CN102680678B - Protein eluent for dried blood spots on filter paper - Google Patents
Protein eluent for dried blood spots on filter paper Download PDFInfo
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- CN102680678B CN102680678B CN201210189829.1A CN201210189829A CN102680678B CN 102680678 B CN102680678 B CN 102680678B CN 201210189829 A CN201210189829 A CN 201210189829A CN 102680678 B CN102680678 B CN 102680678B
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- albumen
- filter paper
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- hydroxypropyl
- protein eluent
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Abstract
The invention discloses an efficient protein eluent, which comprises a surfactant, a stabilizer, an assistant stabilizer and a buffer. Preferably, the protein eluent disclosed by the invention further comprises an antiseptic. The protein eluent can effectively elute the proteins on the dried blood spots on filter paper, can remarkably reduce the interference of other substances in whole blood on immunodetection, and has the characteristics of strong specificity, low cost and good preparation easiness, therefore, the protein eluent has extensive application prospect in newborn disease screening featured by the use of protein as detection marker and of immunodetection techniques.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of albumen eluent for Filter Paper Dry Blood sheet.
Background technology
Universal and the popularization of the examination of newborn infant diseases makes some harm children life, causes the congenital hereditary metabolic disease of children's physique and intellectual developmental disorder can be able to early diagnosis, and can before neonate's clinical symptoms does not occur or when symptom is not obvious, just can give timely treatment, the irreversible damage of avoiding or reduce the each organ of children patient to be subject to produces.
At present, existing chemiluminescence immunoassay detection, time-resolved fluoroimmunoassay detection, Enzyme-linked Immunosorbent Assay, radiommunoassay etc. are multinomial is used in the examination of newborn infant diseases take immune detection as basic detection method.But due to the singularity of neonate's blood sampling, the examination of newborn infant diseases is all that the mark that its Heel blood is dropped in the Filter Paper Dry Blood sheet forming on S & S903 filter paper detects, therefore need mark to be again dissolved in particular solution from Filter Paper Dry Blood sheet, carry out again correlation analysis detection, such as: the examination of Neonatal Congenital Hypothyroidism, need to from Filter Paper Dry Blood sheet, thyrotropic hormone (TSH) be dissolved out, then carry out immune detection.
At present, examination of newborn infant diseases kit is generally used the albumen eluent of Tirs-HCl damping fluid as Filter Paper Dry Blood sheet, after on filter paper blood sheet, albumen elutes, carry out again immune detection, not only albumen dissolution rate is low, reduce the sensitivity of immune detection, and the existence of interfering material, the accuracy detecting reduced again.In addition, Tris-HCl wash-out Filter Paper Dry Blood sheet needs the time also longer, generally will repeatedly wash plate, has increased detection time.Therefore, become for the efficient protein eluent of Filter Paper Dry Blood sheet the key factor improving for the sensitivity of the immunologic function test reagent of the examination of newborn infant diseases, accuracy, detection speed.
Summary of the invention
The object of this invention is to provide a kind of albumen wash-out composition for Filter Paper Dry Blood sheet and albumen eluent.
Albumen wash-out composition of the present invention, is made up of NP40, (2-hydroxypropyl)-beta-schardinger dextrin-, (hydroxypropyl) methylcellulose and calf serum.By the efficient eluent of albumen of described albumen wash-out composition preparation, comprise surfactant, stabilizing agent, auxiliary stabilizer, described surfactant is 10 ‰-65 ‰ NP40, described stabilizing agent is (2-hydroxypropyl)-beta-schardinger dextrin-of 0.1 ‰-1 ‰, and described auxiliary stabilizer is (hydroxypropyl) methylcellulose of 0.1 ‰-1 ‰.
NP40 is a kind of non-ionic surfactant of gentleness, generally, by gather-(oxo-1, the 2-second dimethylene)-α-nonyl phenyl-ω-hydroxyl that exceedes 97%, is less than 1% side chain dinonyl phenyl polyoxyethylene ether, is less than 3% polyglycol formation.In biomedicine, standing being used in cell pyrolysis liquid, for destroying cell membrane.1% concentration NP40 can destroy after birth substantially, and just can obtain plasmosin in conjunction with specific damping fluid.
(2-hydroxypropyl)-beta-schardinger dextrin-is a kind of hydroxyalkylation derivant of cycloheptaamylose, and its structural formula is as follows:
(2-hydroxypropyl)-beta-schardinger dextrin-has good envelope effect, can improve by the stability of envelope material, and have water-solublely, can improve in vivo by the release rate of envelope material.
(hydroxypropyl) methylcellulose is a kind of nonionic cellulose, has and keeps pH value of solution stability and improve dispersed ability, and its aqueous solution also has surfactivity, is a kind of conventional stabilizing agent, and its structural formula is as follows:
Inventor finds through lot of experiments, the mixing of NP40, (2-hydroxypropyl)-beta-schardinger dextrin-and (hydroxypropyl) methylcellulose is used, the elution efficiency of albumen on Filter Paper Dry Blood sheet can be effectively improved, and the impact of interfering material can be reduced.With the eluent that contains 10 ‰ to 65 ‰ NP40, (2-hydroxypropyl)-beta-schardinger dextrin-of 0.1 ‰ to 1 ‰, (hydroxypropyl) methylcellulose of 0.1 ‰ to 1 ‰, albumen on wash-out Filter Paper Dry Blood sheet, adopt chemiluminescence immunoassay detection technique to carry out immune detection, can on the coated plate of coated good antibody, directly detect, wash-out and detection can realize by single step reaction, only need 3 hours detection time, and at room temperature just can realize detection, detect background values lower 24.8 to 52.7 times than common albumen eluent, detection sensitivity can be significantly increased.
In the specific embodiment of the present invention, described surfactant is 10%, 50 ‰ or 65 ‰ NP40, described stabilizing agent is (2-hydroxypropyl)-beta-schardinger dextrin-of 0.1 ‰ or 1 ‰, and described auxiliary stabilizer is (hydroxypropyl) methylcellulose of 0.1 ‰ or 1 ‰.
As preferably, the efficient eluent of albumen of the present invention also comprises antiseptic.In the specific embodiment of the present invention, the Proclin-300 that antiseptic is 1 ‰.
What the examination of newborn infant diseases detected is the destination protein in neonatal heel blood Filter Paper Dry Blood sheet, need to from Filter Paper Dry Blood sheet, destination protein be eluted, carry out again immunoassay detection, and the materials such as a large amount of haemoglobins that exist in whole blood, lipid, lipoidis, fibrous matters on filter paper etc. all can produce interference to the accuracy of the dissolution rate of albumen in Filter Paper Dry Blood sheet, immune detection and sensitivity, and the advantage of immune detection analytical technology can not be embodied completely.
Albumen eluent of the present invention is high and stable for the eluting rate of the albumen wash-out of examination of newborn infant diseases Filter Paper Dry Blood sheet, and cost lower, be easy to preparation, can be widely used in take albumen as detecting mark, and be the examination of newborn infant diseases product based on immunoassay technology, thereby greatly shorten detection time, improve detection sensitivity and accuracy.Through repeatedly test, prove that the present invention has following several advantage:
(1) wash-out and immune response one step just can realize, and detect and can realize at 3h T.T., can meet examination of newborn infant diseases needs.
(2) detection at room temperature just can be carried out, do not need extras.
(3) can effectively reduce the interference of the interfering material in whole blood and filter paper, the common eluent of background ratio is low 24.8 to 52.7 times.
The present invention is a kind of albumen eluent that is specifically designed to Filter Paper Dry Blood sheet, can effectively improve the dissolution rate of albumen on Filter Paper Dry Blood sheet, and can shield interfering material, the advantage of immuno analytical method is given full play to, improve the accuracy and efficiency of the examination of newborn infant diseases, be applicable to, take albumen as detecting mark, adopt the examination of newborn infant diseases project of immunoassay technology, have a good application prospect
Embodiment
The invention discloses a kind of albumen eluent for Filter Paper Dry Blood sheet, those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.The present invention is described by preferred embodiment for albumen eluent and the application of Filter Paper Dry Blood sheet, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.Be below four examples of this invention, this example is only for the present invention is described, but the content of invention is not limited to following instance.
Embodiment 1:
In 50% calf serum, add 10 ‰ NP40 as basic albumen eluant, eluent, add (hydroxypropyl) methylcellulose of 0.1 ‰ (2-hydroxypropyl)-beta-schardinger dextrin-and 0.1 ‰ respectively as stabilizing agent and auxiliary stabilizer simultaneously, then add 1 ‰ Proclin-300 as antiseptic.With the TSH concentration that examination of newborn infant diseases special-purpose punching device is laid respectively diameter 3mm be 0,3,6,15,50,150mIU/L Filter Paper Dry Blood sheet sample, the Filter Paper Dry Blood sheet sample of laying is put into corresponding micropore, add respectively this albumen eluent 100 μ L/ holes, also add another kind to be marked with the TSH antibody-solutions 50 μ L/ holes of horseradish peroxidase (HRP) simultaneously, on 96 orifice plate oscillators, shake elution of reactive 3h, wash after plate 5 times, add luminol luminescent solution, measure luminous value with light-emitting appearance.
| Sample TSH concentration (mIU/L) | 0 | 3 | 6 | 15 | 50 | 150 |
| Luminous counting (RLU/ second) | 730 | 1700 | 3541 | 9113 | 33338 | 97855 |
Embodiment 2:
In 50% calf serum, add 50 ‰ NP40 as basic albumen eluant, eluent, add (hydroxypropyl) methylcellulose of 1 ‰ (2-hydroxypropyl)-beta-schardinger dextrin-and 1 ‰ respectively as stabilizing agent and auxiliary stabilizer simultaneously, then add 1 ‰ Proclin-300 as antiseptic; With the TSH concentration that examination of newborn infant diseases special-purpose punching device is laid respectively diameter 3mm be 0,3,6,15,50,150mIU/L Filter Paper Dry Blood sheet sample, the Filter Paper Dry Blood sheet sample of laying is put into corresponding micropore, add respectively this albumen eluent 100 μ L/ holes, also add another kind to be marked with the TSH antibody-solutions 50 μ L/ holes of horseradish peroxidase (HRP) simultaneously, on 96 orifice plate oscillators, shake elution of reactive 3h, wash after plate 5 times, add luminol luminescent solution, measure luminous value with light-emitting appearance, see the following form.
| Sample TSH concentration (mIU/L) | 0 | 3 | 6 | 15 | 50 | 150 |
| Luminous counting (RLU/ second) | 590 | 1595 | 3253 | 8040 | 27776 | 80008 |
Embodiment 3:
In 50% calf serum, add 65 ‰ NP40 as basic albumen eluant, eluent, add (hydroxypropyl) methylcellulose of 1 ‰ (2-hydroxypropyl)-beta-schardinger dextrin-and 1 ‰ respectively as stabilizing agent and auxiliary stabilizer simultaneously, then add 1 ‰ Proclin-300 as antiseptic; With the TSH concentration that examination of newborn infant diseases special-purpose punching device is laid respectively diameter 3mm be 0,3,6,15,50,150mIU/L Filter Paper Dry Blood sheet sample, the Filter Paper Dry Blood sheet sample of laying is put into corresponding micropore, add respectively this albumen eluent 100 μ L/ holes, also add another kind to be marked with the TSH antibody-solutions 50 μ L/ holes of horseradish peroxidase (HRP) simultaneously, on 96 orifice plate oscillators, shake elution of reactive 3h, wash after plate 5 times, add luminol luminescent solution, measure luminous value with light-emitting appearance, see the following form.
| Sample TSH concentration (mIU/L) | 0 | 3 | 6 | 15 | 50 | 150 |
| Luminous counting (RLU/ second) | 635 | 1642 | 3477 | 8729 | 26394 | 77980 |
Embodiment 4:
With reference to the disclosed content of this instructions and embodiment 1,2,3, take the examination of Neonatal Congenital Hypothyroidism as example, detection mark is TSH, method is that chemiluminescence immunoassay detects, by the albumen eluent of the present invention and conventional Filter Paper Dry Blood sheet (0.1mol/l Tris-HCl damping fluid, pH7.0, spend the night at 4 ℃) relatively, repeatedly test, and observations, sees the following form.
Prove that the present invention has following several advantage:
(1) wash-out and immune response one step just can realize, and detect and can realize at 3h T.T., can meet examination of newborn infant diseases needs.
(2) detection at room temperature just can be carried out, do not need extras.
(3) can effectively reduce the interference of the interfering material in whole blood and filter paper, the common eluent of background ratio is low 24.8 to 52.7 times.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. an albumen wash-out composition, is made up of NP-40, (2-hydroxypropyl)-beta-schardinger dextrin-, hydroxypropyl methylcellulose and calf serum.
2. an albumen eluent, comprise surfactant, stabilizing agent, auxiliary stabilizer and damping fluid, described surfactant is NP-40, its working concentration is 10 ‰-65 ‰, described stabilizing agent is (2-hydroxypropyl)-beta-schardinger dextrin-, and its working concentration is 0.1 ‰-1 ‰, and described auxiliary stabilizer is hydroxypropyl methylcellulose, its working concentration is 0.1 ‰-1 ‰, and described damping fluid is calf serum.
3. albumen eluent according to claim 2, is characterized in that: the NP-40 that described surfactant is 50 ‰.
4. albumen eluent according to claim 2, is characterized in that: described stabilizing agent is 1 ‰ (2-hydroxypropyl)-beta-schardinger dextrin-.
5. albumen eluent according to claim 2, is characterized in that: the hydroxypropyl methylcellulose that described auxiliary stabilizer is 1 ‰.
6. albumen eluent according to claim 2, is characterized in that: also comprise antiseptic.
7. albumen eluent according to claim 6, is characterized in that: the Proclin-300 that described antiseptic is 1 ‰.
8. the application of albumen eluent in preparation examination of newborn infant diseases reagent described in claim 2-7 any one.
9. the application of albumen wash-out composition in preparation examination of newborn infant diseases reagent described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210189829.1A CN102680678B (en) | 2012-06-08 | 2012-06-08 | Protein eluent for dried blood spots on filter paper |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210189829.1A CN102680678B (en) | 2012-06-08 | 2012-06-08 | Protein eluent for dried blood spots on filter paper |
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| CN102680678A CN102680678A (en) | 2012-09-19 |
| CN102680678B true CN102680678B (en) | 2014-06-04 |
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| CN113267390B (en) * | 2021-05-11 | 2023-05-05 | 北京丹大生物技术有限公司 | Detection marker eluent for filter paper dried blood slices and application thereof |
Citations (3)
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| US5427953A (en) * | 1993-11-08 | 1995-06-27 | The Detroit Medical Center | Blood testing method |
| CN1122614A (en) * | 1993-05-14 | 1996-05-15 | T细胞诊断公司 | Method and device for detecting or measuring the amount of a cell-associated molecule |
| CN102419373A (en) * | 2010-09-28 | 2012-04-18 | 广州市达瑞抗体工程技术有限公司 | Insulin and C peptide double-tagging determination kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU686577B2 (en) * | 1992-10-30 | 1998-02-12 | Innogenetics, Inc. | Measurement of total molecule in a sample and methods based thereon |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122614A (en) * | 1993-05-14 | 1996-05-15 | T细胞诊断公司 | Method and device for detecting or measuring the amount of a cell-associated molecule |
| US5427953A (en) * | 1993-11-08 | 1995-06-27 | The Detroit Medical Center | Blood testing method |
| CN102419373A (en) * | 2010-09-28 | 2012-04-18 | 广州市达瑞抗体工程技术有限公司 | Insulin and C peptide double-tagging determination kit |
Non-Patent Citations (5)
| Title |
|---|
| Hydroxypropyl--β-cyclodextrin and its combination with hydroxypropyl-methylcellucose increases aqueous solubility of ⊿9-tetrahydrocannabinol;Pekka Jarho et al;《Life Sciences》;19981231;第63卷(第26期);381-384页 * |
| Pekka Jarho et al.Hydroxypropyl--β-cyclodextrin and its combination with hydroxypropyl-methylcellucose increases aqueous solubility of ⊿9-tetrahydrocannabinol.《Life Sciences》.1998,第63卷(第26期), |
| The effect of hydroxypropyl methylcellulose on the release of dexamethasone from aqueous 2-hydroxypropyl-β-cyclodextrin formulations;Thorsteinn Loftsson et al;《International Journal of Pharmaceutics》;19941231;第104卷;181-184页 * |
| Thorsteinn Loftsson et al.The effect of hydroxypropyl methylcellulose on the release of dexamethasone from aqueous 2-hydroxypropyl-β-cyclodextrin formulations.《International Journal of Pharmaceutics》.1994,第104卷 |
| 刘霞等.滤纸干血片法筛查孕中期唐氏综合征的可行性.《山东大学学报（医学版）》.2009,第47卷(第12期),86-94页. * |
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