CN1196394A - Enzyme immuno spot method for determining virus titre - Google Patents

Abstract

An enzyme immunospotassay for measuring virus titer includes such steps as single-layer cells preparation, diluting virus, culturing infection cells, fixing, enclosing, adding-in specific antibody of the virus, addition of enzyme-linked second antibody, and adding enzyme substrate. The coloured sports appear after several minutes. If CPE appears in each hole can be directly determined by eyes without microscope. Its advantages include high speed, saving labour, and high accuracy.

Description

Measure the enzyme immune spot-ing of virus titer

The present invention relates to the measuring method of virus titer, particularly enzyme immune spot-ing (EIS) is measured virus titer.

The mensuration that virological research and production process all relate to virus quantity, the i.e. mensuration of virus titer are used in the virusology theoretical investigation.Mainly contained animal law, plaque method and cytopathy political reform with existing measuring method in the past, wherein commonly used is plaque method and cytopathy political reform, and particularly cytopathy political reform (CPE) is easy than the plaque method because of it, be beneficial to the mensuration of multiple sample, adopt in many mensuration in batches of being everlasting.But the CPE method is to serve as to judge index with microscopically observation of cell pathology, and calculates half cell infection dosage (TCID according to this ₅₀); Test is to operator's technical requirements height, and the cytopathy that different virus causes is variant also to be had identically, causes the pathology result to be difficult for recognizing.Therefore, it is bigger that test-results is influenced by subjective factor, and operate miss is big, and in addition, the CPE workload is big, the working strength height, and the operating time is long.

The measuring method that the purpose of this invention is to provide a kind of virus titer, this method is easy, quick, be convenient to write down the result, and operate miss is little.

For achieving the above object, the present invention takes following technical scheme: a kind of enzyme immune spot-ing of measuring virus titer, and it comprises following step:

(1) monolayer cell is prepared, and adds cell suspension in each hole of Tissue Culture Plate;

(2) viral dilution, and the virus of different concns is added to respectively in the cell cultures plate hole of measuring usefulness after will diluting;

(3) cultivation of cells infected;

(4) cells infected in the step (3) is fixed, sealed;

(5) add this viral specific antibody;

(6) add the second antibody of enzyme labelling;

(7) add enzyme substrates, have the position of pathology dyeing speck promptly to occur after several minutes.

In the said determination step, preparing to cultivate to cells infected from cell is the routine techniques method, most viruses all are suitable for, in the mensuration of making concrete virus, only need change this viral specific antibody and get final product, in all the other each cultivations, the reactions steps, temperature is controlled between 36-37 ℃, the antibody response time be controlled at 20 minutes to 60 minutes (dezymotize with the substrate-function time short outside, the substrate antibody response at room temperature carries out).

The present invention utilizes immunoreactive specificity and zymochemistry reaction amplification characteristics, creatively form dyeing speck, making microscopically just can debate the cytopathy of not coming out shows with macroscopic form, and guaranteed the specificity (combination of Ag-Ab specificity) of this manifestation mode, enzyme linked immunosorbent assay in the past (EIA) is all measured the colour-change that the OD value is come result of determination or observed liquid with color reaction, its antigen, antibody response carries out under solubilized and dispersive state, the substrate colour developing also shows in being dissolved in liquid again, and EIS is that with the different of EIA the former reaction and performance all take place at the cytopathy position, be localized, tangible, immunohistochemistry technology compares with enzyme, EIS directly detects dyeing speck on Tissue Culture Plate, important difference is arranged.

Advantage of the present invention: make the CPE position that occurs on the micro plate form special dyeing speck by introducing the enzyme immunological technique, thereby can judge directly with the naked eye whether each hole CPE occurs, remove microscopically from and observe the workload increase that CPE causes, make the result objective, more directly perceived, fast, be convenient to record, operate miss is little.

The invention will be further described below in conjunction with embodiment:

Embodiment 1:

1, get cultivation 3-5 days, well-grown FL cell (the Changchun life is ground institute and provided) is after the cell dissociation buffer processing, abandon dried Digestive system, (the MEM substratum contains 5% calf serum, and penicillin, each 10,000 u/ml of Streptomycin sulphate are diluted to 30-50 * 10 in cell culture fluid with cell ⁴/ ml concentration.Every hole adds cell suspension 100 μ l on 96 porocyte culture plates.

2, get Measles virus (50 batches of Changchun Biological Products Institute's (long 47 strains)) and in test tube, be diluted to 10 ^-4, 10 ^-5Concentration adds respectively in the cell cultures plate hole, and each concentration adds 8 holes, and every hole 100 μ 1 continue to do cell and cultivate.

3, culture plate places 5%CO ₂In the incubator, 37 ℃, 8 days,

The visible planned immunization technical supervision of above-mentioned several steps rules (Ministry of Health 1987).

4, fixing: the culture plate that will cultivate 8 days is abandoned its nutrient solution, and each hole adds stationary liquid (70% acetone/PBS (phosphate buffered saline buffer)) 150 μ l, puts ice chest interior 15 minutes, discards stationary liquid, the ventilation airing, put-20 ℃ standby or directly carry out next step.

5, sealing: it (is PBS (0.01M that each hole adds diluent (PBS/BT), PH7.2)+0.5% gelatin and 0.15% tween 20 (Tween-20)) 150 μ l, 37 ℃, 30 minutes, use washing lotion (PBS/T) (PBS+0.05% tween 20 (Tween-20)) to wash again 3 times.

6, add anti-Measles virus monoclonal antibody (M _cA _b) V ₁₇Strain, this antibody was with PBS/GT dilution 1: 3000, and every subsequently hole adds 75 μ l, and 37 ℃, 40 minutes, (washing lotion was the same to wash 6 times.

7, add anti-mouse IgG-peroxidase labelling thing (Ab-E) (U.S. ZYmed company product)

Every hole added 75 μ l and put 37 ℃, 40 minutes, washes 6 times (washing lotion is the same) with PBS/GT dilution 1: 3000 time spent.

Add substrate: add 200 μ lTMB (be Sigma company product, 100mgTMB is dissolved among the 20mlDmso, is distributed into the 1ml/ bottle, put-20 ℃ standby) with the substrate diluent of 10ml (with the 1.0M citric acid with the 0.1M sodium-acetate to PH5.5) and add 20%H again ₂O ₂2 μ l, every hole adds 75 μ l behind the mixing, places 5-20 minute under the room temperature.

The result judges: visual inspection under the white background, and it is positive blue color spot point to occur, and dyeing speck does not appear in the control wells that does not add virocyte, writes down each hole situation and sees picture (accompanying drawing 1).

Two " S " refer to two duplicate samples among Fig. 1, and " 4 ,-5 " refer to 10 ^-4With 10 ^-5Two extent of dilution, NO.V.Contr refer to not have the contrast of virus, promptly do not have the cell of infective virus not occur dyeing speck, negative control after treatment.

Virus titer calculates (Karber method): calculate half cell infection concentration (TCID ₅₀), represent titration of virus with its logarithm, $\mathrm{LogTCI}{D}_{50}=X_m+d\left(\frac{50-\mathrm{\Σpi}}{100}\right)$

The logarithm of Xm=virus maximum concentration

D=extension rate logarithm

Σ P _i=each extent of dilution pathology (spot) hole count occurs and accounts for percentage ratio with the used hole count of extent of dilution, in formula, and Xm=-4, d=1, denominator was 8 when Pi calculated, as 10 ^-4There are 6 holes spot to occur under the concentration, then P _-4=6/8 * 100%=75%, promptly 10 ^-4There is 75% hole spot to occur under the concentration.

The result:

1, Shi Yan 50 crowdes of virus titer (TCID ₅₀/ ml), minimum less than 4.5, the highest by 6.5, distributing sees Table 1,50 batch of virus titer and compares with the CPE method, and 49 parts conform to, coincidence rate 98.0% (49/50), a copy of it virus titer is inconsistent, and EIS method titre is 6.50, and the CPE method is 6.375.

2,50 batches of shared 800 test holess of virus titer, the total coincidence rate of each hole result (+/-) is 99.88% (799/800).

Spot does not appear in all negative control holes.

3, calculate specificity with 800 test holes results (+/-), calculate by following mode: true positives is counted several 510 0 510 false positives of false negative and is counted this test of true negative number 1 289 290

Sum 800 susceptibility=510/510 * 100%=100% specificity=289/290 * 100%=99.66%

50 crowdes of Measles virus EIS of table 1 method titre distributes

On continuous	??5.50	??5.625	??5.75	??5.85	????6.0	??6.125	??6.25	??6.375	??6.50
										???3	?????5	???6	????7	?????3	????5	????4	????4	????2

Claims (6)

1, a kind of enzyme immune spot-ing of measuring virus titer, it is characterized in that: it comprises following step:

(1) monolayer cell is prepared, and adds cell suspension in each hole of Tissue Culture Plate;

(2) viral dilution, and the virus of different concns is added to respectively in the cell cultures plate hole of measuring usefulness after will diluting;

(3) cultivation of cells infected;

(4) cells infected in the step (3) is fixed, sealed;

(5) add this viral specific antibody;

(6) add the second antibody of enzyme labelling;

(7) add enzyme substrates, have the position of pathology dyeing speck promptly to occur after several minutes.

2, the enzyme immune spot-ing of mensuration virus titer according to claim 1, it is characterized in that: described virus is Measles virus, specific antibody is anti-Measles virus monoclonal antibody, and the second antibody of enzyme labelling is anti-mouse IgG one peroxidase labelling thing.

3, the enzyme immune spot-ing of mensuration virus titer according to claim 2 is characterized in that: described anti-Measles virus monoclonal antibody is diluted with PBS/GT, adds on the cells infected after the sealing, and temperature is controlled at 36-37 ℃.

4, the enzyme immune spot-ing of mensuration virus titer according to claim 2 is characterized in that: the described antibody response time was controlled at 20-60 minute, 37 ℃ of temperature.

5, mensuration virus titer enzyme immune spot-ing according to claim 1, it is characterized in that: described adding enzyme labelling thing dilutes with PBS/GT, and temperature is controlled at 36-37 ℃.

6, mensuration virus titer enzyme immune spot-ing according to claim 1, it is characterized in that: the described antibody response time was controlled at 20-60 minute, 37 ℃ of temperature.

Images