CN106404683A - Stable and strong anti-interference phospholipid detection reagent and detection method thereof - Google Patents

Abstract

The present invention relates to the technical field of phospholipid detection, particularly to a phospholipid detection reagent. According to the present invention, a sodium citrate buffer solution and a Bis-Tris Propane (1,3-bis[tris(hydroxymethyl)methylamino]propane) buffer solution are respectively used in reagents R1 and R2, wherein the Bis-Tris Propane buffer solution used in the reagent is a biological buffer solution, provides good protection effects for the peroxidase used in the reagent, and does not adversely affect the reaction system, the reagent R1 contains phospholipase D, DAOS, a Triton X-305 solution, Emulgen 707, a reducing protection agent, a chelating protection agent and a preservative, and the reagent R2 contains a buffer solution, 4-aminoantipyrine, peroxidase, choline oxidase, and a preservative; and the phospholipid detection reagent has advantages of good stability and low cost, and can be widely used in the field of phospholipid detection.

Description

A kind of stable, phospholipid detectable of strong interference immunity and its detection method

Technical field

The present invention relates to phospholipid detection field is and in particular to a kind of detectable detecting phospholipid using enzymatic measurement and detection method.

Background technology

Phospholipid（Phospholipid）, also referred to as phospholipid, phospholipid, refer to the lipid containing phosphoric acid, belong to complex liped.Phospholipid is to form biomembranous main component, is divided into phosphoglyceride and sphingomyelins two big class, is made up of glycerol and sphingol respectively.Phospholipid is amphiphatic molecule, and one end is the head of hydrophilic nitrogenous or phosphorus, and the other end is hydrophobic（Oleophylic）Long hydrocarbyl chain.Due to this reason, phospholipid molecule water-wet side is close to each other, and hydrophobic side is close to each other, and normal and other molecule such as protein, glycolipid, cholesterol collectively forms lipid bilayer, i.e. the structure of cell membrane.

Serum phospholipidses mainly include lecithin, LYSOLECITHIN SUNLECITHIN A, lipid sphyngomyelin and cephalin etc. four part.In clinical position, the general serum total phospholipidses that measure have chemical method and enzymatic assays two class method, such as need to check that the composition of serum phospholipidses then needs by the items technology such as thin layer chromatography, gas chromatography or high performance liquid chroma- tography further.Raise reason：Obstructive jaundice, primary biliary cirrhosiss, primary sclerosing cholangitiss, Ziere syndrome, glycogen storage disease, obesity, diabetes, acute and chronic pancreatitis, nephrotic syndrome, Hypothyroidism, fatty liver, lipodystrophy, gestation, oral contraceptive etc..Reduce reason：Hepatitis gravis, decompensated cirrhosiss, Tangier (Tangier) disease, hyperthyroidism, malabsorption syndrome, myeloproliferative diseases, multiple myeloma, Wolman disease, Leye syndrome, multiple sclerosis etc..

The method of detection phospholipid is more at present, predominantly detects the methods such as method chromatographic method, Mass Spectrometry detection method, chemical method, enzyme process.Wherein chromatograph and Mass Spectrometry detection method are the most accurate, but both approaches are higher to instrument and operator's requirement, and use cost is too expensive, is not suitable for large-area popularization.Chemical cost method is relatively low, but this method accuracy is not high, is easily interfered.Enzyme process is then relatively conventional on the market, this method is mainly carried out by means of TRINDER reaction, generate H2O2 using phospholipase and Choline dehydrogenase, carry out dye-forming reaction by chromogen, this method application is wider, low price, easy to operate, the supporting the use of suitable automatic clinical chemistry analyzer device, but this law is more because being related to enzyme, and be related to TRINDER reaction capacity of resisting disturbance inherently strong the problems such as, the problems such as existing phospholipid detectable mostly is unstable and poor anti jamming capability on the market, affects and normally use.The present invention is to solve these problems, and a kind of strong interference immunity, the enzymatic measurement phospholipid detectable of good stability and its using method have been invented in research.

Content of the invention

Problem to be solved by this invention is the detectable providing a kind of strong utilization enzymatic measurement of stability to detect serum phospholipidses.The detectable simultaneously additionally providing a kind of utilization present invention detects the detection method of serum phospholipidses.

Phospholipid hydrolyzes generation choline and phosphatidic acid in the presence of Choline phosphatase, and choline is aoxidized by Choline dehydrogenase and generates hydrogen peroxide, and hydrogen peroxide and 4-AA, DAOS react generation blue dyess.This dyestuff has maximum absorption band in 600nm, and absorption intensity is directly proportional to content of phospholipid in serum.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method, including reagent R1 and reagent R2.It is characterized in that：

Described reagent R1 comprises, and biological buffer, the wherein solute concentration in reagent R1 is 100-150mmol/L：

Choline phosphatase 0.5KU/L-2KU/L

DAOS 1.2mmol/L-3.2 mmol/L

Triton X-305 solution 1ml/L-3ml/L

Emulgen 707 0.5ml/L-2ml/L

Reduction protectants 0.5mmol/L-2mmol/L

Chelating protective agent 1mmol/L-5mmol/L

Preservative 1g/L-5g/L

Described reagent R2 comprises, and buffer, the wherein solute concentration in reagent R2 is 100-200mmol/L：

4- amino antipyrine 0.75mmol/L-1.5 mmol/L

Peroxidase 10KU/L-15 KU/L

Choline dehydrogenase 8KU/L-12KU/l

Preservative 1g/L-5g/L.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that also include in described reagent R1：Ascorbic acid oxidase 1-5KU/L.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that also include in described reagent R2：FAD2-5 mg/L and sucrose 5g/L-10g/L.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method it is characterised in that in described reagent R1 buffer be citrate buffer solution, concentration in reagent R1 for its solute is 100mmol/L, and when 25 DEG C, the pH value of buffer is 6.0.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method, it is characterized in that, in described reagent R2, buffer is Bis-Tris Propane biological buffer, and concentration in reagent R1 for its solute is 100mmol/L, when 25 DEG C, the PH of buffer is 7.1.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that described preservative is natamycin.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that chelating protective agent in described R1 reagent is HEDTA (hydroxyethylethylene diamine tri-acetic acid).

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method it is characterised in that in described R1 reagent reduction protectants be DTT (dithiothreitol, DTT).

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that the volume ratio of described reagent R1 and reagent R2 is 4:1.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that comprise the steps of：

（1）Measure reagent R1 and reagent R2；

（2）Test serum is mixed with reagent R1, records the absorbance A 1 that wavelength is during 600nm；

（3）Reagent R2 is added step（2）Mixed solution in, after 3-8min, record wavelength be 600nm when absorbance A 2.

A kind of stable, phospholipid detectable of strong interference immunity of the present invention and its detection method are it is characterised in that step（3）In response time be 5min.The preparation method of reagent R1 and reagent R2 is to measure each raw material, be added in container, mix homogeneously.

The present invention employs sodium citrate buffer solution and Bis-Tris in reagent R1 and R2 respectively Propane（1,3- bis- [three (methylol) methylamino] propane）Buffer.Bis-Tris Propane used in reagent（1,3- bis- [three (methylol) methylamino] propane）Buffer is biological buffer, has preferable protective effect to the peroxidase used in reagent, and reaction system will not be had a negative impact.707 two kinds of surfactants of Triton X-305 solution and Emulgen are added in reagent R1; both surfactants can have the preferable emulsification improving reagent; and important toolenzyme this to Choline phosphatase has very strong protective effect, has preferable potentiation simultaneously.In order to strengthen the stability of key enzyme Choline dehydrogenase in R2 reagent, spy adds sucrose and enzymatic protective reagent FAD in R2 reagent, substantially increases the stability of enzyme.

The present invention also adds ascorbic acid oxidase and chelating agen HEDTA (hydroxyethylethylene diamine tri-acetic acid) in reagent; the interference of ascorbic acid and heavy metal ion can effectively be removed; and appropriate chelating agen HEDTA (hydroxyethylethylene diamine tri-acetic acid) protects toolenzyme Choline phosphatase, DTT with specificity（Dithiothreitol, DTT）Addition then can prevent the oxidation to enzyme for the air.

The detectable of this phospholipid has the advantages that good stability, cheap, can extensively be promoted in phospholipid detection field.

The detection method of this phospholipid, its principle is, the difference of A2 and A1, and the ratio of the difference of the numerical value being recorded using same method with titer is multiplied by the concentration of the PLIP of titer it is simply that the concentration of test serum, has the advantages that speed is fast, stability is strong.

Brief description：

Fig. 1 is the stability trendgram of formula 1, formula 2 and formula 3.

Specific embodiment：

With reference to specific embodiment, the present invention is further described.

（1）Formula 1（A kind of phospholipid detectable of existing enzymatic measurement）：

The component of R1：

Citrate buffer solution（PH7.2） 100mmol/L

Choline phosphatase 0.46U/mL

DAOS 1.2mmol/L

Ascorbinase 0.6U/mL

The component of R2：

Citrate buffer solution（PH7.2） 100mmol/L

4- amino antipyrine 0.75mmol/L

Peroxidase 17U/mL

Choline dehydrogenase 7.2U/mL.

（2）Formula 2（A kind of stable enzymatic measurement phospholipid detectable of optimization）：

The component of R1：

Citrate buffer solution 100mmol/L

Choline phosphatase 0.5KU/L

DAOS 1.2mmol/L

Triton X-305 solution 1ml/L

Emulgen 707 0.5ml/L

DTT（Dithiothreitol, DTT） 0.5mmol/L

HEDTA (hydroxyethylethylene diamine tri-acetic acid) 1mmol/L

Ascorbic acid oxidase 1KU/L

Preservative（Natamycin） 1g/L

The component of R2：

Bis-Tris Propane biological buffer 200mmol/L

4- amino antipyrine 0.75mmol/L

Peroxidase 10KU/L

Choline dehydrogenase 8KU/L

FAD 2mg/L

Sucrose 5g/L

Preservative（Natamycin） 1g/L.

（3）Formula 3（A kind of phospholipid detectable of the stable enzymatic measurement after key raw material increase）：

The component of R1：

Citrate buffer solution 200mmol/L

Choline phosphatase 2KU/L

DAOS 3.2mmol/L

Triton X-305 solution 3ml/L

Emulgen 707 2ml/L

DTT（Dithiothreitol, DTT） 2mmol/L

HEDTA (hydroxyethylethylene diamine tri-acetic acid) 5mmol/L

Ascorbic acid oxidase 5KU/L

Preservative（Natamycin） 5g/L

The component of R2：

Bis-Tris Propane biological buffer 200mmol/L

4- amino antipyrine 1.5mmol/L

Peroxidase 15KU/L

Choline dehydrogenase 12KU/L

FAD 5mg/L

Sucrose 10g/L

Preservative（Natamycin） 5g/L.

（4）Detection method：When using using the automatic clinical chemistry analyzer with double reagent function, this detection test adopts Hitachi 7180 fully-automatic analyzer, is measured using end-point method, and detection dominant wavelength is 600nm, is measured using performance rate method.Respectively by R1 and R2 according to 4:1 ratio is placed on corresponding reagent position, places distilled water, standard substance and sample in the correspondence position of specimen disc, operating procedure such as table 1.

Table 1

Computational methods：Content of phospholipid（U/L）=（A measures ÷ A standard）× C standard.

（5）Interference is tested：Take fresh mix serum, classify in three categories part, every part is separated into 5 equal portions, add different interfering materials so as to the concentration in serum reaches the requirement of table 2, then use the reagent of formula 1, formula 2 and formula 3 respectively, measure the content of phospholipid in contrast serum simultaneously.After adding disturbance material, the measurement result of each group is shown in Table 2, mensure average × 100% of relative deviation (%)=(the mensure average of the sample of mensure average-noiseless material of interference sample)/noiseless material.

As can be seen from Table 2, ascorbic acid, bilirubin, hemoglobin and triglyceride do not substantially interfere with to the test result of formula 2 and formula 3, and the impact to formula 1 is larger, illustrates that this detectable has stronger capacity of resisting disturbance.

Table 2

.

（6）The stability test of reagent：

By the reagent in formula 1, formula 2 and formula 3,13 groups of uniform subpackage.Every group of amount of reagent：R1 is 20mL, and R2 is 5mL.It is placed in 2-8 DEG C of refrigerator, a taking-up on the same day group reagent monthly detects phospholipid quality-control product（Target value is 200mg/dL）, as shown in figure 1, formula 2 and formula 3 are more or less the same, reagent is more stable than the phospholipid determination test kit in formula 1 under 2-8 DEG C of condition of storage for testing result.

Claims (10)

1. a kind of detectable of phospholipid, including reagent R1 and reagent R2, the composition of described reagent R1 and R2 is as follows：

1）The group of reagent R1 is divided into：Concentration in reagent R1 for the solute is 100-150mmol/L：

Choline phosphatase 0.5KU/L-2KU/L

DAOS 1.2mmol/L-3.2 mmol/L

Triton X-305 solution 1ml/L-3ml/L

Emulgen 707 0.5ml/L-2ml/L

Reduction protectants 0.5mmol/L-2mmol/L

Chelating protective agent 1mmol/L-5mmol/L

Preservative 1g/L-5g/L

2）The group of reagent R2 is divided into：Concentration in reagent R2 for the solute is 100-200mmol/L

4- amino antipyrine 0.75mmol/L-1.5 mmol/L

Peroxidase 10KU/L-15 KU/L

Choline dehydrogenase 8KU/L-12KU/l

Preservative 1g/L-5g/L.

2. the detectable of phospholipid according to claim 1 is it is characterised in that also include in described reagent R1：Ascorbic acid oxidase 1-5KU/L.

3. the detectable of phospholipid according to claim 1 is it is characterised in that also include in described reagent R2：FAD2-5 mg/L and sucrose 5g/L-10g/L.

4. detectable according to the arbitrary described phospholipid of claim 1-3 it is characterised in that in described reagent R1 buffer be citrate buffer solution, concentration in reagent R1 for its solute is 100mmol/L, and when 25 DEG C, the pH value of buffer is 6.0.

5. phospholipid according to claim 4 detectable it is characterised in that in described reagent R2 buffer be Bis-Tris Propane biological buffer, concentration in reagent R1 for its solute is 100mmol/L, and when 25 DEG C, the PH of buffer is 7.1.

6. the detectable of phospholipid according to claim 5 is it is characterised in that described preservative is natamycin.

7. the detectable of phospholipid according to claim 6 is it is characterised in that chelating protective agent in described R1 reagent is HEDTA (hydroxyethylethylene diamine tri-acetic acid).

8. phospholipid according to claim 7 detectable it is characterised in that in described R1 reagent reduction protectants be DTT (dithiothreitol, DTT).

9. the detectable of phospholipid according to claim 6 is it is characterised in that the volume ratio of described reagent R1 and reagent R2 is 4:1.

10. a kind of detectable of the phospholipid described in claim 7 is used for detecting the detection method of phospholipid it is characterised in that comprising the steps of：

（1）Measure reagent R1 and reagent R2；

（2）Test serum is mixed with reagent R1, records the absorbance A 1 that wavelength is during 600nm；

（3）Reagent R2 is added step（2）Mixed solution in, after 3-8min, record wavelength be 600nm when absorbance A 2.