CN102768277B - Sample diluent of oral mucosal transudate for detecting HIV antibody by colloidal gold method, and preparation method thereof - Google Patents
Sample diluent of oral mucosal transudate for detecting HIV antibody by colloidal gold method, and preparation method thereof Download PDFInfo
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- CN102768277B CN102768277B CN201210259100.7A CN201210259100A CN102768277B CN 102768277 B CN102768277 B CN 102768277B CN 201210259100 A CN201210259100 A CN 201210259100A CN 102768277 B CN102768277 B CN 102768277B
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- transudate
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Abstract
The invention discloses a sample diluent of oral mucosal transudate for detecting HIV antibody by colloidal gold method, and a preparation method thereof, belonging to the technical field of biological detection. The diluent comprises disodium hydrogen phosphate 17.3-19.2 g/L, sodium dihydrogen phosphate 2.6-2.9 g/L, sodium chloride 8.3-9.2 g/L, 10% tween 80 9.5-10.5 mL/L, serum 142.5-157.5 mL/L, streptavidin 28.5-31.5 mg/L, biotin 19-21 mL/L, EDTA-2Na 19-21 g/L, NaN3 2.9-3.2 mL/L, and the balance of purified water. The inventive sample diluent can accelerate reaction speed and enable detection to be smoother.
Description
technical field
The present invention relates to field of biological detection, be specifically related to a kind of mucous membrane of mouth transudate colloidal gold method and detect sample dilution of HIV antibody and preparation method thereof.
Background technology
Immune colloidal gold technique is grow up the nineties in last century a kind of easy, detection technique fast and accurately, its principle is similar to two sandwich ELISAs, different is to use the synthetic compound of collaurum conjugated protein as probe, and the reagent using is all fixed on the nitrocellulose filter with micropore in advance.During use, one end is placed in and detects liquid, nitrocellulose filter can be passed by liquid, if detect in liquid, contains relevant antigen or antibody.In detection zone, just there will be pale red lines positive, otherwise negative.This technology has constantly been used since coming out clinically, the method does not rely on accurate instrument, does not rely on those skilled in the art, simplified operational procedure, compared with current conventional euzymelinked immunosorbent assay (ELISA), have advantages of larger, whole course of reaction just can complete in 15-20 minutes, the sample size needing is few, detects more responsive and accurate.
In recent years the research about acquired immune deficiency syndrome (AIDS) shows, HIV the infected's mucous membrane of mouth transudate contains HIV antibody.Mucous membrane of mouth transudate detects the same with blood testing, follows same examination and makes a definite diagnosis step, and in take a blood sample impracticable or unsafe situation, the advantage that has Noninvasive collecting sample is detected in oral cavity.Constantly perfect along with technology, the mucous membrane of mouth transudate of take is that the quick colloid gold immune diagnostic reagent that sample detects aids antibody has fabulous market outlook.
Mucous membrane of mouth transudate is exposed in air and can solidifies, and causes sampling to carry out, thereby causes detecting unsuccessfully, therefore should as early as possible mucous membrane of mouth transudate be collected with dilution.As can be seen here, the preparation of sample dilution is most important to carrying out smoothly of detecting.But the method that the mucous membrane of mouth transudate of take is at present pattern detection aids antibody also belongs to the starting stage, have not yet to see so far the report that is exclusively used in the sample dilution that detects mucous membrane of mouth transudate HIV antibody.
Summary of the invention
The object of the present invention is to provide a kind of mucous membrane of mouth transudate colloidal gold method that is exclusively used in to detect sample dilution of HIV antibody and preparation method thereof, can add the speed of fast response, make to detect more smooth.
For achieving the above object, the present invention adopts following technical scheme:
The sample dilution that mucous membrane of mouth transudate colloidal gold method detects HIV antibody comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 17.3-19.2g
Sodium dihydrogen phosphate 2.6-2.9g
Sodium chloride 8.3-9.2g
10% POLYSORBATE 80 9.5-10.5ml
Serum 142.5-157.5ml
Avidin 28.5-31.5mg
Biotin 19-21ml
EDTA-2Na 19—21g
NaN3 2.9—3.2ml
Surplus is purified water.
Described serum is cow's serum or chicken serum.
Preferably, described cow's serum is standard NBCS.
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 3-5 minutes;
C measures respectively serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 3-5 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with filtrator;
The bottling of F filtrate, labelled, 2-8 ℃ of stored under refrigeration.
Serum in described step C is cow's serum or chicken serum.
Preferably, described cow's serum is standard NBCS, adopts standard NBCS testing result better.
Preferably, the filtrator that the filtrator in described step e is 0.22um, can effectively reach the object of impurity screening and degerming.
Beneficial effect of the present invention is:
(1) sample dilution provided by the invention contributes to creeping of collaurum, be osmotic pressure, pH value all with the same buffer solution of Human Oral Cavity mucous membrane transudate, it makes red blood cell can keep original shape, can not produce aggegation and haemolysis, and maintain reaction homeostasis, shielding non-specific binding, thus testing process is carried out smoothly.
(2) mucous membrane of mouth transudate is joined in dilution provided by the invention, form and detect liquid, detect the HIV antibody in liquid, first be marked at the colloidal gold reaction on test paper, the compound of reaction moves up under chromatography effect, at this moment can produce a larger fluid pressure, just can make the speed that compound moves up increase, reduce the time of reaction.
Embodiment
Below in conjunction with embodiment, essentiality content of the present invention is described in further detail.
Embodiment 1
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 17.3g
Sodium dihydrogen phosphate 2.9g
Sodium chloride 8.3g
10% POLYSORBATE 80 10.5ml
Cow's serum 142.5ml
Avidin 28.5mg
Biotin 19ml
EDTA-2Na 21g
NaN3 2.9ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 3 minutes;
C measures respectively cow's serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 5 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with filtrator;
The bottling of F filtrate, labelled, 2 ℃ of stored under refrigeration.
The using method of sample dilution:
1, the test tube that sample dilution is housed is inverted three times gently, is made solution fully mix and open test tube cap.
2, from bag, take out clean buccal swab, hand-held buccal swab handle one end, with medium dynamics, with the cloth pad end of swab along the slowly wiping back and forth of upper gum line, from a jiao of mouth, slowly wipe another angle, then still along the wiping of upper gum line to section start (approximately 5 ~ 6 seconds used times).
3, upset buccal swab, with the another side wiping lower gum line of buccal swab cloth pad end, from a jiao of mouth, slowly wipes another angle and finishes, then still along the wiping of lower gum line to section start (about 5-6 of used time second).
4, immediately swab is inserted and to be equipped with in the test tube of dilution, clutch the handle of swab, swab is immersed in vitro in sample dilution, test tube wall is close on the two sides of first relief swab, swipes up and down 2-3 times, extrudes the liquid in buccal swab as far as possible.
5, from test tube, take out swab and lose in biochemical waste receptacle, sampling completes.
Described sample dilution in use, the speed that can add fast response, make to detect more smooth, but dilution is sure not to add too much, add 0.6-0.8ml, dilution can not surpass 1ml at most, if dilution adds excessively, can cause artificial reduction sample concentration, its testing result may be invalid; Add time of dilution should several seconds to one minute after mucous membrane of mouth transudate gathers within, to guarantee the stability of sample.
Embodiment 2
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 19.2g
Sodium dihydrogen phosphate 2.6g
Sodium chloride 9.2g
10% POLYSORBATE 80 9.5ml
Chicken serum 157.5ml
Avidin 31.5mg
Biotin 21ml
EDTA-2Na 19g
NaN3 3.2ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 5 minutes;
C measures respectively chicken serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 3 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 8 ℃ of stored under refrigeration.
Embodiment 3
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 18.5g
Sodium dihydrogen phosphate 2.8g
Sodium chloride 8.5g
10% POLYSORBATE 80 10.0ml
Chicken serum 145.3ml
Avidin 30.2mg
Biotin 20ml
EDTA-2Na 20g
NaN3 3.0ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 4 minutes;
C measures respectively chicken serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 5 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with filtrator;
The bottling of F filtrate, labelled, 3 ℃ of stored under refrigeration.
Embodiment 4
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 19.2g
Sodium dihydrogen phosphate 2.9g
Sodium chloride 8.3g
10% POLYSORBATE 80 9.5ml
Standard NBCS 157.5ml
Avidin 31.5mg
Biotin 20.5ml
EDTA-2Na 19.6g
NaN3 3.2ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 4 minutes;
C measures respectively standard NBCS, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 4 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 5 ℃ of stored under refrigeration.
Embodiment 5
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 18.8g
Sodium dihydrogen phosphate 2.7g
Sodium chloride 9.0g
10% POLYSORBATE 80 10.1ml
Chicken serum 150.5ml
Avidin 29.6mg
Biotin 20.3ml
EDTA-2Na 19.8g
NaN3 3.1ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 3 minutes;
C measures respectively chicken serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 4 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 4 ℃ of stored under refrigeration.
Embodiment 6
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, it is characterized in that: comprise following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 19.0g
Sodium dihydrogen phosphate 2.8g
Sodium chloride 8.4g
10% POLYSORBATE 80 9.7ml
Standard NBCS 144.6ml
Avidin 29.6mg
Biotin 19.3ml
EDTA-2Na 19.5g
NaN3 3.0ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 5 minutes;
C measures respectively standard NBCS, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 3 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 6 ℃ of stored under refrigeration.
Embodiment 7
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, comprises following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 18.6g
Sodium dihydrogen phosphate 2.7g
Sodium chloride 8.6g
10% POLYSORBATE 80 9.8ml
Cow's serum 152.6ml
Avidin 28.8mg
Biotin 20.6ml
EDTA-2Na 19.7g
NaN3 2.9ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 3 minutes;
C measures respectively cow's serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 4 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 7 ℃ of stored under refrigeration.
Embodiment 8
Mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, it is characterized in that: comprise following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 17.5g
Sodium dihydrogen phosphate 2.7g
Sodium chloride 8.8g
10% POLYSORBATE 80 9.9ml
Chicken serum 146.5ml
Avidin 29.7mg
Biotin 20.4ml
EDTA-2Na 19.8g
NaN3 3.2ml
Surplus is purified water
Described mucous membrane of mouth transudate colloidal gold method detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and opens and stir, and wherein, the purified water volume measuring accounts for 0.6 times of the fixed molten cumulative volume of dilution;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 4 minutes;
C measures respectively chicken serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 3 minutes;
D measures NaN3, adds in stirrer, is stirred to completely and dissolves;
E adds purified water to dilution and reaches surely molten cumulative volume, and then, solution filters with 0.22um filtrator;
The bottling of F filtrate, labelled, 5 ℃ of stored under refrigeration.
Claims (5)
1. mucous membrane of mouth transudate colloidal gold method detects the sample dilution of HIV antibody, it is characterized in that: comprise following component:
Material name normal concentration (/L)
Sodium hydrogen phosphate 17.3-19.2g
Sodium dihydrogen phosphate 2.6-2.9g
Sodium chloride 8.3-9.2g
10% POLYSORBATE 80 9.5-10.5ml
Serum 142.5-157.5ml
Avidin 28.5-31.5mg
Biotin 19-21ml
EDTA-2Na 19—21g
NaN
3 2.9—3.2ml
Surplus is purified water.
2. mucous membrane of mouth transudate colloidal gold method according to claim 1 detects the sample dilution of HIV antibody, it is characterized in that: described serum is cow's serum or chicken serum.
3. mucous membrane of mouth transudate colloidal gold method according to claim 2 detects the sample dilution of HIV antibody, it is characterized in that: described cow's serum is standard NBCS.
4. mucous membrane of mouth transudate colloidal gold method according to claim 1 detects the preparation method of the sample dilution of HIV antibody, comprises the following steps:
A measures purified water in stirrer beaker, and open to stir, and wherein, the purified water volume measuring accounts for 0.6 times of cumulative volume of dilution constant volume;
B measures respectively sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 10% POLYSORBATE 80, adds in stirrer, stirs 3-5 minutes;
C measures respectively serum, Avidin, biotin, EDTA-2Na, adds in stirrer, stirs 3-5 minutes;
D measures NaN
3, add in stirrer, be stirred to completely and dissolve;
E adds the cumulative volume that purified water to dilution reaches constant volume, and then, solution filters with filtrator;
The bottling of F filtrate, labelled, 2-8 ℃ of stored under refrigeration.
5. mucous membrane of mouth transudate colloidal gold method according to claim 4 detects the preparation method of the sample dilution of HIV antibody, it is characterized in that: the filtrator in described step e is the filtrator of 0.22 μ m.
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