CN102183636A - Diagnostic kit for anti-strep-A DNase B antibody with chemiluminescence immunoassay and using method thereof - Google Patents
Diagnostic kit for anti-strep-A DNase B antibody with chemiluminescence immunoassay and using method thereof Download PDFInfo
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- CN102183636A CN102183636A CN2010106134285A CN201010613428A CN102183636A CN 102183636 A CN102183636 A CN 102183636A CN 2010106134285 A CN2010106134285 A CN 2010106134285A CN 201010613428 A CN201010613428 A CN 201010613428A CN 102183636 A CN102183636 A CN 102183636A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000003018 immunoassay Methods 0.000 title abstract description 8
- 238000009007 Diagnostic Kit Methods 0.000 title abstract description 4
- 108010004029 deoxyribonuclease B Proteins 0.000 title abstract 3
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 239000000243 solution Substances 0.000 claims abstract description 27
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 19
- 239000013642 negative control Substances 0.000 claims abstract description 14
- 239000013641 positive control Substances 0.000 claims abstract description 13
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 101000582398 Staphylococcus aureus Replication initiation protein Proteins 0.000 claims abstract description 11
- 239000000427 antigen Substances 0.000 claims abstract description 9
- 102000036639 antigens Human genes 0.000 claims abstract description 9
- 108091007433 antigens Proteins 0.000 claims abstract description 9
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 5
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- 238000000576 coating method Methods 0.000 claims abstract description 4
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- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 35
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 35
- 229960004533 streptodornase Drugs 0.000 claims description 35
- 239000000126 substance Substances 0.000 claims description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims description 19
- 102000004190 Enzymes Human genes 0.000 claims description 12
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- 229940088598 enzyme Drugs 0.000 claims description 12
- 239000008363 phosphate buffer Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000007979 citrate buffer Substances 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 4
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 claims description 4
- 239000011697 sodium iodate Substances 0.000 claims description 4
- 229940032753 sodium iodate Drugs 0.000 claims description 4
- 235000015281 sodium iodate Nutrition 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
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- 239000007793 ph indicator Substances 0.000 claims description 3
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- 239000001509 sodium citrate Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
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Abstract
The invention relates to a diagnostic kit for detecting an anti-strep-A DNase B antibody with chemiluminescence immunoassay and a using method thereof. The diagnostic kit comprises horseradish peroxidase (HRP)-labeled staphylococcus aureus protein A, a strep-A DNase antibody B coated microplate, and a chemiluminescence substrate solution. The pre-coated coated microplate is prepared by the steps of: adding a strep-A DNase B recombinant antigen for coating into a buffer solution, uniformly mixing, moving to the interior of a luminous micropored plate, incubating for 18 hours at 4 DEG C, washing the coated microplate, adding a sealing solution, incubating, removing liquid, and fully drying the coated microplate to complete the preparation of the pre-coated coated microplate; and the kit also comprises a sample dilution solution, a concentration washing solution, normal human blood serum as negative control and human blood, containing anti-DNase B antibody positive mixed blood serum, as positive control. Compared with a micro method, the kit provided by the invention has the advantages of higher detection sensitivity, safety, reliability, simplicity and convenience for operation, and low cost, and expensive full-automatic chemiluminescence measuring instruments are not needed.
Description
Technical field
The present invention relates to a kind of anti-A streptodornase B of family antibody diagnosing reagent kit (chemiluminescence) and using method thereof.
Background technology
(Chemiluminescence lmmunoassay's chemiluminescence immune assay CLIA) came out in 1977, and first generation chemiluminescence immunoassay kit was succeeded in developing and put on market in 1985.Enter the nineties, the production of the development of chemical luminescence immune analysis reagent box and automatic measurement instrument has obtained breakthrough, thereby enters the high speed development stage.Chemiluminescence immune assay is a new immuno analytical method that grows up after fluorescence, radioactive isotope and EIA enzyme immunoassay, according to lot of experiment results and clinical practice data, from practicality, stability, accuracy and development prospect, chemiluminescence immune assay maintains the leading position in nonradioactive labeling's analytical technology, the direction and the trend of world today's development have been represented, it not only has immunoreactive specificity, and (detection limit can reach 10 to have the high sensitivity of chemiluminescence reaction
-15~ 10
-18Mol/L).Advantages such as that chemiluminescence immunoassay technology has is highly sensitive, quick, accurate, good reproducibility, the effect phase is long and safety non-toxic is pollution-free become the first-selection that replaces radiommunoassay and EIA enzyme immunoassay technology.
Chemiluminescence enzyme immunoassay is to carry out immune response with enzyme labeling bioactivator (as the antigen or the antibody of enzyme labeling), enzyme on the immune response compound remakes and is used for luminous substrate, luminous under the signal reagent effect, carry out luminescence assays with the luminous signal analyzer.Marker enzyme commonly used at present is horseradish peroxidase (HRP), the substrate that HRP is commonly used is luminol (a 3-amino phthalylhydrazine, luminol) or derivatives thereof such as different luminol (4-amino phthalylhydrazine) etc., the oxidation reaction of luminol is carried out in alkaline buffer, in the presence of peroxidase and active oxygen, generate the excited state intermediate, luminous when it gets back to ground state, its wavelength is 425nm.Luminous intensity depends on the concentration of enzyme in the enzyme immune reaction thing.
Yet, chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, need the expensive luminous measuring instrument of full-automatic chemical, cost an arm and a leg, complicated operation, promote the use of thereby limited, can't effectively be widely used in clinical diagnosis and research work.
Summary of the invention
The object of the present invention is to provide a kind of cheap simple to operate, adopt chemoluminescence method to detect the kit and the using method thereof of the anti-A streptodornase B of family antibody.
The object of the present invention is achieved like this: it comprises staphylococcus aureus protein A, the streptodornase B of A family antigen coated microplate, the chemical luminous substrate liquid of horseradish peroxidase (HRP) mark.
Wherein chemical luminous substrate liquid prepares by the following method:
A liquid: in distilled water, add the Tris-HCl damping fluid that trishydroxymethylaminomethane and dense HCl are made into 0.05 ~ 0.2M, pH6 ~ 10; In the Tris-HCl damping fluid, add luminol reagent, Tween20 and benzofluoranthrene, and mix;
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with the citrate buffer solution of 0.05 ~ 0.2M, pH4 ~ 5, in citrate buffer solution, add 10 ~ 40% superoxols;
Before the use, A liquid and B liquid are waited until chemical luminous substrate liquid according to the 1:1 mixing.
Wherein, the preparation method of horseradish peroxidase-labeled staphylococcus aureus protein A adopts the preparation of sodium iodate method, with the sodium periodate method that improved staphylococcus aureus protein A and horseradish peroxidase is bound up.
Wherein, being prepared as follows of A family streptodornase B antigen coated microplate: will wrap by the A family streptodornase B recombinant antigen that with concentration is 0.1ng/ml ~ 10 μ g/ml and add to mixing in the carbonate buffer solution, the solution of mixing is added to bag by in the plate hole, every hole 100uL, 4 ℃ are incubated overnight, wrap by behind the plate with the phosphate buffer washing that contains Twenn20, add the phosphate buffer that contains bovine serum albumin(BSA) (BSA) again, after room temperature leaves standstill 1.5 ~ 3 hours, discard liquid in the hole, the finish-drying bag is by plate, and 2-8 ℃ of preservation put in factory's aluminide-coating bag vacuum packaging; Bag is lighttight microwell plate by plate, and bag is 48 holes or 96 holes by plate.
The present invention also comprises each 1 bottle of 1 bottle of 20 times of concentrated cleaning solution, negative control and positive control, 1 bottle of sample diluting liquid.
Cleansing solution wherein is the cleansing solution that contains the phosphate buffer of Tween20; The normal human serum of negative control for mixing; Positive control is the anti-A streptodornase B of the family antibody positive human serum of NBCS dilution; Sample diluting liquid is the phosphate buffer that contains the PH indicator.
The using method of the anti-A streptodornase B of family antibody diagnosing reagent kit is as follows:
(1) in 2 ~ 8 ℃ of refrigerators, takes out kit, equilibrium at room temperature 10 ~ 30 minutes;
(2) take out coated slab, insert on the grillage;
(3) negative control 3 holes are established in each experiment, and positive control 2 holes, every hole add negative control or positive control 100 microlitres respectively, every hole adds sample diluting liquid 100 microlitres in the sample well, add sample 10 microlitres again, stick the shrouding film behind the vibration mixing, put 37 ℃ of incubations 20 ~ 40 minutes;
(4) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(5) remove the every hole of blank well and add enzyme labeling thing 100 microlitres, stick the shrouding film, put 37 ℃ of incubations 30 minutes;
(6) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(7) before the use chemical luminous substrate liquid A and B are mixed with 1:1, each hole all adds the chemical luminous substrate mixed liquor of 50 microlitres, mixes, and puts room temperature lucifuge reaction 5 minutes;
(8) on luminous measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn.
Judge according to the RLU value of sample to be tested and the ratio S/CO value of critical value; If S/CO value 〉=1, then positive reaction illustrates and contains the anti-A streptodornase B of family antibody in the sample; If S/CO value＜1, then negative reaction illustrates and does not contain the anti-A streptodornase B of family antibody in the sample.
What kit of the present invention adopted is chemiluminescence immunoassay technology, has higher detection sensitivity than ELISA, safe and reliable, easy and simple to handle, with low cost, do not need the expensive luminous measuring instrument of full-automatic chemical, for the clinical anti-A streptodornase B of family detection of antibodies provides a kind of new using method.
Description of drawings
Fig. 1 is the molecular structure of benzofluoranthrene among the present invention.
Embodiment
Below the invention will be further described by specific embodiment.
The present invention mainly comprises staphylococcus aureus protein A, the streptodornase B of A family antigen coated microplate, the chemical luminous substrate liquid of horseradish peroxidase (HRP) mark.
Chemical luminous substrate liquid is by preparing by the following method:
A liquid: in distilled water, add the Tris-HCl damping fluid that trishydroxymethylaminomethane and dense HCl are made into 0.1M, pH8.5; In the Tris-HCl damping fluid, add luminol reagent, Tween20 and benzofluoranthrene (the benzofluoranthrene molecular structure as shown in Figure 1), and mix;
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with the citrate buffer solution of 0.1M, pH4.6, in citrate buffer solution, add 30% superoxol;
Before the use, A liquid and B liquid are waited until chemical luminous substrate liquid according to the 1:1 mixing.
The preparation method of horseradish peroxidase (HRP) mark staphylococcus aureus protein A adopts the preparation of sodium iodate method, " sodium iodate method " is classic method, with the sodium periodate method that improved staphylococcus aureus protein A and horseradish peroxidase is bound up.
Being prepared as follows of A family streptodornase B antigen coated microplate: will wrap is that the streptodornase B of 10ng/ml A family recombinant antigen adds to mixing in the carbonate buffer solution with concentration, add to bag by in the plate hole, every hole 100uL, 4 ℃ are incubated overnight, wrap by behind the plate 3 times with the phosphate buffer washing that contains Twenn20, add the phosphate buffer that contains bovine serum albumin(BSA) (BSA) again, after room temperature leaves standstill 2 hours, discard liquid in the hole, the finish-drying bag is by plate, 2-8 ℃ of preservation put in factory's aluminide-coating bag vacuum packaging., wherein wrapping by plate is lighttight microwell plate, bag is 48 holes or 96 holes by plate.
Generally on the basis of the staphylococcus aureus protein A of horseradish peroxidase (HRP) mark, the streptodornase B of A family antigen coated microplate, the combination of chemical luminous substrate liquid, also increase following substances, be respectively:
Each 1 bottle of 1 bottle of 20 times of concentrated cleaning solution, negative control and positive control, 1 bottle of sample diluting liquid.Cleansing solution wherein is the cleansing solution that contains the phosphate buffer of Tween20; The normal human serum of negative control for mixing, positive control are the anti-A streptodornase B of the family antibody positive human serum of NBCS dilution.Negative control be the normal human serum that mixes; Sample diluting liquid is the phosphate buffer that contains the PH indicator.
So far the described anti-A streptodornase B of family antibody diagnosing reagent kit preparation is finished.
The present invention detects the using method of the anti-A streptodornase B of family antibody diagnosing reagent kit:
(1) in 2 ~ 8 ℃ of refrigerators, takes out kit, equilibrium at room temperature 10 ~ 30 minutes;
(2) take out coated slab, insert on the grillage;
(3) negative control 3 holes are established in each experiment, and positive control 2 holes, every hole add negative control or positive control 100 microlitres respectively, every hole adds sample diluting liquid 100 microlitres in the sample well, add sample 10 microlitres again, stick the shrouding film behind the vibration mixing, put 37 ℃ of incubations 20 ~ 40 minutes;
(4) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(5) remove the every hole of blank well and add enzyme labeling thing 100 microlitres, stick the shrouding film, put 37 ℃ of incubations 30 minutes;
(6) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(7) before the use chemical luminous substrate liquid A and B are mixed with 1:1, each hole all adds the chemical luminous substrate mixed liquor of 50 microlitres, mixes, and puts room temperature lucifuge reaction 5 minutes;
(8) on luminous measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn.
The result judges: the relative light unit value (RLU) and the ratio S/CO value of critical value according to sample to be tested are judged; If S/CO value 〉=1, then positive reaction illustrates and contains the anti-A streptodornase B of family antibody in the sample; If S/CO value＜1, then negative reaction illustrates and does not contain the anti-A streptodornase B of family antibody in the sample.
Claims (9)
1. the anti-A streptodornase B of family antibody diagnosing reagent kit, it is characterized in that: it comprises staphylococcus aureus protein A, the streptodornase B of A family antigen coated microplate, the chemical luminous substrate liquid of horseradish peroxidase (HRP) mark.
2. a kind of anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 1 (chemoluminescence method) is characterized in that: wherein chemical luminous substrate liquid prepares by the following method:
A liquid: in distilled water, add the Tris-HCl damping fluid that trishydroxymethylaminomethane and dense HCl are made into 0.05 ~ 0.2M, pH6 ~ 10; In the Tris-HCl damping fluid, add luminol reagent, Tween20 and benzofluoranthrene, and mix;
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with the citrate buffer solution of 0.05 ~ 0.2M, pH4 ~ 5, in citrate buffer solution, add 10 ~ 40% superoxols;
Before the use, A liquid and B liquid are waited until chemical luminous substrate liquid according to the 1:1 mixing.
3. the described anti-A streptodornase B of the family antibody diagnosing reagent kit of claim 1, it is characterized in that: the preparation method of horseradish peroxidase-labeled staphylococcus aureus protein A adopts the preparation of sodium iodate method, with the sodium periodate method that improved staphylococcus aureus protein A and horseradish peroxidase is bound up.
4. the anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 1, it is characterized in that: being prepared as follows of A family streptodornase B antigen coated microplate: will wrap by the A family streptodornase B recombinant antigen that with concentration is 0.1ng/ml ~ 10 μ g/ml and add to mixing in the carbonate buffer solution, the solution of mixing is added to bag by in the plate hole, every hole 100uL, 4 ℃ are incubated overnight, wrap by behind the plate with the phosphate buffer washing that contains Twenn20, add the phosphate buffer that contains bovine serum albumin(BSA) (BSA) again, after room temperature leaves standstill 1.5 ~ 3 hours, discard liquid in the hole, the finish-drying bag is by plate, and 2-8 ℃ of preservation put in factory's aluminide-coating bag vacuum packaging.
5. the anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 4 is characterized in that: bag is lighttight microwell plate by plate, and bag is 48 holes or 96 holes by plate.
6. the anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 1 is characterized in that: it also comprises each 1 bottle of 1 bottle of 20 times of concentrated cleaning solution, negative control and positive control, 1 bottle of sample diluting liquid.
7. the anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 6, it is characterized in that: cleansing solution wherein is the cleansing solution that contains the phosphate buffer of Tween20; The normal human serum of negative control for mixing; Positive control is the anti-A streptodornase B of the family antibody positive human serum of NBCS dilution; Sample diluting liquid is the phosphate buffer that contains the PH indicator.
8. an application rights requires the using method of the described anti-A streptodornase B of the family antibody diagnosing reagent kit of 1-7, and it is characterized in that: its using method is:
(1) in 2 ~ 8 ℃ of refrigerators, takes out kit, equilibrium at room temperature 10 ~ 30 minutes;
(2) take out coated slab, insert on the grillage;
(3) negative control 3 holes are established in each experiment, and positive control 2 holes, every hole add negative control or positive control 100 microlitres respectively, every hole adds sample diluting liquid 100 microlitres in the sample well, add sample 10 microlitres again, stick the shrouding film behind the vibration mixing, put 37 ℃ of incubations 20 ~ 40 minutes;
(4) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(5) remove the every hole of blank well and add enzyme labeling thing 100 microlitres, stick the shrouding film, put 37 ℃ of incubations 30 minutes;
(6) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does;
(7) before the use chemical luminous substrate liquid A and B are mixed with 1:1, each hole all adds the chemical luminous substrate mixed liquor of 50 microlitres, mixes, and puts room temperature lucifuge reaction 5 minutes;
(8) on luminous measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn.
9. the using method of the anti-A streptodornase B of family antibody diagnosing reagent kit according to claim 8 is characterized in that: judge according to the RLU value of sample to be tested and the ratio S/CO value of critical value; If S/CO value 〉=1, then positive reaction illustrates and contains the anti-A streptodornase B of family antibody in the sample; If S/CO value＜1, then negative reaction illustrates and does not contain the anti-A streptodornase B of family antibody in the sample.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106290866A (en) * | 2016-08-16 | 2017-01-04 | 海南大学 | The indirect ELISA detection method of bovine papilloma virus 13 type antibody |
| CN109100512A (en) * | 2018-08-02 | 2018-12-28 | 宁波奥丞生物科技有限公司 | A kind of chemical luminescence reagent kit detecting EGFR |
| CN109490530A (en) * | 2018-11-21 | 2019-03-19 | 长沙金域医学检验所有限公司 | A kind of AsAb detection method |
| CN111218438A (en) * | 2019-11-22 | 2020-06-02 | 东方海洋(北京)医学研究院有限公司 | Streptococcus DNase B antigen and application thereof |
| CN117092344A (en) * | 2023-07-19 | 2023-11-21 | 武汉睿奇生物工程有限公司 | Kit for detecting staphylococcus aureus protein A and application thereof |
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| EP0726957B1 (en) * | 1994-08-18 | 2004-11-17 | Beckman Coulter, Inc. | RECOMBINANT DNase B DERIVED FROM STREPTOCOCCUS PYOGENES |
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2010
- 2010-12-30 CN CN2010106134285A patent/CN102183636A/en active Pending
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|---|---|---|---|---|
| EP0660874A1 (en) * | 1993-06-23 | 1995-07-05 | Beckman Instruments, Inc. | Recombinant dnase b derived from streptococcus pyogenes |
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| CN111218438B (en) * | 2019-11-22 | 2022-11-15 | 东方海洋(北京)医学研究院有限公司 | Streptococcus DNase B antigen and application thereof |
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