably, the present invention also provides the measuring method of Application of Sialic Acid Measurement kit, include the following steps：Separate serum sample
Reagent R1 is added into blood serum sample for product, mixes, and after being incubated for 3~5min, reagent R2 is added, mixes, after being incubated for 1~2min,
The speed that the decline of NADH absorbance is measured at 405nm, measures sialic acid concentration.
Sialic acid in sample is formed N-acetyl-neuraminate, and then in N- second by the effect of neuraminidase (NRH)
The effect of acyl neuraminic acid aldolase (NAL) generates pyruvic acid and ManNAc.Pyruvic acid has cream in the presence of NADH
Acidohydrogenase (LDH) effect is lower to generate lactic acid and NAD+, and the speed of measurement NADH absorbance decline can acquire the saliva in sample
Acid concentration.
Compared with prior art, beneficial effects of the present invention include：The present invention in Application of Sialic Acid Measurement reagent by adding
Certain density sucrose and triethanolamine can pass through the enzyme activity stability of Neu 5 Ac aldolase in raising reagent R2
Enhance Application of Sialic Acid Measurement kit stability, assay kit in 2~8 degree of conditions long shelf-life up to 12 months or more, together
When significantly enhance anti-interference ability, the measurement accuracy for sialic acid is good.
Specific embodiment
The present invention is described in further details below with reference to embodiment.
In the present invention, related component is conventional commercial product, or can be obtained by ordinary skill in the art means
?.
One Application of Sialic Acid Measurement kit of embodiment (formula two)
Application of Sialic Acid Measurement kit includes reagent R1 and reagent R2, the reagent R1 include following component and its concentration：Three
Hydroxymethyl aminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic dehydrogenase 2KU/L, NADH 0.5mmol/
L, potassium sorbate 1g/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 12g/L, HCL 8ml/
L, Neu 5 Ac aldolase 2KU/L, sucrose 50g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
Two Application of Sialic Acid Measurement kit of embodiment
Application of Sialic Acid Measurement kit includes reagent R1 and reagent R2, the reagent R1 include following component and its concentration：Three
Hydroxymethyl aminomethane 10g/L, HCL 6ml/L, neuraminidase 1KU/L, lactic dehydrogenase 1KU/L, NADH 0.5mmol/
L, potassium sorbate 0.8g/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 10g/L, HCL
6ml/L, Neu 5 Ac aldolase 3KU/L, sucrose 70g/L, triethanolamine 1.2ml/L, potassium sorbate 0.8g/L.
Three Application of Sialic Acid Measurement kit of embodiment
Application of Sialic Acid Measurement kit includes reagent R1 and reagent R2, the reagent R1 include following component and its concentration：Three
Hydroxymethyl aminomethane 15g/L, HCL 10ml/L, neuraminidase 2KU/L, lactic dehydrogenase 2KU/L, NADH 1mmol/
L, potassium sorbate 1.2g/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 15g/L, HCL
10ml/L, Neu 5 Ac aldolase 2KU/L, sucrose 40g/L, triethanolamine 1ml/L, potassium sorbate 1.2g/L.
1 Application of Sialic Acid Measurement kit of comparative example (formula one)
Application of Sialic Acid Measurement kit includes reagent R1 and reagent R2, the reagent R1 include following component and its concentration：Three
Hydroxymethyl aminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic dehydrogenase 2KU/L, NADH 0.5mmol/
L, potassium sorbate 1g/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 12g/L, HCL 8ml/
L, Neu 5 Ac aldolase 2KU/L, potassium sorbate 1g/L.
Formula one and the difference of formula two are：Without sucrose and triethanolamine.
2 Application of Sialic Acid Measurement kit of comparative example
Application of Sialic Acid Measurement kit includes reagent R1 and reagent R2, the reagent R1 include following component and its concentration：Three
Hydroxymethyl aminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic dehydrogenase 2KU/L, NADH 0.5mmol/
L, potassium sorbate 1g/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 12g/L, HCL 8ml/
L, Neu 5 Ac aldolase 2KU/L, sucrose 50g/L, potassium sorbate 1g/L.
Comparative example 2 and the difference of embodiment one are：Without triethanolamine.
The sample detection comparison of the different assay kits of test example one
Experiment reagent：The SA measurement of above-mentioned formula one and formula two and the production of Shanghai Ju Chuan Pharmaceutical Technology Co., Ltd
Kit (including reagent R1 and R2, but ingredient is different from the present invention, and hereinafter referred to as commercially available group).Respectively by above-mentioned experiment reagent
For the detection of sialic acid, 40 blood serum samples are calibrated while measured simultaneously with AU400 Biochemical Analyzer, the results are shown in Table 1.
Result (the unit of 40 blood serum samples of different kit measurements of table 1：mmol/L)
Through counting, correlation coefficient r=0.9991 of one measurement result of commercially available group of measurement result and formula, commercially available group of measurement knot
Two measurement result correlation coefficient r=0.9995 of fruit and formula, two measurement result correlation coefficient rs of one measurement result of formula and formula=
0.9992, illustrate that correlation is good, a certain amount of sucrose and triethanolamine are added in reagent R2 to be caused to do to testing result
It disturbs, formula two and the product no significant difference through State Food and Drug Administration's approval listing, as a result accurately and reliably.
The accelerated stability of the different assay kits of test example two is investigated
37 degree of accelerated shelf life tests are carried out respectively to formula one and formula two, to investigate the stability of its component, as a result
As shown in table 2.
Sample detection result (the unit of the formula of table 2 one and two accelerated destructive tests of formula after 1 week：mmol/L)
37 degree of accelerated shelf life tests：Refer to and reagent is mounted in bottle and is sealed, is placed on inside 37 degree of water baths, into
Row accelerated shelf life test, 1 week time of 37 degree of destructive tests are equivalent to (2~8 degree) of general storage temperature and save 1 year.
As known from Table 2, when not accelerating, 40 sample detection result average values for being formulated one are 65.6, are formulated two detection
As a result average value is 65.2, substantially quite with formula one；After accelerated shelf life test 1 week, one testing result is formulated through 37 degree
Average value is 38.9, and the testing result average value for being formulated two is 63.7.
It is formulated the sample detection result after destructive test 1 week to be remarkably decreased, average fall reaches
40.7%, and the sample detection result for being formulated two due to containing a certain amount of sucrose and triethanolamine, after destructive test 1 week
Significant change does not occur, average fall is only 2.3%, belongs to zone of reasonableness.Average fall=(when not accelerating
Mean value-mean value of acceleration 1 week)/mean value when not accelerating.
To investigate the concrete reason that the testing result being formulated after accelerating 1 week once 37 degree is remarkably decreased, following experiment is carried out：
Its main component neuraminidase NRH or N- are added respectively in the R1 and R2 in formula one after 37 degree accelerate 1 week respectively
Acetylneuraminate aldolase NAL, additional amount are respectively the 50% of corresponding initial concentration, then respectively detection reagent 1 to reagent 4
The results are shown in Table 3.
3 reagent 1 of table to reagent 4 sample detection result (unit：mmol/L)
| Sample serial number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Reagent 1 | 94.5 | 64.2 | 23.1 | 82.1 | 19.4 | 72.6 | 101.9 | 58.5 | 38.8 | 118.7 |
| Reagent 2 | 63.2 | 45.6 | 11.4 | 41.4 | 10.2 | 44.3 | 49.7 | 48.2 | 18.6 | 71.2 |
| Reagent 3 | 67.8 | 43.9 | 11.3 | 45.2 | 12.1 | 45.0 | 40.6 | 43.4 | 12.1 | 65.1 |
| Reagent 4 | 90.5 | 63.8 | 22.8 | 80.1 | 19 | 79.5 | 106.9 | 55.2 | 35.1 | 112.8 |
| Sample serial number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Reagent 1 | 64.1 | 104.9 | 57.8 | 23.5 | 37.5 | 85.7 | 92.8 | 10.3 | 96.2 | 70.5 |
| Reagent 2 | 41.3 | 69.7 | 41.6 | 10.8 | 17.6 | 44.4 | 73.1 | 5.6 | 70.7 | 40.1 |
| Reagent 3 | 48.1 | 63.2 | 43.1 | 10.6 | 18.7 | 42.7 | 63.4 | 5.4 | 64.6 | 42.6 |
| Reagent 4 | 62.3 | 102.1 | 55.4 | 22.9 | 36.4 | 82.3 | 91.2 | 10.1 | 90.2 | 73.5 |
| Sample serial number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Reagent 1 | 81.5 | 23.0 | 33.1 | 55.2 | 94.7 | 64.5 | 116.4 | 71.3 | 41.9 | 21.9 |
| Reagent 2 | 43.1 | 10.9 | 12.6 | 45.5 | 68.2 | 41.3 | 71.0 | 49.6 | 20.2 | 10.8 |
| Reagent 3 | 46.7 | 12.6 | 11.4 | 49.2 | 63.4 | 46.5 | 66.2 | 41.8 | 21.7 | 11.3 |
| Reagent 4 | 85.5 | 21.8 | 30.3 | 54.6 | 93.9 | 60.2 | 111.8 | 78.6 | 39.6 | 20.1 |
| Sample serial number | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| Reagent 1 | 63.5 | 55.6 | 97.1 | 78.3 | 19.8 | 46.9 | 102.4 | 89.2 | 32.8 | 115.4 |
| Reagent 2 | 44.6 | 46.9 | 60.4 | 42.1 | 10.9 | 27.4 | 68.1 | 41.8 | 16.5 | 62.3 |
| Reagent 3 | 46.7 | 43.8 | 74.5 | 40.9 | 11.4 | 28.6 | 65.4 | 40.4 | 13.2 | 71.1 |
| Reagent 4 | 58.9 | 54.1 | 94.6 | 76.2 | 19.7 | 45.6 | 100.2 | 88.6 | 31.0 | 113.2 |
Reagent 1：R1 and R2 is not accelerated；
Reagent 2：R1 and R2 accelerates 1 week through 37 degree；
Reagent 3：R1 and R2 accelerates 1 week through 37 degree, and adds in R1 into NRH 0.5KU/L；
Reagent 4：R1 and R2 accelerates 1 week through 37 degree, and adds in R2 into NAL 1KU/L.
As known from Table 3：1. measured value and reagent 1 have notable difference after reagent 2 (not adding NRH and NAL) accelerated experiment；2.
Measured value and reagent 1 have notable difference after reagent 3 (individually adding NRH) accelerated experiment, almost the same with 2 measured value of reagent, say
NRH is not inactivated in bright reagent R1；3. measured value is with reagent 1 without obvious poor after reagent 4 (individually adding NAL) accelerated experiment
It is different, it is significantly increased compared with the measured value of reagent 2, illustrates that NAL has obvious inactivation in reagent R2.
Further, the R2 reagent of the R2 reagent and formula two that detect formula one respectively by activation measurement not accelerating and
The vigor of NAL at 37 degree acceleration 1 week, each detection 20 times, as a result as shown in table 4 and table 5.
NAL vigor (the unit of the R2 reagent detection of the formula of table 4 one：KU/L)
NAL vigor (the unit of the R2 reagent detection of the formula of table 5 two：KU/L)
From table 4 and table 5 as can be seen that being formulated one accelerated 1 week, NAL inactivation is serious, and inactivation ratio has reached 47.5%,
And two are formulated since a certain amount of sucrose and triethanolamine is added, NAL inactivation is only 4.0%, illustrates sucrose and triethanolamine pair
NAL has preferable protective effect.
The dosage of three sucrose of test example and triethanolamine in reagent R2 is investigated
Formula one is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, potassium sorbate 1g/L.
Formula two is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 50g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
Formula three is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 10g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
Formula four is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 25g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
Formula five is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 100g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
To investigate dosage of the sucrose in reagent R2, detects formula one to formula five respectively by activation measurement and do not adding
The vigor of NAL when speed accelerates 1 week with 37 degree, the results are shown in Table 6.
Table 6 is formulated one to the NAL when not accelerating to accelerate 1 week with 37 degree of formula five vigor (unit：KU/L)
As can be seen from Table 6, be formulated one NAL inactivation it is serious, inactivation ratio has reached 46.9%, and a certain amount of sugarcane is added
Sugar and triethanolamine after formula two to formula five be respectively：3.5%, 23.4%, 11.6%, 4.1%.Illustrate sucrose in reagent
Preferable protective effect can be played when the additional amount 1ml/L of 25~100g/L of additional amount and triethanolamine in R2.
In addition, being detected respectively to the formula response curve of formula one to formula five, the reaction of formula one to formula five
Rapidly, illustrate that sample-adding amount will not inhibit the quick progress of reaction 100g/L.
Therefore, it is formulated the resultant effect of two (additional amount of sucrose is 50g/L, the additional amount of triethanolamine is 1ml/L) most
It is good.
The anti-interference capability testing of four assay kit of test example
Human serum is complex in composition, and bilirubin and hemoglobin therein etc. can cause the interference of kit measurement, causes to examine
Survey the difference of result.Clinical sample bilirubin, hemoglobin concentration are detected, suitable concentration sample is screened.Take portion without haemolysis blood
Final proof product, are divided into several pieces, sequentially add the chaff interferent (bilirubin or hemoglobin) of various concentration, respectively using formula one,
Formula two and the kit of comparative example 2 carry out detection sialic acid, residue 2 to the blood serum sample of addition bilirubin or hemoglobin
Part does not add chaff interferent, carries out anti-interference capability testing.Relative deviation (%)=(interfering substance-is added not add)/does not add.
Formula one is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, potassium sorbate 1g/L.
Formula two is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 50g/L, triethanolamine 1ml/L, potassium sorbate 1g/L.
Comparative example 2 is as follows：R1:Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic acid
Dehydrogenase 2 KU/L, NADH 0.5mmol/L, potassium sorbate 1g/L；R2:Trishydroxymethylaminomethane 12g/L, HCL8ml/L, N-
Acetylneuraminate aldolase 2KU/L, sucrose 50g/L, potassium sorbate 1g/L.
Testing result is as shown in table 7 and table 8.
The anti-bilirubin interference performance test result of 7 kit of table
Note：Bilirubin 0 refers to that the concentration of bilirubin is 0 μm of ol/L, and bilirubin 50 refers to that the concentration of bilirubin is 50 μm of ol/L.
The anti-hemoglobin interference performance test result of 8 kit of table
Note：Hemoglobin 0 refers to that the concentration of hemoglobin is 0g/L, and hemoglobin 1.0 refers to that the concentration of hemoglobin is 1.0g/L.
By table 7 and table 8 it is found that as the chaff interferent bilirubin or hemoglobin concentration contained in serum increases, detection knot
The relative deviation of fruit rises.Wherein, minimum to be formulated two relative deviation, when the concentration of chaff interferent bilirubin is 200 μm of ol/L
Relative deviation be 7.4%, far below the relative deviation of formula one and comparative example 2, formula one and 2 relative deviation of comparative example are super
Cross 12%；The relative deviation when concentration of chaff interferent hemoglobin is 2.0g/L is 6.9%, far below formula one and comparative example 2
Relative deviation, formula one and 2 relative deviation of comparative example more than 15%.As it can be seen that formula two provided by the invention can significantly drop
The interference of low bilirubin and hemoglobin, anti-interference ability are stronger.
In conclusion the Application of Sialic Acid Measurement kit that the present invention is supplied to significantly enhances while improving stability
Anti-interference ability, reaction speed is fast, and detection accuracy is good.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (6)
1. a kind of Application of Sialic Acid Measurement kit, it is characterised in that：It include such as the following group including reagent R1 and reagent R2, the reagent R1
Point and its concentration：8~20g/L of trishydroxymethylaminomethane, 5~15ml/L of HCL, 0.5~5KU/L of neuraminidase, lactic acid
0.5~5KU/L of dehydrogenase, 0.1~2mmol/L of NADH；The reagent R2 includes following component and its concentration：Trihydroxy methyl ammonia
8~20g/L of methylmethane, 5~15ml/L of HCL, 0.5~5KU/L of Neu 5 Ac aldolase, 25~100g/L of sucrose, three
0.6~1.2ml/L of ethanol amine.
2. Application of Sialic Acid Measurement kit according to claim 1, it is characterised in that：The reagent R1 include following component and
Its concentration：10~15g/L of trishydroxymethylaminomethane, 6~10ml/L of HCL, 0.5~2KU/L of neuraminidase, lactic acid are de-
1~3KU/L of hydrogen enzyme, 0.2~1mmol/L of NADH；The reagent R2 includes following component and its concentration：Trihydroxy methyl amino first
10~15g/L of alkane, 6~10ml/L of HCL, 1~3KU/L of Neu 5 Ac aldolase, 50~70g/L of sucrose, triethanolamine
0.8~1.2ml/L.
3. Application of Sialic Acid Measurement kit according to claim 2, it is characterised in that：The reagent R1 include following component and
Its concentration：Trishydroxymethylaminomethane 12g/L, HCL 8ml/L, neuraminidase 1KU/L, lactic dehydrogenase 2KU/L, NADH
0.5mmol/L；The reagent R2 includes following component and its concentration：Trishydroxymethylaminomethane 12g/L, HCL8ml/L, N- second
Acyl neuraminic acid aldolase 2KU/L, sucrose 50g/L, triethanolamine 1ml/L.
4. Application of Sialic Acid Measurement kit according to any one of claim 1 to 3, it is characterised in that：The reagent R1 is also
Including following component and its concentration：0.8~1.2g/L of potassium sorbate；The reagent R2 is also comprised the following components and its concentration：Mountain
0.8~1.2g/L of potassium sorbate.
5. Application of Sialic Acid Measurement kit according to any one of claim 1 to 3, it is characterised in that：The reagent R1 is also
Including following component and its concentration：Potassium sorbate 1g/L；The reagent R2 is also comprised the following components and its concentration：Potassium sorbate
1g/L。
6. the measuring method of Application of Sialic Acid Measurement kit according to claim 1, it is characterised in that：Include the following steps：
Blood serum sample is separated, reagent R1 is added into blood serum sample, is mixed, after being incubated for 3~5min, reagent R2 is added, mixes, is incubated for 1
After~2min, the speed of NADH absorbance decline is measured at 405nm, measures sialic acid concentration.
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| CN201710330265.1A CN108866153A (en) | 2017-05-11 | 2017-05-11 | A kind of Application of Sialic Acid Measurement kit and its measuring method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110554179A (en) * | 2019-09-10 | 2019-12-10 | 四川新健康成生物股份有限公司 | Anti-heparin interference CKMB determination kit |
| CN111334557A (en) * | 2020-03-20 | 2020-06-26 | 中拓生物有限公司 | Stable serum sialic acid determination kit with strong anti-interference capability and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63160600A (en) * | 1986-12-24 | 1988-07-04 | Fujirebio Inc | Reagent for determining pyruvic acid |
| CN101469344A (en) * | 2007-12-29 | 2009-07-01 | 上海铂锦诊断用品有限公司 | Double reagent for detecting sialic acid liquid by stable enzyme method and use thereof |
| CN102072953A (en) * | 2010-12-30 | 2011-05-25 | 北京九强生物技术有限公司 | Method and kit for stably detecting sialic acid by enzyme method |
| CN102399851A (en) * | 2011-10-25 | 2012-04-04 | 宁波美康生物科技有限公司 | Sialidase detection kit |
| CN106092942A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | Sialic test kit of a kind of mensuration and preparation method thereof |
| CN106324234A (en) * | 2016-08-08 | 2017-01-11 | 上海睿康生物科技有限公司 | Modified N-acetylneuraminic acid aldolase and preparation method and application thereof |
-
2017
- 2017-05-11 CN CN201710330265.1A patent/CN108866153A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63160600A (en) * | 1986-12-24 | 1988-07-04 | Fujirebio Inc | Reagent for determining pyruvic acid |
| CN101469344A (en) * | 2007-12-29 | 2009-07-01 | 上海铂锦诊断用品有限公司 | Double reagent for detecting sialic acid liquid by stable enzyme method and use thereof |
| CN102072953A (en) * | 2010-12-30 | 2011-05-25 | 北京九强生物技术有限公司 | Method and kit for stably detecting sialic acid by enzyme method |
| CN102399851A (en) * | 2011-10-25 | 2012-04-04 | 宁波美康生物科技有限公司 | Sialidase detection kit |
| CN106092942A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | Sialic test kit of a kind of mensuration and preparation method thereof |
| CN106324234A (en) * | 2016-08-08 | 2017-01-11 | 上海睿康生物科技有限公司 | Modified N-acetylneuraminic acid aldolase and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| GOKMEN, SS 等: "Serum total and lipid-bound sialic acid levels following acute myocardial infarction", 《CLINICAL CHEMISTRY AND LABORATORY MEDICINE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110554179A (en) * | 2019-09-10 | 2019-12-10 | 四川新健康成生物股份有限公司 | Anti-heparin interference CKMB determination kit |
| CN110554179B (en) * | 2019-09-10 | 2022-06-28 | 四川新健康成生物股份有限公司 | Anti-heparin interference CKMB determination kit |
| CN111334557A (en) * | 2020-03-20 | 2020-06-26 | 中拓生物有限公司 | Stable serum sialic acid determination kit with strong anti-interference capability and preparation method and application thereof |
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