CN1064550A

CN1064550A - One multinomial novel membrane type quick spot immune diafiltration detection method of inspection and device

Info

Publication number: CN1064550A
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: 1992-09-16
Anticipated expiration: 2007-03-24
Also published as: CN1029574C

Abstract

An a kind of inspection is multinomial, novel membrane type, quick spot immune diafiltration detection method and device, belonging to this method of biochemical field simultaneously can obtain multinomial testing result and promptly obtain several qualitative conclusions simultaneously or obtain comprising that the wide-range of several ranges quantitatively indicates by once adding sample, the reaction film that this method is used can be indicated by different graphic at the different parts bag according to different testing goals, simultaneously the reagent composition is carried out difference adjustment, the present invention takes horseradish peroxidase to join immune Color Appearance System to replace the alkaline phosphatase demonstration in addition, in reaction unit, be positioned on the reaction film, filtering membrane is set, to remove large granular impurity and jelly.

Description

One multinomial novel membrane type quick spot immune diafiltration detection method of inspection and device

Fast spot immune diafiltration detection method (DIBA) is quick, easy to be highly sensitive and be used widely gradually with it, trend ， And that enzyme connection solid-phase immunity absorption before replacing in many aspects detects (ELISA) is arranged 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 garden shape 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 center round spot, 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) And through a large amount of tests and is equipped with the citric acid phosphoric acid damping fluid that contains hydrogen peroxide and can solves an above difficult problem.

The preparation of substrate chromophoric solution: TMB 5mg/100ml.Hci-Na ₂

(reagent 2) HPO ₄0.2M PH 5.0

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 the 0.05%TMS(thimerosal) as mordant, enzyme len antibody compound is diluted to proper proportion with this solution

Cleaning fluid is 0.05% Tween, 20～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 the LH(progesterone growth hormone 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, it is air-dry that bag is promptly closed And with BSA-PBS solution envelope by intact film, and this film is promptly used as the reaction film in the device.

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 1 expression point is shallower than whippletree among the figure

Among the figure ● the color depth of some expression point 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 HB in the urine sample (or blood sample) _S ^A _g(the anti-source of HBS) displaying symbol such as Fig. 3.

Reaction film is handled: at first use certain amount of H CG or HB _SA _gBag is made its colored intensity equal sample colored intensity at the peripheral first range sensitive spot when waiting range concentration by the center round spot, the peripheral first range sensitive spot anti-HCG or the HB of high concentration high-affinity _SA _gAntibody wraps quilt, 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.

Claims

1, multinomial, the novel membrane type of an inspection, quick, spot immune diafiltration detection method, at first add sample to be tested in giving on the earlier treated reaction film, then on this film, add chromogenic reagent with display result, it is characterized in that: join immune display system with horseradish peroxidase and replace alkaline phosphatase display system or the full display system of colloid, obtain several qualitative results simultaneously or obtain the test result of a plurality of sectional quantitative (or sxemiquantitative) of a wide-range simultaneously by application of sample;

2, a kind of quick, spot immune diafiltration pick-up unit comprises reaction film, filtering membrane, barrier film, water absorbing agent and pick-up unit shell, it is characterized in that:

(1) one deck filtering membrane is set with graininess impurity and jelly in effective removal specimen above reaction film;

(2) different parts at the reaction film body carries out different disposal to show different qualitative or quantitative results by detecting requirement;

3, spot immune diafiltration detection method as claimed in claim 1 is characterized in that: horseradish peroxidase joins the qualitative results that immune display system adopts spot-whippletree graphic presentation to detect, and shows the quantitative result that detects with many speckle pattern;

4, spot immune diafiltration pick-up unit as claimed in claim 2 is characterized in that filter membrane is made of two layers of soft toilet paper and one deck filter paper.