CN102662051A - Confining liquid for enzyme linked immunosorbent assay in vitro diagnostic reagent - Google Patents

Abstract

The invention belongs to the technical field of biological in vitro diagnostic reagents and particularly relates to a confining liquid for an enzyme linked immunosorbent assay in vitro diagnostic reagent. The confining liquid is prepared by the following technological steps: preparing PBS (Phosphate Buffer Solution); adding albumin bovine serum to be dissolved fully; adding trehalose to be dissolved fully; adding protein stabilizing agent to be dissolved fully, adding L-lysine to be dissolved fully; adding Proclin 300 to be mixed fully; adding sodium hydroxide solution or hydrochloric acid solution for adjusting the pH value to be mixed fully; and standing for more than 30 min at room temperature and preserving at 2-8 DEG C. Compared with the prior art, the confining liquid has a broad spectrum activity, can inhibit the growth of microbe like bacteria, fungi and microzyme for a longer time, and meanwhile can retain the activity of enzyme in the system.

Description

Confining liquid in the enzyme linked immunological external diagnosis reagent

[technical field]

The invention belongs to biological external diagnosis reagent technical field, be specifically related to confining liquid in a kind of enzyme linked immunological external diagnosis reagent.

[background technology]

The ELISA basis is the enzyme labeling of immobilization and the antigen or the antibody of antigen or antibody, and ultimate principle has: 1. antigen or antibody capable with physical property be adsorbed in surface of solid phase carriers, and keep its immunologic competence; 2. antigen or antibody can be connected to form enzyme conjugates through covalent bond and enzyme, and this kind enzyme conjugates still can keep its immunology and the enzyme activity; 3. after enzyme conjugates and corresponding antigens or the antibodies; Can judge whether immunoreactive existence is arranged according to the color reaction that adds substrate; And the depth of color reaction be with sample in the amount of corresponding antigens or antibody directly proportional, can show test findings by the degree of substrate colour developing.

ELISA has fast, responsive, easy, be easy to advantages such as standardization, be widely used in the detection of antigen, antibody, cell factor and biochemical substances.There are a lot of Related products to be used for the serodiagnosis of multiple disease on the market, like HIV, hepatitis, cardiovascular, cancer etc.

During very biological detection is analyzed; Many reagent or sample component will be adsorbed in solid phase carrier or be fixed in carrier surface, and this combination is not the identifying of ligands specific/acceptor (like Ag-Ab), are called non-specific binding (non-specific binding usually; NSB); This non-specific binding particularly when detecting antibody, may cause false positive; On the other hand, the non-specific identification that may disturb part and acceptor.False positive can reduce the specificity of detection.In addition, non-specific binding also might cause the reduction of signal to noise ratio (S/N ratio) (signal-to-noise ratio), thereby produces false negative, and this can reduce the sensitivity of detection again.A very general method of avoiding non-specific binding is to add sealing reagent.Usually, the surface of solid phase carriers that is used to adsorb biomaterial is very gentle, is to encapsulate in the first step of analyzing, and analyzes component for one and is attached to surface of solid phase carriers, and this component that encapsulates can not all override the whole surface of solid phase carrier usually.Therefore, seal this step to be very important, override the remaining free site of surface of solid phase carriers with so-called closed reagent.After encapsulating, the surface of solid phase carriers of handling with closed reagent should be saturated, has so just avoided the absorption of subsequent material.Used closed reagent during sealing effect is decided by to analyze to a great extent.

Now, the kit that in-vitro diagnosis is used not only has requirement to specificity and sensitivity, and stability also is very important.The closed reagent less stable of single component, the term of validity is very short, and this makes the effect phase of kit also become very short to a certain extent.

How reducing non-specific binding in the enzyme linked immunological external diagnosis reagent, can prolong the term of validity of kit again, is that numerous external diagnosis reagent manufacturer makes great efforts one of problem that solves for a long time.

[summary of the invention]

The present invention provides confining liquid in a kind of enzyme linked immunological external diagnosis reagent in order to overcome the deficiency of prior art.

To achieve these goals, the present invention has designed confining liquid in a kind of enzyme linked immunological external diagnosis reagent, adopts following processing step to prepare:

A) preparation 0.01M～0.2M PBS damping fluid, the pH value is 6.5～7.8;

B) add bovine serum albumin(BSA) in the damping fluid in steps A, final concentration is 1g/L～30g/L, stirs, and it is fully dissolved;

C) add trehalose, final concentration is 0.2g/L～25g/L, stirs, and it is fully dissolved;

D) add protein stabiliser, final concentration 0.2g/L～5g/L stirs, and it is fully dissolved;

E) add L-lysine, final concentration 0.2g/L～10g/L stirs, and it is fully dissolved;

F) add Proclin 300, final concentration is 0.01%～1%, stirs, and makes its abundant mixing;

G) regulate pH value to 6.5～7.4, mixing with 5M sodium hydroxide solution or 5M hydrochloric acid solution;

H) room temperature leaves standstill more than the 30min, 2 ℃～8 ℃ preservations.

Said confining liquid can be used for the microwell plate or the microballoon solid phase carrier of material such as polystyrene or polypropylene.

The present invention compares with prior art, has changed the constituent of traditional close liquid, has selected joining in the confining liquid as protective agent of trehalose, has significantly improved the stability of confining liquid.Added protein stabiliser and L-lysine, more effective maintenance is protein stabilized.Changed antiseptic into Proclin 300 by thimerosal commonly used, the latter has broad spectrum activity, can suppress microbial growths such as bacterium, fungi and saccharomycete in a long time, simultaneously again can the maintenance system in the activity of enzyme, more be applicable to external diagnosis reagent.

[embodiment]

Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment and only be used to explain that the present invention is not used in restriction scope of the present invention.

Embodiment:

The preparation process of confining liquid of the present invention is following:

A) preparation 0.01M～0.2M PBS damping fluid, the pH value is 6.5～7.8;

B) add bovine serum albumin(BSA) in the damping fluid in steps A, final concentration is 1g/L～30g/L, stirs, and it is fully dissolved;

C) add trehalose, final concentration is 0.2g/L～25g/L, stirs, and it is fully dissolved;

D) add protein stabiliser, final concentration 0.2g/L～5g/L stirs, and it is fully dissolved;

E) add L-lysine, final concentration 0.2g/L～10g/L stirs, and it is fully dissolved;

F) add Proclin 300, final concentration is 0.01%～1%, stirs, and makes its abundant mixing;

G) regulate pH value to 6.5～7.4, mixing with 5M sodium hydroxide solution or 5M hydrochloric acid solution;

H) room temperature leaves standstill more than the 30min, 2 ℃～8 ℃ preservations.

Experimental result adopts double antibody sandwich method to detect the content of AFP in the serum, verifies confining liquid, and concrete steps are following:

1) encapsulates AFP antibody, 4 μ g/ml, 100 μ l/ holes, 2 ℃～8 ℃ refrigerator overnight.

2) according to present embodiment preparation confining liquid; Other prepares the one-component confining liquid.

3) dry coating buffer, clap and do, seal with different confining liquids respectively, 300 μ l/ holes, 37 ℃ of incubations 2 hours.

4) wash plate, clap and do, dry 5 hours of vacuum drying chamber.

5) with placing 37 ℃ of constant temperature ovens after the lath Vacuum Package respectively, after 3 days, 5 days, 7 days, take out respectively, deposit and encapsulate the parallel comparison of plate with 2 ℃～8 ℃.

6) preparation 0ng/ml, 5ng/ml, 25ng/ml, 100ng/ml, 400ng/ml, 800ng/ml series A FP standard items.

7) in the ELISA Plate hole, add the AFP standard items successively, adding has value serum, and multiple hole is all established in 100 μ l/ holes, 37 ℃ of incubation 60min.

8) wash plate, clap and do, add certain density HRP mark AFP antibody, 60min is hatched for 37 ℃ in 100 μ l/ holes.

9) wash plate, clap and do, add colour developing liquid, 100 μ l/ holes, room temperature reaction 15min.

10) add stop buffer, 50 μ l/ holes.

11) ELIASA reading detects wavelength 450nm, reference wavelength 630nm, and the result is as shown in the table:

Claims (2)

1. confining liquid in the enzyme linked immunological external diagnosis reagent, adopt following processing step to prepare:

A) preparation 0.01M～0.2M PBS damping fluid, the pH value is 6.5～7.8;

B) add bovine serum albumin(BSA) in the damping fluid in steps A, final concentration is 1g/L～30g/L, stirs, and it is fully dissolved;

C) add trehalose, final concentration is 0.2g/L～25g/L, stirs, and it is fully dissolved;

D) add protein stabiliser, final concentration 0.2g/L～5g/L stirs, and it is fully dissolved;

E) add L-lysine, final concentration 0.2g/L～10g/L stirs, and it is fully dissolved;

F) add Proclin 300, final concentration is 0.01%～1%, stirs, and makes its abundant mixing;

G) regulate pH value to 6.5～7.4, mixing with 5M sodium hydroxide solution or 5M hydrochloric acid solution;

H) room temperature leaves standstill more than the 30min, 2 ℃～8 ℃ preservations.

2. confining liquid in the enzyme linked immunological external diagnosis reagent according to claim 1 is characterized in that: said confining liquid can be used for the microwell plate or the microballoon solid phase carrier of material such as polystyrene or polypropylene.