CN1228634C - Enzyme immunological method for quickly detecting rubella virus antibody - Google Patents
Enzyme immunological method for quickly detecting rubella virus antibody Download PDFInfo
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Abstract
The present invention discloses an enzyme immunological method for rapidly detecting rubella virus antibodies. PEG is mainly utilized to accelerate antigen-antibody reaction, so that the reaction time of conventional ELISA is shortened. Firstly, soluble antigens are coated in an enzyme labeling plate. Secondly, PBS washing liquid with the pH of 9.6 and diluting solution as a sample containing PEG are prepared. Thirdly, serum to be detected, positive control serum and negative control serum are diluted by the diluting solution as a sample containing PEG. Fourthly, the diluted serum is added into the hole of the enzyme labeling plate for incubation. Fifthly, antigen solution in the hole of the enzyme labeling plate is thrown off, and the PBS washing liquid is used for washing. Sixthly, known enzyme labeled antibodies are diluted by the diluting solution containing PEG. Seventhly, the diluted enzyme labeled antibodies are added into the hole of the enzyme labeling plate for incubation. Eighthly, the enzyme labeling plate is taken out and is washed by PBS. Finally, substrates are added for color development. The technology of the present invention has the advantages of convenient operation, high sensitivity, high specificity, short detecting time and low cost and is mainly used for the rapid detection of rubella virus infection during the gestation period as well as epidemiologic survey.
Description
Technical field
The invention belongs to the clinical medicine detection range, the enzyme immunological method that more specifically relates to a kind of improvement detects the method for rubella virus.
Background technology
Rubella virus is one of two kinds of viruses that cause the fetus severe deformities.If no matter the conceived the first four months primary infection of gravid woman rubella virus is dominance or subclinical infection, all can cause miscarriage, stillborn foetus and congenital rubella syndrome (CRS).Therefore, the early diagnosis of rubella virus infection and prenatal and postnatal care, improve the overall quality of newborns very important.At present the early diagnosis of rubella relies on mainly that specific antibody is the detection of immunoglobulin M (IgM) in the serum clinically, the conventional indirect enzyme-linked immunosorbent adsorption experiment of domestic extensive employing (ELISA), because its complex operation, long flow path, the step that how to simplify the operation, shortening detection time is the important topic that each scholar inquires into for many years.At present the enzyme immunological method of the various detection rubella virus IgM antibodies of research such as enzyme join prize law (EIA), mix sandwich enzyme test (Indirect mix enzyme immunosorbent assay indirectly, IMEIA), though traditional indirect elisa method has been done a lot of improvement at aspects such as specificity, susceptibility, ubiquity shortcomings such as complex operation, length consuming time or price height.
The step that conventional indirect enzyme-linked immunosorbent adsorption experiment detects rubella virus IgM antibody is:
1. the preparation carbonate buffer solution is dissolved in 1.59 gram sodium carbonate and 2.93 gram sodium bicarbonates in 1000 milliliters of (ml) distilled waters.
2. the rubella soluble antigen is done the suitableeest antigen concentration dilution (the suitableeest antigen concentration can be determined according to the chessboard titrimetry) with carbonate buffer solution.
3. with the antigen sensibilization polystyrene ELISA Plate of dilution, every hole adds 100 microlitres (ul), 4 ℃ of overnight incubation.
4. the phosphate buffer of secure ph 9.6 (PBS) in an amount of distilled water, adds 4.3 gram potassium dihydrogen phosphates and 68 gram sodium chloride with 4.8 gram sodium hydrogen phosphate heating for dissolving again, behind the stirring and evenly mixing, supplies solution total amount to 10000 milliliter.
5. get above-mentioned PBS500ml, add the Tween-20 of 50 microlitres, be made into the PBS washing lotion that contains 1/1000 Tween-20.
6. get PBS500ml, add the calf serum of 25ml, be made into the PBS dilution that contains 5% cow's serum.
7. get rid of the antigenic solution in the ELISA Plate hole, with above-mentioned PBS washing lotion washing 3 times, each 3～5 minutes.
8. after test serum and positive control and negative control sera were done 1: 200 (or 1: 100) dilution with above-mentioned PBS dilution, every hole added 100ul in ELISA Plate, put in the wet box 37 ℃ of effects 1 hour.Take out with the washing of PBS washing lotion, method is the same.
With the goat anti-human immunoglobulin MIgM of horseradish peroxidase (HRP) mark with dilution (or 1: 2000,1: the 3000) dilution of doing 1: 1500, every hole adds 100ul, puts in the wet box 37 ℃ of effects 1 hour.
10. preparation substrate buffer solution adds the citric acids of 3.4 gram sodium acetates and 5.25 grams mixing in the distilled water of 500ml, adjust pH to 4.8.
11. the preparation substrate solution, tetramethylethylenediamine (TMB) and the 10ul hydrogen peroxide (H of adding 100ul 1% in the above-mentioned substrate buffer solution of 10ml
2O
2) mixing.
12. the taking-up ELISA Plate, with PBS washing 3 times, method is the same.Every hole adds this potpourri of 100ul, puts in the warm box 37 ℃ of effects 15 minutes, absorbance or visual inspection result when reading wavelength being 450 nanometers on microplate reader.Criterion is positive with ratio P/N 〉=2.1 of known negative control serum (N) with serum to be checked (P).
This method is removed the envelope antigen time, and each Buwen was respectively 1 hour the time of educating, and 1 hour, 15 minutes, total consuming timely reach 2～3 hours, be unsuitable for and should promptly detect and large-scale epidemiology survey.The step that enzyme connection prize law (EIA) detects rubella virus IgM antibody is:
1. prepare antibody sandwich liquid.
2. goat-anti people IgM is diluted with coating buffer.
With goat-anti people IgM solution bag by in ELISA Plate, 37 ℃ of incubations 2 hours.
4. the PBS of system pH value 9.6 in an amount of distilled water, adds 1.48 gram sodium hydrogen phosphate heating for dissolving again 0.43 gram potassium dihydrogen phosphate and 6.80 and restrains sodium chloride, behind the stirring and evenly mixing, supplies solution total amount to 1000 milliliter.
5. above-mentioned PBS500ml adds 50 microlitre Tween-20s, is made into the PBS washing lotion that contains 1/1000 Tween-20.
6. get the PBS of 250ml, add the cow's serum of 50ml, be made into the PBS confining liquid of 20% cow's serum.
7. get PBS250ml, add the calf serum of 12.5ml, be made into the sample dilution that contains 5% cow's serum.
8. get rid of the goat-anti people IgM solution in the ELISA Plate hole, with above-mentioned PBS washing lotion washing 3 times, each 3～5 minutes.
9. every hole adds confining liquid 100ul, and ambient temperature overnight is washed 3 times, and method is the same.
10. every hole adds rabbit wind resistance exanthema virus antibody, and 37 ℃ of effects were washed plate after 1 hour, and method is the same.
11. every hole adds goat-anti rabbit IgM enzyme labelled antibody, 37 ℃ of effects were washed plate after 1 hour, and method is the same.
12. every hole adds rabbit wind resistance exanthema virus antibody, 37 ℃ of effects were washed plate after 1 hour, and method is the same.
13. after test serum and positive control and negative control sera were done 1: 200 (or 1: 100) dilution with the sample dilution, every hole added 100ul in ELISA Plate, put in the wet box 37 ℃ of effects 1 hour.Take out with the washing of PBS washing lotion, method is the same.
13. the preparation substrate buffer solution adds the citric acids of 3.4 gram sodium acetates and 5.25 grams mixing in the distilled water of 500ml, adjust pH to 4.8.
14. the preparation substrate solution, tetramethylethylenediamine (TMB) and the 10ul hydrogen peroxide (H of adding 100ul 1% in the above-mentioned substrate buffer solution of 10ml
2O
2) mixing.
15. the taking-up ELISA Plate, with PBS washing 3 times, method is the same.Every hole adds this potpourri of 100ul, puts in the warm box 37 ℃ of effects 15 minutes, absorbance or visual inspection result when reading wavelength being 450 nanometers on microplate reader.Criterion is positive with ratio P/N 〉=2.1 of known negative control serum (N) with serum to be checked (P).
Enzyme connection prize law (EIA) is used to detect rubella virus IgM antibody, and more conventional indirect elisa method specificity obviously improves, and can eliminate the influence of the RF factor that may occur in the ELISA method, but this method susceptibility is low than indirect ELISA, and complex steps.
Utilize the indirect elisa method of monoclonal antibody in addition in addition, also can improve the specificity and the susceptibility of classic method greatly as enzyme labelled antibody, but because Monoclonal Antibody process complexity, and cost an arm and a leg, should not popularize.
Summary of the invention
The object of the present invention is to provide a kind of enzyme immunological method of fast detecting rubella virus antibody, make it easy and simple to handle, detection time is short, and expense is cheap, susceptibility and specificity height.
For achieving the above object, the present invention adopts following technical measures:
Polyglycol (PEG) is the different compound of molecule amount that 1,2 ethylene glycol is polymerized, and uses the more Macrogol 4000 (molecular weight is 4000) that has at present, Macrogol 6000, cetomacrogol 1000 0, polyglycol 12000, Macrogol 2000 0 etc.Recent study shows that the polyglycol compounds can improve the susceptibility of antigen-antibody reaction, accelerates the speed of antigen-antibody combination.The polyglycol of molecular weight 6000 is used for fast detecting rubella virus antibody, and add simultaneously in serum dilution and the enzyme labeling thing dilution (the enzyme labeling substrate concentration improves than indirect elisa method), to impel the acceleration of antigen-antibody reaction,, simplify the operation to shorten the reaction time.
Concrete steps:
1. the preparation carbonate buffer solution is dissolved in 1.59 gram sodium carbonate and 2.93 gram sodium bicarbonates in 1000 milliliters of (ml) distilled waters.
The rubella soluble antigen with carbonate buffer solution with 1: 200 or 1: 400 or 1: 800 dilution back bag by in 96 hole polystyrene reaction plates, every hole added 100 microlitres (ul), 4 ℃ of overnight incubation or 37 ℃ of incubations 2～4 hours.
3. the phosphate buffer of secure ph 9.6 (PBS) in an amount of distilled water, adds 4.3 gram potassium dihydrogen phosphates and 68 gram sodium chloride with 4.8 gram sodium hydrogen phosphate heating for dissolving again, behind the stirring and evenly mixing, supplies solution total amount to 1000 milliliter.
4. get above-mentioned phosphate buffer (PBS) 500ml, add the Tween-20 of 50 microlitres, be made into phosphate buffer (PBS) washing lotion that contains 1/1000 Tween-20.
5. get phosphate buffer (PBS) 500ml, add the calf serum of 25ml and the polyglycol (PEG) 6000 of 20 grams, be made into the sample dilution that contains 4% polyglycol (PEG).
6. get rid of the antigenic solution in the ELISA Plate hole, with above-mentioned phosphate buffer (PBS) washing lotion washing 3～5 times, each 3～5 minutes.
7. after test serum and positive control and negative control sera were done 1: 200 (or 1: 100) dilution with the sample dilution that contains 4% polyglycol (PEG), every hole added 100ul in ELISA Plate, put in the wet box 37 ℃ of effects 5～30 minutes.Take out with the washing of phosphate buffer (PBS) washing lotion, wash 3-5 time, each 3-5 minute.
8. the goat anti-human immunoglobulin M (IgM) of horseradish peroxidase (HRP) mark was done 1: 1000 or 1: 500 or dilution in 1: 2000 with the sample dilution that contains 4% polyglycol (PEG), every hole adds 100ul, puts in the wet box 37 ℃ of effects 5～30 minutes.
9. preparation substrate buffer solution adds the citric acids of 3.4 gram sodium acetates and 5.25 grams mixing in the distilled water of 500ml, adjust pH to 4.8.
10. preparation substrate solution adds tetramethylethylenediamine (TMB) and the 10ul hydrogen peroxide (H of 100ul 1% in the above-mentioned substrate buffer solution of 10ml
2O
2) mixing.
11. the taking-up ELISA Plate, with phosphate buffer (PBS) washing 3-5 time, each 3-5 minute, every hole added the 100ul substrate solution, put in the warm box 37 ℃ and acted on 5～15 minutes, absorbance or visual inspection result when reading wavelength being 450 nanometers on microplate reader.Criterion is positive with ratio P/N 〉=2.1 of known negative control serum (N) with serum to be checked (P).
Advantage of the present invention and effect: the characteristics of this method maximum are that the conventional indirect ELISA reaction time was contracted in the shortest 30 minutes by original 2-3 hour.Two Buwen of conventional indirect ELISA can be shortened the time of educating simultaneously, the incubation time was by 5 minutes to 30 minutes, background changes not obvious, all can draw satisfied result, therefore in practical operation, the incubation time can select for use arbitrarily, hurry up, can grasp flexibly slowly, near 5 minutes,, be specially adapted to instant detection slowly by 30 minutes.The present invention can also shorten developing time to 5 minute, sometimes even immediately reading; Can improve absorbance, negative background does not have obvious rising, and reaction pattern is clear, and the yin and yang attribute contrast is obvious, with conventional indirect ELISA relatively, positive colour developing obviously improves and negative background raises not obvious; Prolong the reaction time, positive hyperchromicly obviously be better than negative hyperchromicly, do not hinder the result and judge; Be that range estimation or microplate reader reading calculate P/N value and all be easy to judge, and stable and repeatability is all better.
The fast detecting that is established as the pregnancy period rubella virus infection and the epidemiology survey of this method provide new means.
Embodiment
Blood serum sample: 39 parts of positive serums pick up from the acute stage rubella patient that hospital outpatient is gone to a doctor, and detect through kit (brilliant U.S. bioengineering company limited produce) to be the rubella virus IgM antibody positive; 184 parts of negative serums pick up from child-bearing period schoolgirl health check-up serum, detect negative through the mentioned reagent box.
Implementation step:
1. the system carbonate buffer solution is dissolved in 1.59 gram sodium carbonate and 2.93 gram sodium bicarbonates in 1000 milliliters of (ml) distilled waters.
2. the rubella soluble antigen wraps quilt in 96 hole polystyrene reaction plates with carbonate buffer solution with 1: 400 dilution back, and every hole adds 100 microlitres (ul), 4 ℃ of overnight incubation.
3. the phosphate buffer of secure ph 9.6 (PBS) in an amount of distilled water, adds 4.3 gram potassium dihydrogen phosphates and 68 gram sodium chloride with 4.8 gram sodium hydrogen phosphate heating for dissolving again, behind the stirring and evenly mixing, supplies solution total amount to 1000 milliliter.
4. get above-mentioned PBS500ml, add the Tween-20 of 50 microlitres, be made into the PBS washing lotion that contains 1/1000 Tween-20.
5. get PBS500ml, add the calf serum of 25ml and the PEG 6000 of 20 grams, be made into the sample dilution that contains 4%PEG.
6. get rid of the antigenic solution in the ELISA Plate hole, with above-mentioned PBS washing lotion washing 3 times, each 3 minutes.
7. after test serum and positive control and negative control sera were done dilution in 1: 100 with the sample dilution that contains 4%PEG, every hole added 100ul in ELISA Plate, put in the wet box 37 ℃ of effects 10 minutes.Take out with the washing of PBS washing lotion, method is the same.
8. the goat-anti people IgM of horseradish peroxidase (HRP) mark is done dilution in 1: 1500 with the sample dilution that contains 4%PEG, every hole adds 100ul, puts in the wet box 37 ℃ of effects 10 minutes.
9. preparation substrate buffer solution adds the citric acids of 3.4 gram sodium acetates and 5.25 grams mixing in the distilled water of 500ml, adjust pH to 4.8.
10. preparation substrate solution adds tetramethylethylenediamine (TMB) and the 10ul hydrogen peroxide (H of 100ul 1% in the above-mentioned substrate buffer solution of 10ml
2O
2) mixing.
11. the taking-up ELISA Plate, with PBS washing 3 times, method is the same.Every hole adds this potpourri of 100ul, puts in the warm box 37 ℃ of effects 10 minutes, the absorbance when reading wavelength being 450 nanometers on microplate reader.Criterion is positive with ratio P/N 〉=2.1 of known negative control serum (N) with serum to be checked (P).
A. compare indirect ELISA and the result who improves the ELISA method
Table 1 indirect ELISA and the testing result of improvement ELISA method to the rubella virus IgM antibody positive serum
| Indirect ELISA | Add up to | |||
| Positive | Negative | |||
| Improvement ELISA | Positive negative the total | 35 1 36 | 3 0 3 | 38 1 39 |
α＝0.05，χ
2＝0.25，ν＝1，P＞0.05
Table 2 indirect ELISA and the testing result of improvement ELISA method to the rubella virus IgM antibody negative serum
| Indirect ELISA | Add up to | |||
| Negative | Positive | |||
| Improvement ELISA method | Negative positive the total | 152 9 161 | 23 0 23 | 175 9 184 |
α＝0.05，χ
2＝5.28，ν＝1，P＜0.05
From table 1 and table 2 as can be seen, improvement ELISA method is 97.4% (38/39) to the recall rate of positive serum, is higher than the recall rate 92.3% (36/39) of indirect elisa method, but both difference not statistically significants, and the recall rate unanimity of two methods is described; Improvement ELISA method is 95.1% (175/184) to the recall rate of negative serum, and apparently higher than the recall rate 87.5% (161/184) of indirect elisa method, difference has statistical significance.
B. specificity test
(1) replace RV antigen with HSV, EHF antigen, the result is negative.
(2) adopt improvement ELISA method to detect RV-IgM antibody in each 3 parts of RV, CBV, HSV-1, HSV-2, the RSV positive serums respectively, the result has only measured corresponding RV-IgM antibody in the sample of RV positive serum, its P/N value descends with extension rate, and all the other sample results are all negative, illustrate that this law is special, sees Table 3.
The specificity test findings of table 3 rapid ELISA
| Serum dilution | RV-IgM | CBV-IgM | HSV-1IgM | HSV-2IgM | RSV-IgM |
| 1∶100 1∶200 1∶400 1∶800 1∶1600 | 6.2 5.0 2.8 2.1 1.5 | 1.8 1.5 1.6 1.2 0.9 | 1.2 1.3 1.1 1.1 0.8 | 1.4 1.3 1.2 1.4 1.1 | 0.9 1.0 1.1 0.9 0.8 |
(3) stability and replica test
With each 5 parts of rubella IgM antibody positive serum and rubella IgM negative antibody serum, on same block of plate, repeat application of sample 5 times (hole), measure its repeatability.Measure A value basically identical, interassay coefficient of variation is less than 10% (seeing Table 4); Select each 1 part of positive serum and negative serum to carry out 5 times and measure at different plates and different time, positive serum CV=10.3% as a result, negative serum CV=9.92% is all in allowed band.This shows native system stability and repeatability better.See Table 4.
The repeatable measurement result of table 4 rubella IgM
| Number of times | Positive serum A value | Negative serum A value | ||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 1 2 3 4 | 0.43 0.42 0.45 0.43 | 0.33 0.35 0.35 0.32 | 0.60 0.60 0.60 0.62 | 0.37 0.38 0.39 0.38 | 0.66 0.68 0.69 0.65 | 0.08 0.10 0.10 0.09 | 0.12 0.14 0.13 0.14 | 0.08 0.08 0.10 0.09 | 0.11 0.13 0.12 0.11 | 0.09 0.07 0.07 0.08 |
In addition, do not add the contrast of PEG simultaneously, only improve enzyme labeling substrate concentration or serum dilution, the same this method of other condition, the result can not detect positive sample, illustrate that the reaction time shortening is not owing to due to the more conventional indirect ELISA raising of enzyme labeling substrate concentration, can not shorten by improving serum dilution among the present invention.
Claims (1)
1, a kind of enzyme immunological method that detects rubella virus antibody may further comprise the steps:
A, preparation carbonate buffer solution are dissolved in 1.59 gram sodium carbonate and 2.93 gram sodium bicarbonates in 1000 milliliters of distilled waters;
With 1: 200 or 1: 400 or dilution in 1: 800, bag was by in 96 hole polystyrene reaction plates with carbonate buffer solution for B, rubella soluble antigen, and every hole adds 100 microlitres, 4 ℃ of overnight incubation or 37 ℃ of incubations 2～4 hours;
The phosphate buffer of C, secure ph 9.6 in an amount of distilled water, adds 4.3 gram potassium dihydrogen phosphates and 68 gram sodium chloride with 4.8 gram sodium hydrogen phosphate heating for dissolving again, behind the stirring and evenly mixing, supplies solution total amount to 1000 milliliter;
D, get phosphate buffer 500ml, add the Tween-20 of 50 microlitres, be made into the phosphate buffer washing lotion that contains 1/1000 Tween-20;
E, get phosphate buffer 500ml, add the calf serum of 25ml and the Macrogol 6000s of 20 grams, be made into the sample dilution that contains 4% polyglycol;
F, get rid of the antigenic solution in the ELISA Plate hole, with phosphate buffer washing lotion washing 3～5 times, each 3～5 minutes;
After G, test serum and positive control and negative control sera diluted with the sample dilution work that contains 4% polyglycol in 1: 200 or 1: 100, every hole adds 100ul in ELISA Plate, put in the wet box 37 ℃ of effects 5～30 minutes, take out with the washing of phosphate buffer washing lotion, wash each 3～5 minutes 3～5 times;
H, the goat anti-human immunoglobulin M of horseradish peroxidase-labeled was done 1: 1000 or 1: 500 or dilution in 1: 2000 with the sample dilution that contains 4% polyglycol, every hole adds 100ul, puts in the wet box 37 ℃ of effects 5～30 minutes;
I, preparation substrate buffer solution add the citric acids of 3.4 gram sodium acetates and 5.25 grams mixing in the distilled water of 500ml, adjust pH to 4.8;
J, preparation substrate solution, tetramethylethylenediamine and the 10ul hydrogen peroxide mixing of adding 100ul 1% in the 10ml substrate buffer solution;
K, taking-up ELISA Plate were with phosphate buffer washing 3～5 times, each 3～5 minutes.Every hole adds the 100ul substrate solution, puts in the warm box 37 ℃ of effects 5～15 minutes, absorbance or visual inspection result when reading wavelength being 450 nanometers on microplate reader.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1328583C (en) * | 2004-06-30 | 2007-07-25 | 中国人民解放军军事医学科学院野战输血研究所 | Serum or plasma sample diluting liquid and its use |
| CN101781360B (en) * | 2010-02-05 | 2012-07-25 | 吉林大学 | Recombinant rubella virus protein and application |
| CN104345143A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Technology for improving activity of enzyme-labeled working fluid anti-human IgA alkaline phosphatase |
| CN104730231B (en) * | 2015-03-26 | 2016-06-08 | 北京乐普医疗科技有限责任公司 | A kind of sample buffer for fluorescence immunoassay detection by quantitative and application thereof |
| CN115097129B (en) * | 2022-08-24 | 2023-03-10 | 山东子峰生物技术有限公司 | Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1 |
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