CN102608328A - TRFIA (Time-resolved Fluorescence Immunoassay) kit for mouse interferon alpha and detection method thereof - Google Patents

Abstract

The invention discloses a TRFIA (Time-resolved Fluorescence Immunoassay) kit for mouse interferon alpha, wherein the interferon alpha is named as IFN-alpha for short, and the kit comprises the following parts: IFN-alpha antibody coated board (1) with micropores, a buffer solution (2), a mouse IFN-alpha standard (3), a lyophilized product of a biotin IFN-alpha antibody (4), a lyophilized product of europium-labeled Eu<3+>-streptavidin (5), a cleaning solution (6) and an enhancement solution (7). A detection method of the kit comprises the steps of: taking the IFN-alpha antibody coated board (1), adding the mouse IFN-alpha standard (3) and other processed samples into respective micropores, adding the biotin IFN-alpha antibody (4), oscillating for reaction, cleaning by using the cleaning solution (6), adding the europium-labeled Eu<3+>-streptavidin (5) for reaction, cleaning by using the cleaning solution (6), adding the enhancement solution (7), oscillating, then measuring fluorescence intensity cps, and calculating the content of IFN-alpha in the samples based on a standard curve. The kit disclosed by the invention has the advantages of long validity period, short operation flow, high sensitivity, wide measuring range of the standard curve, capability of being operated fully automatically, and very wide application range.

Description

A kind of time resolved fluoro-immunoassay kit and detection method thereof of mouse IFN-

Technical field

The present invention relates to time resolved fluoro-immunoassay (TRFIA) technical field, especially relate to detection to IFN-content in mice serum, blood plasma, cell culture supernatant or other associated biomolecule liquid.

Background technology

Interferon (IFN) is a kind of broad-spectrum disease resistance toxic agent, and direct killing or inhibition are not viral, and mainly are to make cell produce antiviral protein through the cell surface receptor effect, thereby suppress duplicating of virus; But also enhanced natural killer cell (NK cell), macrophage and the lymphocytic vigor of T simultaneously, thus play immunoregulation effect, and strengthen anti-virus ability.Interferon is one group of reactive protein (mainly being glycoprotein) with multiple function, is a kind of cell factor that is produced by monocyte and lymphocyte.They on allogenic cell, have wide spectrum antiviral, influence cell growth, and differentiation, regulate multiple biological action such as immunologic function.

At present, detecting interferon method comparatively commonly used is enzyme linked immunosorbent assay (ELISA).Its advantage is high specificity, and is easy and simple to handle, is particularly suitable for the detection of batch samples; But ELISA is owing to adopt macromolecular enzyme labeling, and its sensitivity and stability and measurement range also have certain limitation.TRFIA is emerging detection technique in the ultramicron detection range, is to utilize the high sensitivity of immunoreactive high degree of specificity and mark tracer to combine and one type of micro substance detection technique setting up.

Summary of the invention

To the problems referred to above that prior art exists, the applicant provides a kind of time resolved fluoro-immunoassay kit and detection method thereof of mouse IFN-.This kit term of validity is long, operating process is short, highly sensitive, but the full automatic working of typical curve broad quantum, range of application are very extensive.

Technical scheme of the present invention is following:

A kind of time resolved fluoro-immunoassay kit of mouse IFN-; IFN-is called for short IFN-α; By forming with the lower part: the IFN-Alpha antibodies with micropore encapsulates plate (1), damping fluid (2), mouse IFN-α standard (3); The dried frozen aquatic products of biotin IFN-Alpha antibodies (4), the Eu of europium mark ³⁺The dried frozen aquatic products of-Streptavidin (5), cleansing solution (6) and enhancing liquid (7).

The detection method of kit: get the IFN-Alpha antibodies and encapsulate plate (1); Add mouse IFN-α standard (3) or other samples of handling well in micropore separately, add biotin IFN-Alpha antibodies (4) again, oscillating reactions; With cleansing solution (6) washing, add Eu again ³⁺-Streptavidin (5) reacts, and with cleansing solution (6) washing, adds to strengthen again and measures fluorescence intensity cps, the IFN-alpha content in the reference standard curve calculation sample after liquid (7) vibrates.

Detection method more specifically: get the IFN-Alpha antibodies and encapsulate plate (1); The mouse IFN-α standard (3) or other samples of handling well that add 50 l are in micropore separately; Add the biotin IFN-Alpha antibodies (4) of 50 l again with damping fluid (2) dilution; 25 ℃ vibrated 1 hour, and cleansing solution (6) is given a baby a bath on the third day after its birth inferior, added 100 l Eu with damping fluid (2) dilution again ³⁺-Streptavidin (5), 25 ℃ vibrated 0.5 hour, washed six times with cleansing solution (6), added 200 l enhancing liquid (7) vibration again and measured fluorescence intensity cps, the IFN-alpha content from the typical curve calculation sample after 5 minutes.

The micropore that said IFN-Alpha antibodies with micropore encapsulates plate (1) has 96 or 48 holes.The said sample that other are handled well can be for mice serum, blood plasma, cell culture supernatant etc. the mouse IFN-β of natural or reorganization.

Beneficial technical effects of the present invention is:

The ultimate principle of TRFIA be with trivalent rare earth ions and chelate thereof as tracer, replace fluorescent material, isotope, enzyme and chemiluminescent substance, materials such as labelled antigen, antibody, nucleic acid probe.After immune response takes place, according to the characteristics of the fluorescence spectrum of rare earth ion chelate (the Stokes displacement of high specificity, fluorescence spectrum, life-span long), differentiate fluorescence analyser with the time, measure the fluorescence intensity of immune response end product.According to fluorescence intensity and relative intensity of fluorescence ratio, judge the concentration of analyte in the reaction system, reach the purpose of quantitative test.

The TRFIA technology adopts the atom mark; Method is stable, and sterically hindered little, highly sensitive; The fluorescence counting of TRFIA can be crossed over 4 one magnitude; Overcome the absorbance scope among the ELISA and had only the restriction of 2 one magnitude, the sensitivity and the range of detection be multiplied than ELISA, makes detection sensitiveer, more stable.The TRFIA detectability that adopts the foundation of TRFIA method is 10 ^-18Mol/L is much higher than 10 of ELISA ^-10Mol/L and radiommunoassay (RIA) 10 ^-12Mol/L.

Description of drawings

Fig. 1 is the time resolved fluoro-immunoassay kit synoptic diagram of this mouse IFN-.

Among the figure: 1, the IFN-Alpha antibodies encapsulates plate; 2, damping fluid; 3, mouse IFN-α standard; 4, biotin IFN-Alpha antibodies; 5, Eu ³⁺-Streptavidin; 6, cleansing solution; 7, strengthen liquid.

Fig. 2 is fluorescent value-concentration standard curve.

Embodiment

1, the Eu of europium mark ³⁺The preparation of-Streptavidin 5:

Get solution of streptavidin (the 1mg packing of SIGMA company), its buffer system (Streptavidin generally is kept in the damping fluid with the solution form) is converted into the Na of 50 mmol/L through Sephadex G-50 ₂CO ₃-NaHCO ₃, pH is 8.5 damping fluid, this is means commonly used, purpose is to be transformed under the alkali condition to be beneficial to reaction.The good damping fluid that contains 1 mg Streptavidin adds the Eu that contains 0.2 mg to get conversion then ³⁺-N ₂-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu ³⁺In-DTTA) the bottle; 30 ℃ of magnetic agitation 20 hours; The reactant liquor that obtains through with the Sepharose CL-6B post of 80 mmol/L Tris-HCl (pH is 7.8) damping fluid balance (1 * 40cm) chromatography, Ultraviolet Detector is monitored at A280, first absorption peak is the protein peak of mark; Collect the corresponding solution of protein peak, the dilution packing is subsequent use.

2, the preparation of biotin IFN-Alpha antibodies 4:

Get l mg biotin N hydroxysuccinimide eater (BNHS) and be dissolved in 0.1 ml dimethyl formamide (DMF); Other gets antibody 1 mg of the anti-IFN-α that mark uses, and using pH is 9.0 50 mmol/L Na ₂CO ₃-NaHCO ₃Damping fluid is made into the antibody-solutions of the anti-IFN-α of 5 mg/ml; With antibody (volume ratio l:4) hybrid reaction of BNHS and anti-IFN-α, 25 ℃ of electromagnetic agitation 3 hours, reactant liquor is 7.2 Tris-HCl dialysis 24h to 50 mmol/L pH; Liquid is changed 3 times in the centre; Remove responseless BNHS, obtain biotin biotin IFN-Alpha antibodies 4, further dilution, packing, freeze-drying ,-20 ℃ of preservations.

3, the IFN-Alpha antibodies encapsulates the preparation of plate 1:

It is the Na of 9.6 50 mmol/L that the antibody of the anti-IFN-α that encapsulates use is used pH ₂CO ₃-NaHCO ₃Damping fluid is diluted to the coating buffer of 5 mg/L, and each hole of 96 (or 48) hole microwell plate adds and adds 100 μ l respectively, and 4 ℃ of placements are spent the night.Discard coating buffer, flushing twice adds pH that 150 μ l contain 3 g/L BSA and is the Na of 9.6 50mmol/L ₂CO ₃-NaHCO ₃The damping fluid sealing, 4 ℃ of placements are spent the night.Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.

4, the preparation of other reagent:

(1) preparation of mouse IFN-α standard 3: obtain 0pg/ml, 2pg/ml, 10pg/ml respectively from high concentration IFN-α (the 200uL packing of miltenyi biotec company) dilution; 100pg/ml; 500pg/ml, these 6 kinds of standards of 1000pg/ml, said dilution is a damping fluid 2;

(2) preparation of damping fluid 2: with 8 mmol/L NaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1 ml/LTween-80 and 0.1%NaN ₃PH be that 7.8 Tris-HCl mixes;

(3) preparation of cleansing solution 6: with 14.5 mmol/L NaCl, 0.2 ml/LTween-80 and 0.2%NaN ₃50 mmol/L pH be that 7.8 Tris-HCl mixes;

(4) strengthen the preparation of liquid 7: 1 liter of pH adds 15 mol β-naphthoyltrifluoroacetones (β-NTA), 50 mol TOPOs (TOPO), 1 ml triton x-100 (Triton X-100) in 3.2 the Potassium Hydrogen Phthalate damping fluid.

5, the reagent that provides of the time resolved fluoro-immunoassay kit of this mouse IFN-: the reagent in each box enough carries out 96 measurements, and the material in the box is following:

(1) 1 * 96 hole IFN-Alpha antibodies encapsulates the antibody that plate 1 (8 * 12 hole can be split as single hole) is coated with anti-IFN-α;

(2) 6 * mouse IFN-α titers 3,1.0 ml/ bottles, concentration of standard solution is: 0 pg/ml, 2 pg/ml, 10 pg/ml, 100 pg/ml, 500 pg/ml, 1000 pg/ml;

The dried frozen aquatic products of (3) 1 * biotin IFN-Alpha antibodies 4, during mensuration with 0.5 ml dissolved in distilled water;

(4) 1 * Eu3 ⁺The dried frozen aquatic products of-Streptavidin 5, during mensuration with 0.5 ml dissolved in distilled water;

(5) 1 * enhancing liquid 7:15 ml;

(6) 1 * cleansing solution 6:30 ml, the time spent dilutes with distilled water 1:25;

(7) 1 * damping fluid 2:30 ml.

6, measure points for attention before:

(1) uses before all reagent to be gone up to room temperature (18 ~ 30 ℃);

(2) immediately all reagent are put back to 2 ~ 8 ℃ after the use;

(3) if the hyperchannel pipettor is used in the big suggestion of sample size;

(4) detect in the step process at all, avoid irradiate light, use the cap covers micropore;

(5) taking-up needs microwell plate and the framework with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2 ~ 8 ℃.

7, it is following specifically to detect step:

Choosing mice serum is sample, and the preparation method is at 4000rpm centrifugal 10 minutes, gets supernatant.Get the micropore IFN-Alpha antibodies that is coated with anti-IFN-Alpha antibodies and encapsulate plate 1; The sample that adds the mouse IFN-α standard 3 of 50 l or handle well is in micropore separately; Add 50 l with the biotin IFN-Alpha antibodies 4 of damping fluid 2 with the 1:50 dilution; 25 ℃ vibrated 1 hour, and it is inferior to give a baby a bath on the third day after its birth with cleansing solution 6, added 100 l with the Eu of damping fluid 2 with the 1:50 dilution ³⁺5,25 ℃ of vibrations of-Streptavidin 0.5 hour are washed six times with cleansing solution 6; Add 7 vibrations of 200 l enhancing liquid and measure fluorescence intensity cps after 5 minutes; IFN-alpha content from typical curve (as shown in Figure 2) calculation sample sees shown in the table 1 that this routine sample concentration is 47 pg/ml.

Table 1

Claims (5)

1. the time resolved fluoro-immunoassay kit of a mouse IFN-; IFN-is called for short IFN-α; It is characterized in that by forming with the lower part: the IFN-Alpha antibodies with micropore encapsulates plate (1), damping fluid (2), mouse IFN-α standard (3); The dried frozen aquatic products of biotin IFN-Alpha antibodies (4), the Eu of europium mark ³⁺The dried frozen aquatic products of-Streptavidin (5), cleansing solution (6) and enhancing liquid (7).

2. detection method with the described kit of claim 1; It is characterized in that: get the IFN-Alpha antibodies and encapsulate plate (1); Add mouse IFN-α standard (3) or other samples of handling well in micropore separately, add biotin IFN-Alpha antibodies (4) again, oscillating reactions; With cleansing solution (6) washing, add Eu again ³⁺-Streptavidin (5) reacts, and with cleansing solution (6) washing, adds to strengthen again and measures fluorescence intensity cps, the IFN-alpha content in the reference standard curve calculation sample after liquid (7) vibrates.

3. detection method according to claim 2; It is characterized in that: get the IFN-Alpha antibodies and encapsulate plate (1); The mouse IFN-α standard (3) or other samples of handling well that add 50 l add the biotin IFN-Alpha antibodies (4) of 50 l with damping fluid (2) dilution again in micropore separately, 25 ℃ of vibrations 1 hour; Cleansing solution (6) is given a baby a bath on the third day after its birth inferior, adds 100 l Eu with damping fluid (2) dilution again ³⁺-Streptavidin (5), 25 ℃ vibrated 0.5 hour, washed six times with cleansing solution (6), added 200 l enhancing liquid (7) vibration again and measured fluorescence intensity cps, the IFN-alpha content from the typical curve calculation sample after 5 minutes.

4. the time resolved fluoro-immunoassay kit of mouse IFN-according to claim 1 is characterized in that the micropore that said IFN-Alpha antibodies with micropore encapsulates plate (1) has 96 or 48 holes.

5. according to claim 2 or 3 described detection methods, it is characterized in that the said sample that other are handled well can be natural for mice serum, blood plasma, cell culture supernatant etc. or the mouse IFN-β of reorganization.

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