CN100353166C

CN100353166C - Enzyme tag stabilizing agent

Info

Publication number: CN100353166C
Application number: CNB200410057389XA
Authority: CN
Inventors: 杨晓林; 孙旭东
Original assignee: Yuande Biological Medicine Engineering Co Ltd Beijing
Current assignee: Yuande Biological Medicine Engineering Co Ltd Beijing
Priority date: 2004-08-30
Filing date: 2004-08-30
Publication date: 2007-12-05
Anticipated expiration: 2024-08-30
Also published as: CN1743846A

Abstract

The present invention relates to a novel stabilizing agent of enzyme markers for prolonging the preservation time of the solutions of the enzyme markers, which belongs to immunoassay products capable of using the enzymic markers as tracer agents. The stabilizing agent is prepared from protein, haemoglobin, anti-free radical substances and buffer solutions and can be widely used in a plurality of departments, comprising clinical diagnosis, scientific research, environment and sanitary detection, medicolegal expertise, etc.

Description

Novel enzyme label stabilizing agent

Technical field

The present invention is a kind of can be used for in the immune detection product of enzyme labeling thing as tracer agent, to prolong the novel enzyme label stabilizing agent of enzyme labeling thing solution storage life.

Background technology

The immune detection analytical technology has obtained using widely in current biomedical sector, and detected in clinical diagnosis, scientific research, environment and health, legal medical expert identifies etc. allly becomes indispensable routine techniques means in multidisciplinary, bringing into play irreplaceable effect, therefore relevant testing product also just has huge market.Present detection technique mostly all with the enzyme labeling thing as tracer agent, and wherein common with the connection thing of peroxidase.Because the main biochemical activity of this enzyme is to change the superoxide in the reaction system into superoxide radical, therefore this enzyme labeling thing also can will exist in its solution, perhaps the small amounts material that produces gradually in storing, transporting is converted into superoxide radical, the latter then can attack consumingly enzyme molecule itself and with its bioactive molecule that connects mutually, thereby cause its inactivation.Because this " suicide " phenomenon usually causes the enzyme labeling thing to take the lead in losing efficacy, so the stability of enzyme labeling thing has also just become to improve the bottleneck of such detectable product reliability and the term of validity.Therefore many products are forced to use freeze-drying or concentrate the enzyme labeling thing of refrigeration, but this needs the user by the way of redissolving or diluting preparation work liquid voluntarily, this has not only increased user's workload, and also can cause new operate miss certainly, also be not suitable for the matched reagent of robotization detecting instrument simultaneously.

In recent years, can in enzyme labeling thing solution, consume superoxide radical or the stable thing molecule that connects owing to it is found that some, do not influence simultaneously the material of immune response and enzyme-substrate reactions again, as: some possesses vitamin, some Polyphenols or polyhydroxy class material, some polyamino acid etc. of anti-oxidation characteristics, thereby prolonged the storage life of enzyme labeling thing solution greatly, lay a good foundation for such testing product directly adopts the enzyme labeling thing working fluid of room temperature preservation, these materials are called as label stabilizing agent (Conjugator Stabilizer).In fact, the enzyme labeling thing that contains stabilizing agent working fluid can directly be provided, with the room temperature preservation that realizes product and the high reliability that guarantees this detectable and the long term of validity, the technology content of this series products and the important symbol of manufacturer's research and development ability and level have been become to weigh at present.

Summary of the invention

How to find and use to have on Green Tea Extract or the stable particular matter that connects the thing molecular characterization but the notice of past people mainly concentrates on, and ignored the possibility of eliminating enzyme labeling thing " suicide " phenomenon by other approach.We find that through years of researches and practice two kinds of common reagent are arranged, and promptly albumen (as BSA) and haemoglobin (HB) can delay the deactivation phenomenom of enzyme labeling thing effectively under some particular concentration condition.

Wherein because high concentration BSA can reduce the nonspecific immunity reaction greatly, therefore be present the most frequently used sealer, extensively made an addition in all kinds of immunologic function test reagents, its working concentration is all more than 1%, and in order further to reduce non-specific responding, often use the BSA of higher concentration, for example about 3% to 5%.And we find that BSA has two-phase for the generation and the consumption of free radical, i.e. its attack that both can replace the enzyme labeling thing to bear free radical, thereby as a kind of free radical scavengers for protecting enzyme labeling thing; Can in the process of self degrading, discharge peroxidating material and even free radical again simultaneously, thereby quicken the inactivation of enzyme labeling thing, therefore select suitable BSA concentration extremely important for protective enzyme label activity.We are by a large amount of experiment confirms: BSA concentration is relatively stable on a reduced levels in the active decline rate of 1% enzyme labeling thing when following; And active decline rate increases suddenly when this concentration is above, and as active decline rate doubled by 3% o'clock, active decline rate increased more than ten times by 10% o'clock.Simultaneously, the experiment of using all kinds of other albumen to replace BSA to carry out all demonstrates similar result, illustrates that this effect and albumen kind are irrelevant, and only closely related with its concentration.Take all factors into consideration the requirement that suppresses non-specific responding and protective enzyme label activity, the optium concentration of each albuminoid of experiment confirm in enzyme labeling thing solution is all between 0.1% to 1%.

And, be considered to cause the interfering material of non-special signal simultaneously because HB usually is present among the commercial rough BSA, therefore make every effort to for a long time it is removed from the related immune detectable always.As everyone knows, because HB has the iron porphyrin of containing, therefore have stronger free radical catalytic activity, promptly react the conversion that causes between superoxide radical and the hydroxyl radical free radical, so we think that its tool in the Green Tea Extract process has certain effect by FantonShi reaction and HaberweissShi.Specifically, by above-mentioned reaction the superoxide radical of longer life is converted to more short-life hydroxyl radical free radical exactly, and the latter is consumed very soon by attacking HB self or near molecule, under all lower situation of HB and enzyme labeling substrate concentration, near the probability of the enzyme labeling thing molecule HB molecule is very little, and therefore the possibility of being attacked is also little; Because above-mentioned reaction has reduced the superoxide radical concentration of longer life, attack the possibility of enzyme labeling thing molecule simultaneously thereby further reduced it.We confirm by experiment: the active decline rate of HB concentration enzyme labeling thing when 50 μ g/ml to 200 μ g/ml reduces about 50% than not adding HB, its non-specific responding of while also a little less than; And more than 200 μ g/ml or 50 μ g/ml DeGrain when following, and, HB concentration strengthens rapidly along with increasing non-specific responding.Take all factors into consideration the requirement that suppresses non-specific responding and protective enzyme label activity, we think that the optium concentration of HB in enzyme labeling thing solution should be between 50 μ g/ml to 200 μ g/ml.

In sum, we think that above-mentioned two class materials are under specific concentrations, the label stabilizing effect is obvious for improving, and have the advantages that cost is low, be easy to prepare, can be widely used in in the immune detection product of enzyme labeling thing as tracer agent, thus the storage life that has prolonged enzyme labeling thing solution greatly.Therefore it is all multidisciplinary to be widely used in clinical diagnosis, scientific research, environment and health detection, legal medical expert's evaluation etc.

Embodiment

Below be an embodiment of this invention, this embodiment only is used to illustrate the present invention, but content of the present invention is not limited to following examples.

In the phosphoric acid isotonic buffer solution (PBS) of pH7.2, add the 5mMol/L vitamin C as antioxidant, add 3% or 0.5% BSA simultaneously respectively, and the HB of 50 μ g/ml is as enzyme labeling thing stabilizing agent.After horseradish peroxidase (HRP) that adds dilution in 1: 1000 and anti-prostate specific antigen (PSA) antibody connect thing, place 37 ℃ of water-baths to carry out hot accelerated tests whole solution, to verify its effect.The enzyme labeling thing activity in the above-mentioned solution is measured in the back all around, and the activity of its result before by accelerated tests is 100%, calculates respectively that respectively to organize residual activity as follows:

Stabilizer type residual activity (100%)

(1)3％BSA 26.7

(2)0.5％BSA 51.2

(3)0.5％BSA+50μg/mlHB 97.6

Claims

1. an enzyme labeling thing stabilizing agent is made up of bovine serum albumin(BSA), haemoglobin, antioxidant and damping fluid; It is characterized in that: it is 0.1% to 1% bovine serum albumin(BSA) and the concentration haemoglobin as 50-200 μ g/ml that this stabilizing agent has used concentration.

2. according to the enzyme labeling thing stabilizing agent of claim 1, wherein said antioxidant is vitamin, polyphenols, polyhydroxy class material or the polyamino acid that possesses anti-oxidation characteristics.

3. according to the enzyme labeling thing stabilizing agent of claim 1, wherein said antioxidant is a vitamin C.