CN102628763A

CN102628763A - Reagent for high-efficiency elution of active antibodies in mouth swab and corresponding elution method

Info

Publication number: CN102628763A
Application number: CN2012100651906A
Authority: CN
Inventors: 祁军; 刘智勇; 刘振宇; 李秀平; 刘寅
Original assignee: TIANJIN EXIT-ENTRANCE CHECK AND GUARANTINE BUREAU
Current assignee: TIANJIN EXIT-ENTRANCE CHECK AND GUARANTINE BUREAU
Priority date: 2012-03-14
Filing date: 2012-03-14
Publication date: 2012-08-08

Abstract

The invention discloses a reagent for high-efficiency elution of active antibodies in a mouth swab and a corresponding elution method. The reagent comprises 8g/L of NaCl, 0.2g/L of KCl, 1.15g/L of Na2HPO4, 0.2g/L of KH2PO4, 0.05ml/L of Tween-20, 1g/L of bovine serum albumin (BSA), 0.2g/L of NaN3 and 100 micrograms per milliliter of goat anti-mouse IgG antibodies. Aiming at a mouth swab, the reagent for high-efficiency elution of active antibodies in a mouth swab and the corresponding elution method overcome the defects of the prior art.

Description

A kind of reagent and elution process that is used for high-level efficiency wash-out buccal swab active antibodies

Technical field

The present invention relates to antibody and gather inspection technology, specifically a kind of reagent and elution process that is used for high-level efficiency wash-out buccal swab active antibodies.

Background technology

The mucous membrane of mouth transudate is the important development direction of present immune detection, carried out adopting mostly in the process of antibody test serum as detected object in the past, and its gatherer process causes wound, is not suitable for person under inspection's self check and has also strengthened person under inspection's misery simultaneously.In recent years; Detection to the mucous membrane of mouth transudate is growing; But the AC in the mucous membrane of mouth transudate is lower, particularly carries out can producing a large amount of losses in the process of antibody elution at swab, and it is mainly derived from two aspects; One, the improper antibody that makes of composition is adsorbed on the swab in a large number in the damping fluid, and two, the composition in the solution reduces the activity of antibody.If can being arranged, the method for active antibodies in the high-level efficiency wash-out buccal swab will greatly quicken antibody scope and sensitivity that the mucous membrane of mouth transudate detects.

Summary of the invention

To buccal swab, the present invention has overcome shortcoming of the prior art, and the agent prescription and the method for active antibodies in a kind of high-level efficiency wash-out buccal swab is provided.

In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: configuration solution A and solution B also taked following method wash-out.

Wherein the prescription of solution A is NaCl 8g/L, KCl 0.2g/L, Na ₂HPO ₄1.15g/L, KH ₂PO ₄0.2g/L, tween (tween) 20 0.05ml/L, bovine serum albumin(BSA) (BSA) 1g/L, NaN ₃0.2g/L;

The prescription of solution B is that the prescription of B liquid is 100 μ g/mL sheep anti-mouse igg antibody solution.

Operated to be called earlier that buccal swab was infiltrated in the 2mL A liquid 10 minutes, and added 1mL B liquid again, buccal swab is fully concussion in above-mentioned solution, and the solution that the concussion back is obtained is active antibodies solution in the wash-out buccal swab.

Above-mentioned active antibodies solution end user immunoglobulin (Ig) (IgG) ELISA detection kit is carried out quantitative test, and analysis result is not with using the swab eluent of this method to compare, and this method of comparative result proof can improve 5 to 10 times of elution efficiencies.

Detection method is following:

Get mucous membrane of mouth transudate antibody sample of gathering according to prescription of the present invention and method and each the 100 μ L of mucous membrane of mouth transudate antibody sample that gather according to conventional method respectively.

In refrigerator, take out human immunoglobulin(HIg) (IgG) ELISA detection kit, take out required lath at room temperature in the aluminium foil bag behind the balance 20min, the residue lath is put back to 4 ℃ with the valve bag sealing.

Set up standard article hole and sample aperture, the standard items hole respectively adds the standard items 50 μ L of variable concentrations;

Sample aperture adds sample to be tested 10 μ L earlier, adds sample dilution 40 μ L again; Blank well does not add.

Except that blank well, every hole adds the detection antibody 100 μ L of horseradish peroxidase (HRP) mark in standard items hole and the sample aperture, seals reacting hole with the shrouding film, 37 ℃ of water-baths or constant temperature oven incubation 60min.

Discard liquid, the thieving paper arsis is done, and cleansing solution is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, and the thieving paper arsis is done, and so repeats to wash plate 5 times (the also available plate machine washing plate of washing).

Every hole adds substrate A, each 50 μ L of B, and 37 ℃ of lucifuges are hatched 15min.

Every hole adds stop buffer 50 μ L, in the 15min, measures the OD value in each hole in the 450nm wavelength.

The result judges

The drawing standard curve in the Excel worksheet, is made horizontal ordinate with standard items concentration, and corresponding OD value is made ordinate, draws out standard items linear regression curve, presses curvilinear equation and calculates each sample concentration value.

Above-mentioned antibody sample is analyzed, and analysis result is following:

Table one uses the present invention and does not use the AC in the sample that obtains of the present invention

Unit (10 ²μ g/mL)

	Do not use the present invention	Use the present invention
			1	1.36	33.2
2	2.36	35.7
			3	5.97	28.9

Description of drawings

Fig. 1 uses the present invention and does not use the AC in the sample that obtains of the present invention.

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is done further explain:

The present invention realizes through following technical scheme:

(1) configuration solution A and solution B.

Wherein the prescription of solution A is NaCl 8g/L, KCl 0.2g/L, Na ₂HPO ₄1.15g/L, KH ₂PO ₄0.2g/L, tween (tween) 20 0.05ml/L, bovine serum albumin(BSA) (BSA) 1g/L, NaN3 0.2g/L;

(2) swab of the mucous membrane of mouth transudate of gathering is put into the container that a volume is fit to.

(3) earlier buccal swab was infiltrated in the 2mL A liquid 10 minutes.

(4) add 1mL B liquid again, buccal swab is fully concussion in above-mentioned solution, and the solution that the concussion back is obtained is active antibodies solution in the wash-out buccal swab.

(5) above-mentioned sample solution is detected.

(6) in refrigerator, take out human immunoglobulin(HIg) (IgG) ELISA detection kit, take out required lath at room temperature in the aluminium foil bag behind the balance 20min.

(7) set up standard article hole and sample aperture, the standard items hole respectively adds the standard items 50 μ L of variable concentrations;

(8) except that blank well, every hole adds the detection antibody 100 μ L of horseradish peroxidase (HRP) mark in standard items hole and the sample aperture, seals reacting hole with the shrouding film, 37 ℃ of water-baths or constant temperature oven incubation 60min.

(9) discard liquid, the thieving paper arsis is done, and cleansing solution is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, and the thieving paper arsis is done, and so repeats to wash plate 5 times (the also available plate machine washing plate of washing).

(10) every hole adds substrate A, each 50 μ L of B, and 37 ℃ of lucifuges are hatched 15min.

(11) every hole adds stop buffer 50 μ L, in the 15min, measures the OD value in each hole in the 450nm wavelength.

(12) drawing standard curve in the Excel worksheet, is made horizontal ordinate with standard items concentration, and corresponding OD value is made ordinate, draws out standard items linear regression curve, presses curvilinear equation and calculates each sample concentration value.

The AC that uses this method to gather after measured is 3.1mg/mL.Near the antibody content in the serum.

Claims

1. an agent prescription that is used for high-level efficiency wash-out buccal swab active antibodies comprises a plurality of components in its prescription, comprises NaCl 8g/L, KCl 0.2g/L, Na ₂HPO ₄1.15g/L, KH ₂PO ₄0.2g/L, tween (tween) 20 0.05ml/L, bovine serum albumin(BSA) (BSA) 1g/L, NaN ₃0.2g/L and 100 μ g/mL sheep anti-mouse igg antibodies.

2. a kind of agent prescription that is used for high-level efficiency wash-out buccal swab active antibodies according to claim 1 is characterised in that the elution step of buccal swab is following:

Earlier buccal swab was infiltrated in the 2mL A liquid 10 minutes, the prescription of A liquid is NaCl 8g/L, KCl0.2g/L, Na ₂HPO ₄1.15g/L, KH ₂PO ₄0.2g/L, tween (tween) 20 0.05ml/L, bovine serum albumin(BSA) (BSA) 1g/L, NaN ₃0.2g/L.

Add 1mL B liquid again, the prescription of B liquid is 100 μ g/mL sheep anti-mouse igg antibodies, and above-mentioned solution fully shakes, and the solution that the concussion back is obtained is active antibodies solution in the wash-out buccal swab.