CN1029574C

CN1029574C - Method and device for membrane type fast spot immume percolation test capable of doing multiple items once

Info

Publication number: CN1029574C
Application number: CN 92101890
Authority: CN
Inventors: 祝加贝
Original assignee: Individual
Current assignee: Individual
Priority date: 1992-03-24
Filing date: 1992-03-24
Publication date: 1995-08-23
Anticipated expiration: 2007-03-24
Also published as: CN1064550A

Abstract

The present invention relates to a multiple-detection, new film-type and fast blob immunity infiltration detecting method and a device, which belongs to the field of biochemistry. In the method, a sample is added for once, and a plurality of detection results can be simultaneously obtained; a plurality of characteristic conclusions or wide range quantitative marks containing a plurality of measuring ranges can be simultaneously obtained; a reaction film used by the method can be used for coating different figure marks at different parts according to different detection goals, and simultaneously, reagent constituents are differently adjusted; in addition, the present invention adopt a horseradish peroxidase joined immunity color developing system to replace alkaline phosphatase displaying; a filter film is arranged on a reaction film in a reaction device in order to remove granular impurities and jelly.

Description

Method and device for membrane type fast spot immume percolation test capable of doing multiple items once

The invention belongs to biochemical field

Fast spot immune diafiltration detection method (DIBA) is quick, easy to be highly sensitive and be used widely gradually with it, has enzyme connection solid-phase immunity absorption before replacing in many aspects to detect the trend of (ELISA), and in constantly improving.As changing product film into from the cellulose nitrate membrane portions of the earliest non-bonding combination with chemical bond as the film of solid phase carrier.The display system of this technology also develops into collaurum from single alkaline phosphatase.These two kinds of display systems, its sensitivity is all low and cost is higher, and this has just limited the DIBA The Application of Technology, in addition because the restriction of technology itself, can't be in order to the anti-source of detection bulky grain.

Present immunologic detection method all is that an application of sample can only be tested a kind of result, not only wastes sample so in actual applications, and efficient can not improve.The ELISA method then need be done many such as tired latching operations such as dilutions when detection by quantitative, and it is very not convenient to use, and other method also has this disadvantage.

In addition, some special index is owing to the reason of inherent characteristic own can not be finished in single detection.This had only by the multinomial detection of application of sample repeatedly and just can finish in the past, such as the short progesterone growth hormone of the LH(in the urine) semi-quantitative analysis be a typical example.

The purpose of this invention is to provide a kind of novel immunity percolation detection method and device, not only have high sensitivity but also can obtain the test result of a plurality of sectional quantitative (or semidefinite) of several qualitative results and a wide-range by application of sample simultaneously.

The present invention is achieved through the following technical solutions:

One. show it is relatively independent principle based on spot on membrane carrier, we carry out different disposal (bag quilt) to the different parts of membrane carrier, also will adjust to adapt to this a plurality of bag quilt the reagent composition simultaneously certainly.(implication of bag quilt is that corresponding antibodies or antigen are dissolved among the PBS, and concentration is fitted needs and adjusted, and is added in the corresponding site on the reaction film then.）

(optional φ=12mm) center is contrasted thick stick (i.e. a whippletree shape figure) with certain a certain amount of sample to be tested composition bag in order to reduce commercial cost at a circular membrane area of trying one's best little, make its colored intensity equal the colored intensity of certain two kinds of (or several) sample to be tested when a certain concentration respectively, Here it is qualitative (sxemiquantitative) detects the contrast thick stick, use the high-affinity antibody bag that resists these two kinds of (or several) samples by these two kinds of (or several) sampling points to be tested respectively in the orientation up and down at whippletree then, this has just made qualitative (sxemiquantitative) reaction film.

Can certain a certain amount of composition bag to be measured when detection by quantitative be made its colored intensity equal the colored intensity of this sample when first range by a centre dot, after this range of dividing by the measured intensity incremental order around the central point is handled with the different antibodies (deciding according to self laboratory antibody feature) that reduces the same antibody of concentration successively or reduce affinity successively, makes it to obtain under the sample concentration of its corresponding range the colored intensity the same with central control point.

Two. the present invention adopts the horseradish peroxidase Color Appearance System on Color Appearance System, and this system compares tool economy with alkaline phosphoric acid enzyme system, the advantage that is easy to make, but two difficult problems are arranged when using it for the DIBA technology.

(1) the horseradish peroxidase reaction system has high sensitivity in elisa technique, and DIBA really needs the enzyme reaction system of insoluble substrate that good color developing effect just can be arranged; And the sensitivity of the insoluble substrate Color Appearance System of existing horseradish peroxidase is lower.

(2) the horseradish peroxidase system substrate chromophoric solution of current employing is by two kinds of solution compositions, first kind as OPD, α-tetrachloro naphthols, DAB etc., another kind of for containing the damping fluid of hydrogen peroxide, and these two kinds of solution mixing certain hours lapse, owing to these two reason restrictions, so that prior art can't be used for the horseradish peroxidase system DIBA technology.

The present invention finds to select for use half solubility chromogenic substrate TMB(tetramethyl in the peppery enzyme system to join stupid amine through a large amount of tests) and be equipped with the citric acid phosphoric acid damping fluid that contains hydrogen peroxide and can solve an above difficult problem.

The preparation of substrate chromophoric solution:

TMB 5mg/100ml

(reagent 2) Hci-Na ₂HPO ₄0.2M pH5.0

H ₂O ₂0.004%

Make horseradish peroxidase len antibody compound and can adopt the Routine Test Lab technology, but will notice that the compound that obtains preferably carries out a gel filtration and separates, choose molecular weight and be 20～300,000 component to avoid in the diafiltration reaction, producing too high background.

Three. the preparation main points of reagent 1:

The 0.5%BSA(bovine serum albumin(BSA))-the PBS(phosphate buffer) include 0.05%TMS(sulphur but mercury) as mordant, enzyme len antibody compound is diluted to proper proportion with this solution

Cleaning fluid is 0.05%Tween20～PBS

Four. for proving installation (Fig. 1) has designed economical and practical filter membrane (3) to replace special filtrator or the heavy centrifugal treating in the existing DIBA technology, filter membrane only adds No. 2 filter paper of one deck Xinhua again by the pliable and tough toilet paper of the quality of two-layer yellow and constitutes.

Below in conjunction with description of drawings embodiment:

(1) qualitative, half-quantitative detection: detect yellow this ketone growth hormone of LH(in the urine sample (or blood sample)) and the HCG(human chorionic gonadotrophin), displaying symbol such as Fig. 2.

Reaction film is handled: select the high affinity antibody sandwich thick stick top point that reacts simultaneously with LH and HCG for use, with the antibody sandwich thick stick below point special to HCG, with the HCG bag identical with the colored intensity of the HCG of the LH50mlu of 40mlu by the contrast thick stick, promptly with BSA-PBS solution sealing and air-dry, promptly use as the reaction film in the device by this film by intact film for bag.

Operation: the Xiao Chi (7) that goes into the device top with the urina sanguinis drop fills it, treating that it infiltrates fully adds 3 reagent 1 and left standstill 1 minute after infiltrating fully on the filtering membrane (3), add 4 clean-out systems again, treat to infiltrate fully the back dropper (plastic tube of a headband point, this pipe is double do get urine sample with) peel off filter membrane, add 3 substrate chromophoric solutions and after treating to absorb fully on the reaction film (4), add 2 again, display result such as Fig. 2, each little figure represents among Fig. 2:

1 is all feminine gender for LH and HCG

2 for LH weak positive but be lower than 40mlu

3 is that the LH positive is more than or equal to 40mlu

4 for HCG weak positive but be lower than 50mlu

5 is that the HCG positive is more than or equal to 50mlu

6 for detecting error

The color depth of O point expression point is shallower than whippletree among the figure

The figure mid point represents that a little color depth equals or is better than thick stick

(1) is the pick-up unit lid among Fig. 1, and (2) are device body, and (5) are barrier film, and (6) are absorbent material

(2) detection by quantitative: detect HCG or HBs in the urine sample (or blood sample) ^AThe anti-source of g(HBS) displaying symbol such as Fig. 3.

Reaction film is handled: at first made its colored intensity equal sample colored intensity at the peripheral first range sensitive spot when waiting range concentration with certain amount of H CG or HBsAg bag by centre dot, the peripheral first range sensitive spot wraps quilt with the anti-HCG or the HBsAg antibody of high concentration high-affinity, second to the 6th range sensitive spot is then successively with the same antibody that reduces concentration or wrap quilt with the different antibodies of reduction affinity successively, make it in the sample concentration of its corresponding range, to obtain the colored intensity the same with central control point, Processing of Preparation then is identical with general DIBA, operation is with top qualitative, half-quantitative detection embodiment is identical, and it is as follows that six ranges of setting reach the figure number corresponding with Fig. 3:

1. be that 5mlu 2. is 80mlu for 20mlu 3.

4. be that 320mlu 5. is 5lu for 1.2lu 6.

Description of drawings

Fig. 1 is this contrive equipment cut-open view

Fig. 2 is a displaying symbol qualitative and the half-quantitative detection result

Fig. 3 is detection by quantitative result's a displaying symbol

Claims

1, a kind of immunity percolation detection method comprises the steps:

A. wrap by the qualitative reaction film, become subpackage to be contrasted thick stick with a kind of sample to be tested wherein, make its colored intensity equal the colored intensity of a certain amount of two kinds of sample to be tested compositions respectively at a circular membrane center:

B. use these two kinds of sampling points to be tested of high affinity antibody sandwich of anti-these two kinds of sample constituents respectively in the orientation up and down of contrast thick stick:

It is characterized in that: join immune display system with horseradish peroxidase and replace alkaline phosphatase display system or collaurum display system, the qualitative results that adopts spot one whippletree graphic presentation to detect.

2, a kind of immunity percolation detection method comprises the steps:

The A bag by the centre dot of reaction film, is made its colored intensity equal the colored intensity of this sample when first range with one-tenth subpackage to be measured by a plurality of sectional quantitative reaction films;

B. the range of being divided by the measured intensity incremental order around the central point tested sampling point of different antibodies bag that reduces the same antibody of concentration successively or reduce affinity successively makes it to obtain the colored intensity the same with central control point under the sample concentration of corresponding range.

It is characterized in that: join immune display system with horseradish peroxidase and replace alkaline phosphatase display system or collaurum display system, and show the quantitative result that detects with many speckle pattern.