CN105137098B

CN105137098B - Determination reagent of cholesterols in serum high density lipoprotein

Info

Publication number: CN105137098B
Application number: CN201510518624.7A
Authority: CN
Inventors: 巩晓东; 李宏奎; 王珍娥
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-08-24
Filing date: 2015-08-24
Publication date: 2017-01-25
Anticipated expiration: 2035-08-24
Also published as: CN105137098A

Abstract

The invention discloses a determination reagent of cholesterols in serum high density lipoprotein. The reagent comprises a proper amount of an antiseptic, a stabilizer, a toner, a high affinity enzyme compound, a surfactant and bovine serum albumin, and the high affinity enzyme compound is prepared through the following steps: 1, carrying out enzyme activation: selecting an enzyme, pre-culturing the enzyme, carrying out activation culture on the enzyme, and carrying out a dehydration reaction; and 2, preparing the high affinity enzyme compound: selecting the compound, and carrying out a hybrid reaction. The reagent can be used to detect the cholesterols on a fully-automatic biochemical analyzer with various models without sample centrifuge, precipitation or other complicated operations, so the detection workload is greatly reduced, the detection time is shortened, and the detection cost is reduced; and the reagent has the advantages of good specificity, high sensitivity and high detection accuracy, and compared with a detection result of a nationally recommended Friedewald formula, the result of the reagent shows that the correlation reaches 0.997 or above.

Description

The mensure reagent of cholesterol in serum high-density LP

Technical field

The invention belongs in medical science and technological field of biochemistry, more particularly, to serum high-density LP cholesterol survey Determine reagent.

Background technology

Serum cholesterol concentration can be used as the mark of human body lipid metabolism.HDL-C (hdl) is volume Minimum lipoprotein, compares with other lipoprotein, and hdl contains protein content maximum (> 50%), its main apolipoprotein is apoa- , a- and a small amount of apo-c, e；Phosphatide is its main lipid, also a small amount of cholesterol, cholesteryl ester and triglycerides In the presence of LCA (lcat), CHF becomes cholesteryl ester, will be accumulated in through hdl The CHF of tissue is transported to liver, reduces the concentration of CHF in blood plasma hdl, forms cholesterol and flows to from cell membrane The concentration gradient of plasma lipoprotein, reduces the deposition of tissue cholesterol, thus limiting atherosclerotic generation, development, rises To study of anti-atherogenic effect.So, in blood plasma hdl with atherosclerotic become negatively correlated, clinical medicine can use Hdl-c suffers from the danger of coronary heart disease (chd) to evaluate.

There are supercentrifugation, electrophoresis and the precipitation method to traditional assay method of HDL-C, hypervelocity Centrifugal process is different according to the proportion of lipoprotein, with ultracentrifugation device, lipoprotein is classified, then cholesterol is measured, The method passes through ultracentrifugation and two processes of cholesterol detection, wastes time and energy, and costly；Electrophoresis needs to borrow As Ago-Gel etc. separates as carrier, operate and its numerous and diverse, and the degree of accuracy is low；The precipitation method be using polyanion and The oarse-grained lipoprotein of bivalent metal ion agent precipitate, is then centrifuged for, then detects its content with its supernatant, although the method Simplicity, but it is only limitted to indivedual or a few sample detection, and when primary hypertriglyceridemiapatients patients are detected, result can be caused higher Error, affect final judged result.

In recent years, deeply develop with clinical medical, HDL-C detection (hdl-c) has been achieved with entirely Automation, and hdl-c reagent is the key realizing fully-automated synthesis, its quality height directly affects the accurate of detection data Property.At present, the report about hdl-c reagent is more, and such as patent cn 1632541 a discloses cholesterol in HDL Mensure reagent and preparation method, disclose by " live in appropriate preservative, stabilizer, chromogen, high-affinity enzyme compound and surface Property agent " the hdl-c reagent made, this reagent achieves cholesterol fully-automated synthesis in HDL, solves tradition inspection The deficiency that survey method exists, and improve accuracy in detection, correlation reaches more than 0.98；Cn 1696659 a discloses one Kind measure LDL-C reagent and preparation method, disclose by " appropriate amount of buffer solution, stabilizer, chromogen, high affine The reagent that property enzyme compound and surfactant " is made, this reagent achieves cholesterol in low-density lipoproteins fully-automated synthesis, Solve the deficiency of traditional detection method presence, and improve accuracy in detection, correlation reaches more than 0.98.Above-mentioned two File all discloses the technical scheme being made detection material by high-affinity enzyme compound, but the high-affinity enzyme in this two files Compounds process for production thereof is simple, does not carry out activation process to enzyme during preparation, there is enzyme and the compound reaction time is long, and react Insufficient, and then have impact on overall performance and the yield of enzyme compound.Additionally, the mensure about cholesterol in HDL The report of reagent all adopts HMW chemical composition to prepare, and compound preparation price is high and purity is low, and then affects detection knot Really.A kind of even phase method in-vitro diagnosis of serum LDL cholesterol (ldl-c) as disclosed in patent cn 102041296 a Reagent；Assay method of HDL-C etc. disclosed in cn 101078729 a.

Content of the invention

It is an object of the invention to: provide a kind of mensure reagent of cholesterol in serum high-density LP, by enzyme The steps such as selection, preculture, activation culture and dehydration improve the activity of enzyme, then mix with the compound processing through catalytic activation Close reaction, improve and measure reagent sensitivity, and then improve testing result accuracy.

To achieve these goals, the present invention adopts the following technical scheme that

The mensure reagent of cholesterol in serum high-density LP, including appropriate preservative, stabilizer, chromogen, height is affine Property enzyme compound, surfactant, bovine serum albumin(BSA) it is characterised in that: described high-affinity enzyme compound be according to Lower section method prepares:

1) activation of enzyme:

1. the selection of enzyme: cholesterol esterase, cholesterol oxidase and lipoprotein lipase, three according to 5～1:1～5:1～ 1.5 ratio mixing；

2. the preculture of enzyme: above-mentioned mixed enzyme is placed in 32～39 DEG C of temperature, trains in advance in the cushioning liquid of ph value 5.5～8 Support 3～8 hours, wherein, described cushioning liquid is PBS or citric acid solution；

3. the activation culture of enzyme: proceed to by bovine serum albumin(BSA), glycerine, beta-schardinger dextrin, edta- through pre-incubated mixed enzyme In na solution, penicillin and aseptic water mixed liquid, under the conditions of 34～39 DEG C of temperature, activation culture 5～10 hours, is activated Enzyme cocktail buffer a；

4. dehydration: Filter column is prepared using water absorbing agent, above-mentioned activating enzymes cocktail buffer a is slowly flowed through filtration Post, removes the free water adding in activating enzymes cocktail buffer, and adjusts buffer solution ph value at any time so as to maintain 5.5～8；

2) prepare high-affinity enzyme compound

1. compound selects: one of steroid glycoside, triterpene glucoside, archon and polyenes or many Plant combination of compounds combination, and compound carries out catalytic activation using known approaches, obtains activating compounds composition b；

2. hybrid reaction: by step 1) activating enzymes cocktail buffer a that obtains and step 2) the activating compounds group that obtains Compound b mixes, and the appropriate cushioning liquid of supplement, and under the conditions of 32～39 DEG C, hybrid reaction 3～10 hours, obtain high-affinity Enzyme compound.

Further, in described serum high-density LP cholesterol measure reagent by following percentage component system Become: high-affinity enzyme compound 68～73%, bovine serum albumin(BSA) 23～30%, surfactant 2.4～2.7%, preservative 1.3～1.5%, stabilizer 1.1～1.5%, chromogen 0.2～0.3%, combination of the above total amount is 100%.

Further, described activating enzymes cocktail buffer a with activating compounds composition b mixed proportion is: 1～2:3～ 10.

Further, in the step 3. activation culture of enzyme, described mixed liquor component proportion is: bovine serum albumin(BSA) 50～ 60%th, glycerine 5～8%, beta-schardinger dextrin 1～3%, edta-na solution 1～1.5%, penicillin 0.5～0.8%, sterilized water 34 ～37%.

Compared with the mensure reagent of cholesterol in existing serum high-density LP, the beneficial effects of the present invention is:

1st, this reagent can on the automatic clinical chemistry analyzer of various models to HDL in cholesterol carry out Detection, without numerous and diverse operations such as sample centrifugation, precipitations, significantly reduces detection workload, shortens detection time, reduce inspection Survey cost.

2nd, this reagent properties is stable, stablizes more than 12 months under the conditions of 2～8 DEG C, is not in that reagent is muddy, corruption of going bad Go bad and coacervation, only need gently to vibrate during use to shake up.

3rd, this reagent specificity is good, sensitivity is high, and Detection accuracy is high, the friedewald formula detection recommended with country Results contrast, correlation reaches more than 0.997.

Specific embodiment

With reference to specific embodiment, technical scheme is described further it is intended to help same domain technology people Member understands.

Embodiment 1

The mensure reagent of cholesterol in serum high-density LP, is made up of the component of following percentage: high-affinity enzyme Compound 68～73%, bovine serum albumin(BSA) 23～30%, surfactant 2.4～2.7%, preservative 1.3～1.5%, steady Determine agent 1.1～1.5%, chromogen 0.2～0.3%, combination of the above total amount is 100%.

Described surfactant be nonionic surfactant, can be tween-80, trition x-100, One or more of thesit.

Described stabilizer is edta-na, the composition of beta-schardinger dextrin, and the two according to mol ratio is: 0～5:5～0, should Stabilizer is the supplement to bovine serum albumin(BSA) stabilizer, is in order to strengthen bovine serum albumin(BSA) and to add.

Described chromogen is the bromo- 3- hydroxybenzoic acid (tbhba) of 2,4,6- tri-.

Described high-affinity enzyme compound is to prepare in accordance with the following methods:

1) activation of enzyme:

3. the activation culture of enzyme: proceed to by bovine serum albumin(BSA) 55%, glycerine 6.5%, β-ring through pre-incubated mixed enzyme In the mixed liquor that dextrin 2%, edta-na solution 1.2%, penicillin 0.6%, sterilized water 34.7% are configured to, temperature 34～ Activation culture 5～10 hours under the conditions of 39 DEG C, obtain activating enzymes cocktail buffer a；

2) prepare high-affinity enzyme compound

1. compound selects:, it is catalyzed with known succimide method in aqueous medium taking tea saponin as a example Activation process, concrete activation method is as follows:

A, tea saponin is dissolved in absolute ethyl alcohol (analysis pure), obtains the tea saponin-ethanol solution of 20～35mg/ml；

B, by tea saponin-ethanol solution and activator according to the ratio mixing for 2:8 for the mol ratio after, in temperature 18～24 DEG C, under ph value 5～8.5, and nitrogen protection, continuously stirred reaction 3～8 hours, obtain activating compounds composition b；

2. hybrid reaction: by step 1) activating enzymes cocktail buffer a that obtains and step 2) the activating compounds group that obtains Compound b is that 1～2:3～10 mix according to mol ratio, and the appropriate cushioning liquid of supplement, hybrid reaction under the conditions of 32～39 DEG C 3～10 hours, obtain high-affinity enzyme compound.

The mensure reagent of cholesterol in the serum high-density LP of the present invention, on the automatic clinical chemistry analyzer in test Major parameter such as table 1 below:

Table 1- automatic clinical chemistry analyzer parameter

test	hdl-c
		assay code	2point
assay point	15～34
		wave(sub/main)	700nm/546nm
sample volume	3ul
		r1volume	225ul
r2volume	75ul
		calib.type	linear
calib/span.point	2/2

In order to verify the actual effect of reagent of the present invention, it is contrasted with Precipitation Determination value, its result see table 2.

And correlation is calculated according to table 2 determination data, by table 2 (measured values of reagent measured value of the present invention and the precipitation method) Carry out correlation analysis, result display correlation r reaches 0.997, illustrates that correlation is fabulous.

Table 2- reagent of the present invention measured value and the measured value of the precipitation method

Thus, in the serum high-density LP of the present invention mensure reagent of cholesterol compared to existing technology, its sensitivity Higher, test accuracy is higher, has significant progressive.

Claims

1. in serum high-density LP cholesterol mensure reagent, including appropriate preservative, stabilizer, chromogen, high-affinity Enzyme compound, surfactant, bovine serum albumin(BSA) it is characterised in that: described high-affinity enzyme compound is according to following Method prepares:

1) activation of enzyme:

1. the selection of enzyme: cholesterol esterase, cholesterol oxidase and lipoprotein lipase, three is according to 5～1:1～5:1～1.5 Ratio mixes；

2. the preculture of enzyme: by above-mentioned mixed enzyme be placed in 32～39 DEG C of temperature, preculture 3 in the cushioning liquid of ph value 5.5～8～ 8 hours, wherein, described cushioning liquid was PBS or citric acid solution；

3. the activation culture of enzyme: proceed to molten by bovine serum albumin(BSA), glycerine, beta-schardinger dextrin, edta-na through pre-incubated mixed enzyme In liquid, penicillin and aseptic water mixed liquid, activation culture 5～10 hours under the conditions of 34～39 DEG C of temperature, obtain activating enzymes and mix Close buffer solution a, wherein, described mixed liquor component proportion is: bovine serum albumin(BSA) 50～60%, glycerine 5～8%, β-ring paste Essence 1～3%, edta-na solution 1～1.5%, penicillin 0.5～0.8%, sterilized water 34～37%；

4. dehydration: Filter column is prepared using water absorbing agent, above-mentioned activating enzymes cocktail buffer a is slowly flowed through Filter column, removes The free water adding in enzyme cocktail buffer of deactivating, and adjust buffer solution ph value at any time so as to maintain 5.5～8；

2) prepare high-affinity enzyme compound

1. compound selects: one or more of steroid glycoside, triterpene glucoside, archon and polyenes group Polymerisable compounds combine, and compound carries out catalytic activation using known approaches, obtains activating compounds composition b；

2. hybrid reaction: by step 1) activating enzymes cocktail buffer a that obtains and step 2) the activating compounds composition b that obtains Mixing, and the appropriate cushioning liquid of supplement, hybrid reaction 3～10 hours under the conditions of 32～39 DEG C, obtain high-affinity enzyme Compound.

2. in serum high-density LP as claimed in claim 1 cholesterol mensure reagent it is characterised in that: this reagent by The component of following percentage is made: high-affinity enzyme compound 68～73%, bovine serum albumin(BSA) 23～30%, surfactant 2.4～2.7%, preservative 1.3～1.5%, stabilizer 1.1～1.5%, chromogen 0.2～0.3%, combination of the above total amount is 100%.

3. in serum high-density LP as claimed in claim 1 cholesterol mensure reagent it is characterised in that: described work Changing enzyme cocktail buffer a with activating compounds composition b mixed proportion is: 1～2:3～10.