CN101706499A - FLAG fusion tag colloidal gold test strip and preparation method thereof - Google Patents

Abstract

The invention relates to a preparation method of a colloidal gold reagent and a rapid detection method for a fusion tag. The colloidal gold reagent belongs to a detection reagent of a FLAG fusion tag in the field of biologic research. A double- antibody sandwich method immunoassay principle is adopted in the method, and a Flag monoclonal antibody and a Flag polyclonal antibody which are purified are respectively used as a colloidal gold combined antibody and an envelope antibody, thus the gene engineering expression protein adopting FLAG as the fusion tag is rapidly detected.

Description

FLAG fusion tag colloidal gold test strip and preparation method thereof

Technical field

The invention belongs to biological technical field,, belong to the detectable of FLAG fusion tag in the biological scientific research field in particular to a kind of preparation of colloid gold reagent and to the fast detecting of fusion tag.

Background of invention

Fusion tag is a kind of eight amino acid whose small peptides (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) that contain, as fusion tag widespread use in engineered protein is expressed.Along with the widespread use of immunoassay mouth benefit, immunoassay develops to both direction: a class is full-automatic immunoassay, and is another kind of for being the tachysynthesis analysis of carrier with the nitrocellulose filter.The former needs expensive full-automatic instrument to reach and the strict supporting all ingredients box of instrument, is subjected to conditional request higher.Along with easily and fast, the needs of popular detection, main on the basis of EIA enzyme immunoassay is that the immune colloid gold quick diagnosis technology of carrier grows up rapidly and widely with the nitrocellulose filter.The ultimate principle of this method is to be carrier with the miillpore filter, bag is by known antigens or antibody, after adding sample to be checked, through the capillarity of filter membrane or transudation the antibody or the antigen of bag quilt on antigen in the sample or antibody and the film are combined, reach testing goal with collaurum bond mark again.

Summary of the invention

The purpose of this invention is to provide a kind of FLAG fusion tag colloidal gold test strip.

Another object of the present invention provides a kind of preparation method of FLAG fusion tag colloidal gold test strip.

This test strips adopts double antibody sandwich method immunoassays principle, with the Flag monoclonal antibody of purifying and Flag polyclonal antibody respectively as collaurum binding antibody and coated antibody.Be used for substituting enzyme and exempt from adopting FLAG to carry out fast detecting as the gene engineering expression albumen of fusion tag, detection speed reaches 5 times that enzyme is exempted from, and does not use any detecting instrument.

The present invention is achieved in that this test strips adopts double antibody sandwich method immunoassays principle, with the Flag monoclonal antibody of purifying and Flag polyclonal antibody respectively as collaurum binding antibody and coated antibody.When containing in the sample to be checked when adopting FLAG as the gene engineering expression albumen of fusion tag, first and golden labelled antibody combination, because chromatography action-reaction compound moves forward along nitrocellulose membrane, when running into coated antibody, form the Ab-Ag-Ab-Au compound and be enriched in bag, form the aubergine precipitation line by on the line.On coated film, also have simultaneously a nature controlling line,, when having only a purplish red colo(u)r streak, be judged to feminine gender so when two purplish red colo(u)r streaks, be judged to the positive.

Good effect of the present invention:

1, rapidly quick, shorten greatly the time as a result.The time that colloidal gold immunity chromatography goes out the result, in minutes this was that other method for quick is beyond one's reach at present.

2, sensitive and accurate.The immune colloid gold fast diagnosis method is not because it has sacrificed its specificity and susceptibility fast.

3, safe and simple, do not need any instrument and equipment.The immune colloid gold detection method does not need any instrument and equipment, and only the test-strips or the kit that need prepare gets final product; More, omitted step, simplified operation greatly, be more suitable for rig-site utilization with developer and stop buffer than ELISA because collaurum itself has color.Because do not participate in such as objectionable impuritiess such as radioactive isotope, o-phenylenediamines, thus do not pollute the environment yet, have detection methods such as radioactive isotope or enzyme mark incomparable security.

4, stable reagent, extraneous factor influence is less. and combining between gold grain and the protein molecular belongs to physical adsorption process during colloid gold label protein, so whole labeling process is very little to the biologically active influence of protein, easily obtains higher mark rate; Collaurum does not belong to bioactivator, and the factor of interference detection results will significantly reduce, so reagent is highly stable, it is less influenced by extraneous factors such as temperature, can deposit in room temperature.

5, with low cost, required reagent and sample size are few.Because " immunity concentrates ", sample size can be low to moderate 10-20ul; Add and need not any instrument and equipment, and can detect by single part of sample, cost is declined to a great extent.

Description of drawings

Do not have.

Embodiment

1, the trisodium citrate legal system is equipped with the 12-30nm collaurum and gets one of 250ml triangular flask, adds 100ml distilled water and 1ml 1% chlorauride, ebuillition of heated; 1% sodium citrate of 1.5ml is added in the above-mentioned solution.Mixing keeps boiling 30min again, and solution colour is blackening at first, reddens gradually again.

2, albumen combines best pH mensuration with collaurum

(1) gets several 1.5ml test tubes, add 1ml 10nm collaurum respectively;

(2) with 25mM K2CO3 pH is adjusted to 3,4,5,6,7,8,9,10 respectively;

(3) get one 96 well culture plates, respectively above-mentioned collaurum is got 100ul respectively from low to high by pH and add in the hand-hole triplicate;

Every hole adds the Flag antibody that 3ul concentration is 1mg/ml respectively, mixes, and places 10-15min under the room temperature;

It is 10%NaCl solution that every hole adds 20ul concentration respectively, mixes, and places 10min under the room temperature;

Observing colloid gold change color, record keeps red minimum pH; Observing colloid gold change color was placed under room temperature 2 hours, and record still keeps red minimum pH.

3, minimum protein concentration determines

(1) removes residual thing or polymer in the albumen with 0.22m filtering with microporous membrane or high speed centrifugation;

(2) get one 96 hole titer plates, each repeats to be several holes, adds the collaurum 100ul of best pH respectively, repeats 3 times;

(3) each hole adds albumen (general concentration the is 0.05-0.1mg/ml) 1～20ul of different amounts successively, and mixing is placed 15min under the room temperature;

(4) add 20ul 10%NaCl, place 10min under the room temperature; It is minimum protein concentration that color still keeps red minimum albumen consumption.

4, gold mark pad preparation

(1) gets two 1.5ml test tubes, add 1.2ml 10nm collaurum respectively;

(2) add an amount of 25mM K2CO3 pH is adjusted into 9.0;

(3) adding 10ul concentration respectively is 1mg/ml IgG, mixing, and room temperature is placed 10min;

(4) add 12ul 2%PEG20000 respectively, room temperature is placed 5min;

(5) the centrifugal 20min of 10000rpm absorbs supernatant gently;

(6) with 20ul 50mM PBS, pH 7.0,, include 1%BSA, 0.02%PEG20000,100mMNaCl, the collaurum precipitation that the 1%NaN3 solution weight suspends loose, and focus in the new pipe.

(7) carry out placing 37 ℃ of dryings behind the metal spraying according to every milliliter of gold shop 10cm2 glass fibre, set 120 minutes drying time.

5, draw film

The Flag polyclonal antibody is diluted to 0.5mg/ml with the phosphate buffer of 0.01mol/L pH7.2, draws 100cm according to every milliliter and wrap quilt.

In the clinical detection process, cholerythrin in the sample, haemoglobin and blood fat etc. usually have certain influence to diagnostic result, and this test will verify whether cholerythrin, haemoglobin and blood fat be influential to the testing result of hepatitis b virus s antigen diagnostic kit (colloidal gold method).

6, be assembled into test strips

(1) on the magnetic blank of test strips, paste following component successively:

1. thieving paper,

2. glass fibre membrane is adsorbing dry golden labeling antibody on the film,

3. nitrocellulose filter, bag is by the antibody band and the Quality Control bar thieving paper (handle) that can directly react with label on the film.

4. adhesive sticker about.

(2) be cut into the paper slip of 5mm after assembling is finished with cutting knife, put into paper box, gland is formed test card, connects same drying agent and puts into aluminium foil bag, one bag of portion.

7, detect

(1) from the original packing aluminium foil bag, takes out reagent strip, use as soon as possible 1 hour planted agent.

(2) reagent strip is inserted in the sample in the direction of arrows.Attention: the sample liquid level can not surpass the mark line of reagent strip.

Take out after (3) at least 10 seconds and lie against on the clean smooth table top; Can also not take out, be placed in the specimen collection container always up to reading the result.

(4) appearance of wait aubergine band, test result should read in 5-10 minute.Observations is invalid about 20 minutes.An aubergine band only appears in test strips when negative on nature controlling line (C) position; Test strips an aubergine band respectively occurs when positive on detection line (T) and nature controlling line (C) position; Test strips the aubergine band all do not occur when invalid on detection line (T) and nature controlling line (C) position.

Claims (2)

1. the preparation of a colloid gold reagent and to the fast detecting of fusion tag Flag is characterized by: adopt double antibody sandwich method immunoassays principle, with the Flag antibody of purifying respectively as collaurum binding antibody and coated antibody.When containing in the sample to be checked when adopting FLAG as the gene engineering expression albumen of fusion tag, first and golden labelled antibody combination, because chromatography action-reaction compound moves forward along nitrocellulose membrane, when running into coated antibody, form the Ab-Ag-Ab-Au compound and be enriched in bag, form the aubergine precipitation line by on the line.On coated film, also have simultaneously a nature controlling line,, when having only a purplish red colo(u)r streak, be judged to feminine gender so when two purplish red colo(u)r streaks, be judged to the positive.

2. Flag antibody as claimed in claim 1, comprising the monoclonal antibody of one or more Flag or many anti-mixing or Flag monoclonal antibody ', Fab, F (ab ' through modifying) 2 fragments and genetic engineering Flag antibody, it is characterized in that, can with Flag label idiosyncrasy.