CN105241873A - Lipase detection kit - Google Patents

Abstract

The present invention discloses a lipase detection kit, and belongs to the technical field of clinical in vitro detection reagents. The kit comprises a reagent R1, a reagent R2 and a calibration product. According to the present invention, the composite stabilizer comprising 4-FPBA, glucomannan, NaCl, propylene glycol, Triton 100 and BSA R2 is added to the reagent R2, such that the stability of the kit is effectively improved, the linear range is good, the reagent accuracy is high, and the further promotion application in the market is easily achieved.

Description

A kind of lipase detection kit

Technical field

The present invention relates to clinical vitro detection reagent technique field, particularly a kind of lipase detection kit.

Background technology

Lipase (Lipase, GEH, LPS) is under the jurisdiction of carboxylic ester hydrolase class, is a kind of enzyme of hydrolysis of long chain fatty acid glyceride, and progressively triglyceride hydrolysis can be become glycerine and fatty acid, be one of digestive ferment of pancreatic secretion.Except serum, also lipase active can be measured at places such as stomach, mucous membrane of small intestine, lung, leucocyte, adipocyte, milk.In normal blood, only have a small amount of lipase, in blood, lipase is easily removed by kidney, when pancreatic secretion is hyperfunction, ductus pancreaticus be obstructed pancreas damaged or downright bad time, lipase adverse current or directly access blood, makes lipase activity in blood increase.Under normal circumstances, in pancreas, LPS concentration is 20000 times of serum, be liver, duodenal about 100 times.When there is acute parotidits, LPS secretes increase, the LPS of high concentration is entered in blood also increase in regularity, show as: blood LPS 4 ~ 8h after acute parotidits morbidity starts to increase, 24h reaches peak value, recover normal after continuing 8 ~ 14d, sensitivity: 80% ~ 100%, specificity: 84% ~ 96%.Serum LPS expression becomes remarkable positive correlation with acute parotidits disease progression degree, is the main monitoring index of the acute parotidits state of an illness.

At present, the LPS assay method that laboratory is conventional has titrimetry, pH-Stat method, turbidimetry and fluorometry.Titrimetry is classical way, but the method poor sensitivity, need use a large amount of serum, the reaction time is long, and the quality of olive oil emulsion quality also directly affects the accuracy of lipase activity measurement result in addition, now few use; PH-Stat method, because of its effort, time-consuming, is difficult to robotization and needs special instrument and not being widely adopted; Nephelometry measures in the result of sample about has the absorbance of 3%-5% to occur negative test because molecule assembles; Fluorometry is a kind of convenient, special and sensitive lipase activity determination method, but the apparatus expensive needed, and is difficult to adapt to the application of routine clinical chemical laboratory.

Lipase detection kit is based on enzyme development process, adopt 1, o-two bay ancestor glycerine-3-glutaric acids-(the 6'-methyl resorufin)-ester of 2-is reacted as substrate and lipase and is produced methyl resorufin, measures lipase active at 570nm wavelength place according to the methyl resorufin generating rate of product.The method is a kind of without the need to pre-service sample, and technology and equipment is less demanding, and precision and the higher analytical approach of specificity.Because the method does not need expensive equipment, can robotization be realized, and a large amount of sample can be measured, therefore be subject to clinical extensive popularization.But owing to there is enzyme in common lipase enzyme development process detection reagent, the stability of this reagent can be made to be affected, to be unfavorable for the long-term preservation of reagent, thus to cause the adverse consequences of poor accuracy and waste.

Summary of the invention

For problems of the prior art, the invention provides a kind of lipase detection kit.This kit is compared with the kit of routine, and good stability, accuracy is high, and the range of linearity is good, and sensitivity for analysis is high, is conducive to reagent applying clinically.

The present invention is achieved by the following measures:

A kind of lipase enzyme detection kit, it is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Tauroursodeoxycholic acid 35mmol/L

Sodium chloride 40mmol/L

Reagent R2 consists of:

PH4.0 tartaric acid buffer 9.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-glutaric acid-(6 '-methyl resorufin)-ester 0.2mmol/L

4-formylphenyl boronic acid 0.01%

Glucomannan 0.5-1mg/mL

NaCl5mmol/L

Propylene glycol 20mg/mL

Triton X-100 1%

BSA0.1-1g/L。

Described reagent R1 and reagent R2 ratio are in use R1:R2=2:1.

Kit of the present invention carries out on the automatic biochemistry analyzer with double reagent function, its concrete using method as shown in Figure 1:

Calibration object used in the present invention is the compound calibration object that Landau company of Britain produces.

Add physiological saline, sample or calibration object 3 μ l, after the reagent R2 adding 100 μ l again reacts 5min, read twice absorbance, and calculate Δ A/min after adding R1 reagent 200 μ l preincubate 5min afterwards again.

Beneficial effect of the present invention:

Lipase enzyme development process detection kit provided by the invention, by adding by 4-formylphenyl boronic acid (4-FPBA) in reagent R2, glucomannan, NaCl, propylene glycol, Triton X-100, the compound stabilizer of BSA composition, each component synergy makes stable reagent performance excellent, solve enzyme and preserve this difficult problem unstable for a long time, it can increase the stabilization time of enzyme in test, and the activity of enzyme can not be affected, thus effectively enhance the stability of kit, but can not have an impact to the accuracy of reagent and sensitivity for analysis, be conducive to this reagent further to promote in the market.

Accompanying drawing explanation

The concrete operations schematic diagram of Fig. 1 reagent of the present invention on the automatic biochemistry analyzer with double reagent function;

Fig. 2 embodiment 2 accuracy validation laboratory test results and control group testing result correlativity;

Fig. 3 embodiment 3 accuracy validation laboratory test results and control group testing result correlativity;

Fig. 4 embodiment 4 accuracy validation laboratory test results and control group testing result correlativity.

Embodiment

For a better understanding of the present invention, further describe below in conjunction with specific embodiment.

Embodiment 1

Market obtains the lipase kit of a kind of accuracy excellence of accreditation, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Sodium chloride 40mmol/L

Tauroursodeoxycholic acid 35mmol/L

Reagent R2 consists of:

Tartaric acid buffer pH4.09.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-glutaric acid-(6 '-methyl resorufin)-ester 0.2mmol/L

The kit that the present embodiment describes, in use, its assay method adopts Toshiba 120 automatic analyzer with double reagent function, operates as follows:

Add physiological saline, sample or calibration object 3 μ l, after the reagent R2 adding 100 μ l again reacts 5min, read twice absorbance, and calculate Δ A/min after adding R1 reagent 200 μ l preincubate 5min afterwards again.

The compound calibration object that the calibration object that the present embodiment uses is produced for Landau company of Britain.

Embodiment 2

A kind of lipase detection kit, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Sodium chloride 40mmol/L

Tauroursodeoxycholic acid 35mmol/L

Reagent R2 consists of:

Tartaric acid buffer pH4.09.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-penta 2

Acid-(6 '-methyl resorufin)-ester 0.2mmol/L

4-FPBA0.01%

Glucomannan 0.5mg/mL

NaCl5mmol/L

Propylene glycol 20mg/mL

Triton X-100 1%

BSA0.1g/L

Concrete assay method is with embodiment 1.

Embodiment 3

A kind of lipase detection kit, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Sodium chloride 40mmol/L

Tauroursodeoxycholic acid 35mmol/L

Reagent R2 consists of:

Tartaric acid buffer pH4.09.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-penta 2

Acid-(6 '-methyl resorufin)-ester 0.2mmol/L

4-FPBA0.01%

Glucomannan 0.8mg/mL

NaCl5mmol/L

Propylene glycol 20mg/mL

Triton X-100 1%

BSA0.5g/L

Concrete assay method is with embodiment 1.

Embodiment 4

A kind of lipase detection kit, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Sodium chloride 40mmol/L

Tauroursodeoxycholic acid 35mmol/L

Reagent R2 consists of:

Tartaric acid buffer pH4.09.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-penta 2

Acid-(6 '-methyl resorufin)-ester 0.2mmol/L

4-FPBA0.01%

Glucomannan 1mg/mL

NaCl5mmol/L

Propylene glycol 20mg/mL

Triton X-100 1%

BSA1g/L

Concrete assay method is with embodiment 1.

Experimental verification is carried out to kit assay performance obtained in above-described embodiment 1-4.

accuracy validation is tested:

Using the kit of embodiment 2,3,4 as experimental group, the lipase kit in embodiment 1, market obtaining the accuracy excellence of accreditation carries out contrast experiment as a control group, detects 40 samples, and the result of detection is as Fig. 1-Fig. 3.

Known by the detection data of Fig. 1-Fig. 3, the testing result linearly dependent coefficient r of embodiment 2,3,4 detection kit and control test kit is respectively 0.9971,0.9985,0.9981, correlativity is relatively good, show that kit of the present invention and market obtaining the lipase detection kit with excellent accuracy approved has high consistency, prove that other various compositions that kit of the present invention adds can not impact its accuracy, kit still keeps good accuracy.

linear dependence confirmatory experiment:

Lipase high level sample is found to be 700U/L, serial dilution is carried out with physiological saline, the sample of preparation 6 variable concentrations, be followed successively by the sample of 700U/L, 560U/L, 420U/L, 280U/L, 140U/L, 0U/L concentration, each concentration level various kinds originally measures three times respectively, gets its mean value respectively.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result is as shown in table 1.

Table 1 embodiment 1-4 linear correlation confirmatory experiment testing result

Theoretical concentration (U/L)	Embodiment 1 testing result (U/L)	Embodiment 2 testing result (U/L)	Embodiment 3 testing result (U/L)	Embodiment 4 testing result (U/L)
					0	3	2	4	4
140	140	140	139	146
					280	287	274	280	283
420	425	438	407	398
					560	531	578	571	536
700	722	698	687	693
					Correlation coefficient r	0.9980	0.9993	0.9994	0.9992

Above-mentioned testing result display, embodiment 1-4 testing result correlativity is all greater than 0.990, but the testing result of embodiment 2,3,4 is greater than 0.999, has better linear dependence compared with embodiment 1, and this illustrates that reagent of the present invention has better linear dependence.

stability confirmatory experiment:

2 DEG C ~ 8 DEG C, store reagents in the light protected environment of non-corrosiveness gas, detect the stability of four kinds of embodiment reagent.Four kinds of reagent are monthly chosen same sample and are measured its absorbance three times, average, and contrast, thus determine the stabilization time of reagent with fresh embodiment 1 reagent testing result.Detect data as table 2.

Table 2 stability confirmatory experiment testing result

Time	Embodiment 1 reagent testing result	Embodiment 2 reagent testing result	Embodiment 3 reagent testing result	Embodiment 4 reagent testing result	Fresh embodiment 1 reagent testing result
						12 months	0.01657	0.01606	0.01687	0.01698	0.01679
13 months	0.01568	0.01587	0.01646	0.01632	0.01682
						14 months	0.01656	0.01668	0.01654	0.01674	0.01664
15 months	0.01326	0.01399	0.01387	0.01355	0.01406
						16 months	0.00975	0.01966	0.01967	0.01008	0.01987
17 months	0.00221	0.01734	0.01686	0.01691	0.01729
						18 months	0.00063	0.01826	0.01757	0.01811	0.01843
19 months	0.00002	0.01453	0.01432	0.01463	0.01457
						20 months	0.00001	0.01685	0.01596	0.01623	0.01635
21 months	0.00000	0.01374	0.01421	0.01417	0.01461
						22 months	0.00000	0.01284	0.01314	0.01321	0.01358
23 months	0.00000	0.01263	0.01198	0.01167	0.01265
						24 months	0.00000	0.01198	0.01213	0.01191	0.01235
25 months	0.00000	0.00857	0.00843	0.00830	0.01232
						26 months	0.00000	0.00065	0.00082	0.00097	0.01328

Experimental result shows, embodiment 1 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 15 months, stablize, and embodiment 2,3,4 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 24 months, stablize, the stability that effectively can improve lipase detection kit in reagent by adding compound stabilizer is described.

Comprehensive above analysis, lipase detection kit provided by the invention, effectively can improve the stability of kit in reagent R2 by adding compound stabilizer, the range of linearity is better, and the accuracy of reagent is also better.Therefore, lipase detection kit provided by the invention is conducive to further promoting the use of in the market.

Claims (2)

1. a lipase detection kit, is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of:

PH7.2Tris damping fluid 100mmol/L

Tauroursodeoxycholic acid 35mmol/L

Sodium chloride 40mmol/L

Reagent R2 consists of:

PH4.0 tartaric acid buffer 9.5mmol/L

Colipase 500U/L

1,2 o-two bay ancestor glycerine-3-glutaric acid-(6 '-methyl resorufin)-ester 0.2mmol/L

4-formylphenyl boronic acid 0.01%

Glucomannan 0.5-1mg/mL

NaCl5mmol/L

Propylene glycol 20mg/mL

Triton X-100 1%

BSA0.1-1g/L。

2. kit according to claim 1, is characterized in that, described reagent R1 and reagent R2 ratio are in use R1:R2=2:1.