CN104371981B - Method for preparing antigen tablet for detecting varicella-zoster virus antibody - Google Patents
Method for preparing antigen tablet for detecting varicella-zoster virus antibody Download PDFInfo
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- CN104371981B CN104371981B CN201410551513.1A CN201410551513A CN104371981B CN 104371981 B CN104371981 B CN 104371981B CN 201410551513 A CN201410551513 A CN 201410551513A CN 104371981 B CN104371981 B CN 104371981B
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Abstract
The invention provides a method for preparing an antigen tablet for detecting the varicella-zoster virus antibody. According to the method, a varicella-zoster virus VZV84-7 strain is used as an infected virus strain to prepare the antigen tablet; compared with an Oka strain infected diploid cell, the virus has the characteristics that the virus CPE is in a dispersed manifestation and is widely distributed on the surface of the cell after infecting a sensitive cell; a Vero cell is selected as a cell matrix, and can be dispersed more sufficiently than the diploid cell, and the cellular morphology is more completed; after the infected virus is reproduced, the CPE widely occurs to the cell, so that observation is facilitated and results can be determined when the virus infected cell is used for preparing the antigen tablet. By adopting the method, the problems of low antigen tablet quality and complicated tablet producing process in varicella-zoster antibody detection by an FAMA method can be solved, the preservation time of the antigen tablet can be prolonged, and conditions are provided to massive preparation of the antigen tablet. Compared with a traditional method, the method has the advantages of simplicity and practicality, the antigen tablet can be frozen at -20-70 DEG C and stored for over one year without remarkable quality change, and the result of tablet microscopic examination is not influenced.
Description
technical field:
The present invention relates to a kind of varicella virus antibody test antigen slide preparation method, make cell suspension with digestion after varicella zoster virus cells infected to cytopathy, then carry out the preparation of antigen slide, belong to biological technical field.
background technology:
Varicella zoster virus antibody detection method conventional at present has FAMA method, milk-globule assay method and ELISA method.ELISA method is convenient and swift is applicable to extensive pattern detection, but it is too high to there is false negative rate in actual applications, can not the defect of the good protectiveness of reaginic antibody.Milk-globule assay method operation relative complex, subjectivity is strong, is applicable to pattern detection in a small amount.FAMA method is the gold standard of the varicella zoster virus antibody test of generally acknowledging in the world at present, has highly sensitive, the feature of high specificity.
Traditional FAMA method, each laboratory all adopts varicella zoster virus Oka strain infection diploid cell to prepare antigen slide, after this virus cell, cytopathic manifestation is banded CPE, therefore when preparing antigen slide, occur that virus dispersion on cell is uneven, microscopy and result of determination are had a certain impact.And the antigen slide preservation period using traditional method to prepare is short, can only preserve about one month under-20 ~-70 DEG C of preservation conditions.Quality and the shelf time of the stability of antigen slide and the reproducibility of experimental result and antigen slide are closely related, there is no bibliographical information at present and how to extend the conserving time limit of antigen slide and the novel preparation method of antigen slide.
summary of the invention:
The invention provides a kind of varicella virus antibody test antigen slide preparation method, solution FAMA method detects the of low quality and film-making complicate problem of antigen slide in varicella antibody, extend the shelf time of antigen slide simultaneously, prepare antigen slide for mass and provide condition.
a kind of varicella virus antibody test antigen slide preparation method of the present invention, is characterized in that comprising the steps:
Varicella zoster virus-virus infects VZV permissive cell, put 35 DEG C of incubators and be cultured to virus replication peak, through tryptic digestion, carry out cell counting, suitable cell quantity is selected to prepare virus-cell suspension, cell suspension is instilled on 12 hole slide glasss, after 35 DEG C of cultivations, cleaning, cold acetones fix drying, be stored in-20 ~-70 DEG C.
Infection varicella zoster virus of the present invention strain is preferably varicella zoster virus VZV 84-7 strain.
Cell matrix of the present invention is preferably Vero cell.
The cell density 2.5-3.5 × 10 growing into individual layer of the present invention
5between/ml.
Virus titer for cells infected seed culture of viruses of the present invention is between 4.9-5.2lgPFU/ml; Infection multiplicity (MOI) is 0.01-0.05.
Varicella virus antibody test antigen slide preparation method of the present invention, the trypsinase working concentration for infected cell Digestive system is 0.25%.
Varicella virus antibody test antigen slide preparation method of the present invention, the virocyte for the preparation of (FAMA method) antigen slide counts in 1.2-1.8 × 10
5between/ml.
Varicella virus antibody test antigen slide preparation method of the present invention, in antigen slide preparation process, prepares virus-cell suspension without the need to centrifugal, recyclable after acetone fixed cell sheet.Virocyte sheet after fixing, can be directly frozen without the need to embathing through PBS.
the invention provides a kind of varicella virus antibody test antigen slide preparation method, concrete steps are as follows:
Growth selection is to the Vero cell of individual layer, and density is about 2.5-3.5 × 105/ml; Varicella zoster virus strain VZV 84-7 virus titer is 4.9-5.2lgPFU/ml; With infection multiplicity 0.01-0.05 cells infected; Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, with 5.6%NaHCO3 adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion without the need to centrifugal, carry out cell counting; Add the MEM nutrient solution containing 1% glutamine 5% new-born calf serum pH value 7.4 ± 0.1, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing 1 time, dries naturally, directly fixes 15 minutes, after taking-up by cold acetone room temperature, directly puts into film magazine ,-20 DEG C of preservations.
positively effect of the present invention is:adopt varicella zoster virus VZV84-7 strain to prepare antigen slide as infection strain, compared with infecting diploid cell with Oka strain, after this virus infection sensitive cells, its principal feature is that the manifestation of this viral CPE is dispersed in, and is distributed in cell surface widely; Select Vero cell as cell matrix, more abundant than diploid cell dispersion, and cellular form is more complete, after infecting virus multiplication, CPE extensively occurs in cell, prepares antigen slide be more convenient for observing and judging result with this virus infected cell.
The antigen slide preparation method provided simplifies preparation process, without centrifugal, directly carries out dripping a sheet with growth media suspension cell, under making cell be in a more suitable condition, and cell maintenance integrity highly, the antigen slide qualification rate of preparation is higher; The antigen slide of preparation through dripping a sheet, clean, dry after fix with cold acetone, frozenly before do not need PBS again to clean, save test period and unnecessary trouble; Solve and film-making complicate problem of low quality with antigen slide in FAMA method detection varicella antibody, extend the shelf time of antigen slide simultaneously, prepare antigen slide for mass and provide condition; The present invention is simple compared with traditional method, can more than-20 ~-70 DEG C of freezen protective to year, and quality is without considerable change, and microscopy sheet result is unaffected.
Accompanying drawing explanation
Fig. 1 is that under vero cell and 2BS cell prepare antigen slide fluorescent microscope, observing effect compares picture.
Embodiment
Following embodiment further illustrates content of the present invention, but should not be construed as limitation of the present invention, and without departing from the spirit and substance of the case in the present invention, the amendment do method of the present invention, step or condition or replacement, all belong to scope of the present invention.
embodiment 1
Growth selection is to the Vero cell of individual layer, and density is about 2.5-3.5 × 10
5/ ml; Varicella zoster virus strain VZV 84-7 virus titer is 4.9lgPFU/ml; With infection multiplicity 0.01 cells infected; Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, uses 5.6%NaHCO
3adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion, carry out cell counting; Add the MEM nutrient solution containing 1% glutamine 5% new-born calf serum pH value 7.4 ± 0.1, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing 1 time, dries naturally, directly fixes 15 minutes, after taking-up by cold acetone room temperature, directly puts into film magazine ,-20 DEG C of preservations.
embodiment 2
Growth selection is to the Vero cell of individual layer, and density is about 2.5-3.5 × 10
5/ ml; Varicella zoster virus strain VZV 84-7 virus titer is 5.2lgPFU/ml; With infection multiplicity 0.01 cells infected; Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, uses 5.6%NaHCO
3adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion, carry out cell counting; Add the MEM nutrient solution containing 1% glutamine 5% new-born calf serum pH value 7.4 ± 0.1, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing 1 time, dries naturally, directly fixes 15 minutes, after taking-up by cold acetone room temperature, directly puts into film magazine ,-20 DEG C of preservations.
embodiment 3
Growth selection is to the Vero cell of individual layer, and density is about 2.5-3.5 × 10
5/ ml; Varicella zoster virus strain VZV 84-7 virus titer is 4.9lgPFU/ml; With infection multiplicity 0.05 cells infected; Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, uses 5.6%NaHCO
3adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion, carry out cell counting; Add the MEM nutrient solution containing 1% glutamine 5% new-born calf serum pH value 7.4 ± 0.1, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing 1 time, dries naturally, directly fixes 15 minutes, after taking-up by cold acetone room temperature, directly puts into film magazine ,-20 DEG C of preservations.
embodiment 4
Growth selection is to the Vero cell of individual layer, and density is about 2.5-3.5 × 10
5/ ml; Varicella zoster virus strain VZV 84-7 virus titer is 5.2lgPFU/ml; With infection multiplicity 0.05 cells infected; Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, uses 5.6%NaHCO
3adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion, carry out cell counting; Add the MEM nutrient solution containing 1% glutamine 5% new-born calf serum pH value 7.4 ± 0.1, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing 1 time, dries naturally, directly fixes 15 minutes, after taking-up by cold acetone room temperature, directly puts into film magazine ,-20 DEG C of preservations.
positively effect more of the present invention is carried out by following traditional varicella virus antibody test antigen slide preparation method:
comparative example 1
Varicella zoster virus VZV Oka strain (titre 4.9lgPFU/ml) infects the 2BS cell growing to individual layer with 0.01 MOI, cell density is about between 2.5 ~ 3.5 × 105/ml.Virus infection liquid is the MEM containing 1% glutamine, 2% new-born calf serum, with 5.6%NaHCO3 adjust pH to 7.5 ± 0.1.Put 35 DEG C of constant incubators and be cultured to pathology peak, through 0.25% tryptic digestion, add growth media piping and druming cell, collecting cell carries out centrifugal, and it is resuspended to add PBS in precipitation, through cell counting, regulate cell concn to 1.2 ~ 1.8 × 105/ml, instill in 12 hole slide glasss, every hole 20 ~ 25 μ l, put 35 DEG C, 5%CO2 incubator wet box after 30 minutes cold acetone room temperature fix 15 minutes, after taking-up, PBS cleans three times, puts into film magazine after drying ,-20 ~-70 DEG C of preservations.
comparative example 2
Varicella zoster virus VZV 84-7 strain (titre 4.9lgPFU/ml) infects 2BS cell with 0.01 MOI, and cell density is about between 2.5 ~ 3.5 × 105/ml.Virus infection liquid is the MEM containing 1% glutamine 2% new-born calf serum, adjusts pH value to 7.5 ± 0.1 with 5.6%NaHCO3.Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak, through 0.25% tryptic digestion, carry out cell counting, add MEM nutrient solution, regulate cell concn to 1.2 ~ 1.8 × 105/ml, prepare cell suspension.Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, puts 35 DEG C, after 5%CO2 incubator hatches 30 minutes, PBS cleans 2 times, naturally dries, and directly fixes 15 minutes by cold acetone room temperature, after taking-up, directly put into film magazine ,-20 ~-70 DEG C of preservations.
conclusion:
1,observation of cell form under antigen slide inverted microscope prepared by embodiment 1 and comparative example 1, cellular form size is even, and full person meets next step requirement of experiment.Adopt with a collection of cell, prepare antigen slide by two kinds of methods respectively, each 100 respectively.Basis of microscopic observation, compares antigen slide microscopy qualification rate (%) prepared by two kinds of methods, and antigen slide qualification rate prepared by the method that result the present invention adopts is apparently higher than traditional method.The results are shown in Table 1:
Table 1: antigen slide microscopy qualification rate prepared by two kinds of methods compares
| Batch | 1 | 2 | 3 |
| Comparative example 1 | 77.2±7.7 | 83.5±5.5 | 79.0±8.2 |
| The present invention | 89.1±2.9 | 93.8±4.3 | 94.3± |
2, Microscopic observation after the antigen slide fluorescent dye prepared of embodiment 1,2,3,4 and comparative example 1, the antigen slide cell integrity that Vero cell is prepared as cell matrix is apparently higher than 2BS cell.And vero cell individual cells good dispersion degree, more easily observe.The cell that VZV84-7 infects, the outer green fluorescence of coating is evenly distributed, and is easy to observations and judges.See Fig. 1.
3, the antigen slide that prepared by embodiment 1 and comparative example 1 is used for concrete testing inspection.Serum sample PBS to be detected carries out gradient dilution, 2,4,8,16,32,64,128,256,512,1028 times of dilutions, is added in 12 hole antigen slide respectively, and every hole 20 μ l, adds PBS in addition as negative control.Each sample standard deviation does 10 holes.37 DEG C hatch 20 minutes after, PBS cleans three times, and add two anti-(1:100 dilutions) of FITC mark, every hole 20 μ l, hatches 20 minutes for 37 DEG C.PBS cleans three times, adds azovan blue staining fluid, fluorescence microscopy Microscopic observation.The cell that VZV84-7 infects, the outer green fluorescence of coating is evenly distributed, and is easy to observations and judges.As can be seen from table, adopt VZV84-7 cells infected, result is more stable, reproducible, and the variation coefficient (CV) is much smaller than comparative example 1.Wherein embodiment 1 represents with T-1, and comparative example 1 represents with B-1.
Table 2 two kinds of methods detect varicella zoster antibody comparative result
4, the antigen slide that prepared by embodiment 1 and experimental example 1 takes out respectively at preservation for 1,3,6,9,12 months, observes the shelf time to the impact of antigen slide effect.
Microscopic observation, it is full, complete that antigen slide prepared by embodiment 1 preserves cellular form in the antigen slide of 1-12 month, meets requirement of experiment.And antigen slide prepared by traditional method is preserved and within 1 month, is occurred that cytomorphosis is shrivelled.
Adopt with a serum sample, carry out the detection of antigen slide.Serum sample PBS carries out gradient dilution, 2,4,8,16,32,64,128,256,512,1028 times of dilutions, is added in 12 hole antigen slide respectively, and every hole 20 μ l, adds PBS in addition as negative control.37 DEG C hatch 20 minutes after, PBS cleans three times, and add two anti-(1:100 dilutions) of FITC mark, every hole 20ul, hatches 20 minutes for 37 DEG C.PBS cleans three times, adds the blue staining fluid of Yi Weisi, detects under fluorescent microscope.Fluorescence microscopy Microscopic observation, the cell that VZV84-7 infects, the outer green fluorescence of coating is evenly distributed, and is easy to observations and judges.The results are shown in Figure 1.The antigen slide cryopreservation prepared of improving one's methods still had good antigenicity after 12 months.The results are shown in Table 3.
The antigen slide FAMA method detected result of table 3 different shelf time;
Claims (1)
1. a varicella virus antibody test antigen slide preparation method, is characterized in that comprising the steps:
Adopt VZV84-7 attenuated strain virus titer to be 4.9 ~ 5.2lgPFU/ml, infecting vero cell MOI is 0.01 ~ 0.05, and virus infection liquid is that MEM nutrient solution includes 1% glutamine, 2% new-born calf serum, with 5.6%NaHCO3 adjust pH to 7.5 ± 0.1; Infected cell is put 35 DEG C of constant incubators and be cultured to virus replication peak; Through 0.25% tryptic digestion, add the MEM nutrient solution containing 5% new-born calf serum, regulate cell concn to 1.2 ~ 1.8 × 10
5/ ml, prepares cell suspension; Instilled by cell suspension in 12 hole slide glasss, every hole 20 ~ 25 μ l, is placed in wet box by slide glass, is placed in 35 DEG C, 5%CO
2after incubator hatches 30 minutes, PBS rinsing once, is dried naturally, and cold acetone room temperature directly puts into film magazine after fixing 15 minutes ,-20 DEG C ~-70 DEG C preservations.
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Inventor after: Shen Hongjie Inventor after: Shuang Hui Inventor after: Zhang Yu Inventor after: Wang Wei Inventor after: Zheng Xiaoli Inventor after: Li Haiyan Inventor after: Li Shengjun Inventor after: Zhao Haibo Inventor after: Yan Lei Inventor after: Zhu Xiaowen Inventor before: Shen Hongjie Inventor before: Shou Dao Inventor before: Li Haiyan Inventor before: Zheng Xiaoli Inventor before: Li Shengjun Inventor before: Zhao Haibo Inventor before: Yan Lei Inventor before: Zhu Xiaowen Inventor before: Shuang Hui Inventor before: Zhang Yu |
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Free format text: CORRECT: INVENTOR; FROM: SHEN HONGJIE LI HAIYAN ZHENG XIAOLI LI SHENGJUN ZHAO HAIBO YAN LEI ZHU XIAOWEN SHUANG HUI ZHANG YU SHOU DAO TO: SHEN HONGJIE ZHANG YU WANG WEI ZHENG XIAOLI LI HAIYAN LI SHENGJUN ZHAO HAIBO YAN LEI ZHU XIAOWEN SHUANG HUI |