CN1034975C - Medicinal box for high-sensitivity quick detection of schistosomiasis circulating antigen - Google Patents
Medicinal box for high-sensitivity quick detection of schistosomiasis circulating antigen Download PDFInfo
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- CN1034975C CN1034975C CN92108014A CN92108014A CN1034975C CN 1034975 C CN1034975 C CN 1034975C CN 92108014 A CN92108014 A CN 92108014A CN 92108014 A CN92108014 A CN 92108014A CN 1034975 C CN1034975 C CN 1034975C
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Abstract
The present invention provides a medicine box for detecting a circulating antigen of schistosomiasis, which is composed of solid phase carriers and detecting reagents, wherein the solid phase carriers and the detecting reagents are used in mutual match, each solid phase carrier uses PVC as a supporting matrix, and each detecting reagent is an anti-schistosome circulating antibody enzyme combination amplified by a fluorescein-anti-fluorescein multistage amplification system. The medicine box of the present invention has the advantages of high sensitivity, rapidity and simple and convenient operation, and accords with the practical requirements of popularization and application.
Description
What the present invention relates to is medicine box used when using serological technique detection snail fever circulating antigen.
In prevention and cure of snail fever work, whether the existence of imago of blood fluke or worm's ovum metabolin is arranged in the human body, be the important basis for estimation of diagnosis, treatment and efficacy assessment, it is present main detection means that the utilization serological technique is tested.Because after human body is infected, though also fully recover through treatment, therefore the antibody that is produced in its body still can keep the long period, thereby the method that detects of antagonist and result usually can not truly reflect whether also have the degree that worm alive and activity infect in the body in the past, also inconvenience is accurately judged and the examination result of treatment, and the detection to circulating antigen that now in the direct antimer worm parasitism alive is arranged gradually thereby have more diagnostic significance replaces.Through studying for a long period of time, now understood these circulating antigen and can comprise as adult pellicle antigen, intestines related antigen etc. and drain the worm source property antigen be released in patient's blood and enter the circulation system and several big classes such as worm's ovum source property antigen of forming by miracidium secretion by imago of blood fluke.Show the very complicated spectrotype that they are made up of tens of kinds of antigen components of a few class macromolecular substances such as protein, polysaccharide and RNA (ribonucleic acid) through electrophoretic analysis.Because the concentration of these circulating antigen in infected patient serum is generally all very low, whether this numerous antigenic component has on the size of diagnostic significance and diagnostic value and has nothing in common with each other again in addition, thereby has increased difficulty and difficulty to the circulating antigen testing with etiological diagnosis meaning and value.
In present snail fever circulating antigen detects, report that a more class is to use the monoclonal antibody that is obtained by different antigen preparation to carry out the method for specific detection.For example, the tight self-service people of grade had once reported the method that detects with the dot enzyme-linked test of the monoclonal antibody direct method of the relevant negative electrode antigen of intestines in " Chinese parasitology and parasitic disease magazine " 1990.Vol.8 (3); " reporting again among the Shanghai IMMUNOLOGY KEY WORDS INDEX 1991.Vol.11 (3) that the dot enzyme-linked test of monoclonal antibody direct method of adopting circulation worm's ovum soluble glycoprotein antigen detects the method for circulation egg antigen.The same interim method of having reported that the monoclonal antibody that adopts adult pellicle antigen detects that people such as Qiu Lishu plant magazine before above-mentioned.People such as Zhou Rui are in the 4th interim detection method of also having reported with the dot-ELISA of the monoclonal antibody of bilharzial excretory-secretory antigen (S.ESA) preparation of this magazine nineteen ninety.Though this can have the higher specific selectivity of getting rid of other disturbing factor by the resulting monoclonal antibody of certain specific antigen to this kind antigen, but as can be known by aforementioned content, in the numerous complicated spectrotype of composition, its this species specific reach is too limited to after all, and the diagnostic significance of the antigen that detects has or not or size also still belongs to be measured.These have problems, and some is also touched upon in above-mentioned relevant document to some extent.Do not caused its recall rate and accuracy in actual use to be difficult to reach as reported in the literature level because the susceptibility that in detection the circulating antigen that is in low-level state is detected is high, especially more obvious to the detection influence of low-grade infection.In addition, spotting method is adopted in the detection of above-mentioned bibliographical information more, its operation steps is many, require and strictness of controlled condition, for some remote poverty-stricken area even can claim it is making excessive demands of being difficult to realize, and it is long to detect the used time, and waste of material is also bigger, and these have also caused the difficulty in generally applying.
The inventor of teachings herein " once reported among the IMMUNOLOGY KEY WORDS INDEX 1988.Vol.4 (1) a kind of by can be simultaneously at prepared polyclonal antibody serum of multiple antigen such as worm source property and worm's ovum source property, the method for utilizing the antigen probe of the multistage amplification principle of biotin-avidin system (BAS) to detect.Though with regard to a certain specific antigen, polyclonal antibody in some aspects may be not as good as the specific selectivity height of monoclonal antibody, but, it obtains by prepared in laboratory because being animal through special selection, and be can be at the multiple circulating antigen of snail fever, therefore it is not only being got rid of other parasitic infection interference and is having necessary specificity on only at schistosome antigen, and the total susceptibility and the recall rate of various circulating antigen obviously improved.But since its detection method still fail to overcome spotting method intrinsic shortcoming, for example using necessary simplify the operation and to the extensive adaptability of various simple and crude or severe environmental conditions for extensively popularizing, and further improving detection accuracy, especially aspects such as susceptibility that low infectiosity is detected and recall rate all also have and need further improve and improve part.
Purpose of the present invention also can all have extensive adaptability that a kind of detection circulating antigen of schistosome is provided to various environmental baselines for further improving detection level and simplifying the operation just, especially with the polyclonal antibody testing tool that circulating antigen of schistosome is detected on the basis, promptly common alleged medicine box.
Detection medicine box of the present invention still includes solid phase carrier and the detectable two parts that use that should cooperatively interact and forms.Solid phase carrier wherein is a supporting substrate with Polyvinylchloride (PVC) material.Because present known PVC material itself just has the effect of the anti-circulating antigen antibody of absorption that this carrier should possess, therefore said solid phase carrier can just adopt the supporting substrate itself that this material constitutes.At present except that the PVC material, the solid phase carrier that adopts as cellulose nitrate film or other suitable material forms is also arranged, therefore the said solid phase carrier of the present invention is except that above-mentioned form, also can be for be coated with the complex form of other suitable film layer with various forms on PVC host material surface, for example the preferential recommendation of the present invention is the complex carrier form that is coated with the cellulose nitrate rete.
The said detectable of the present invention is the mixing material form that A, B two components are arranged.Wherein component A is the anti-fluorescein antibody with horseradish peroxidase (KRP) mark, and B component is with fluorescein-labeled anti-schistosome circulating antigen antibody.Anti-fluorescein antibody among the component A can be by the height that the routine immunization injection the prepares animal anti-fluorescein antibody of tiring, behind the immune serum purifying with HRP on the sodium periodate method mark.Experimental results show that, with the HRP mark time, so that each anti-fluorescein antibody molecule on average in conjunction with 1.5-2 HRP molecule for well, being below or above this value can be because of HRP quantity not sufficient or excessive adverse effect such as not enough or non-specific colour developing, the accuracy of influence colour developing to some extent of may causing in the colour developing when the chromogenic reagent.Anti-schistosome circulating antigen antibody in the B component by aforementioned content as can be known, though needn't get rid of the use monoclonal antibody fully, but through using individual plant monoclonal antibody and polyclonal antibody relatively, under the good situation of test method specificity, the latter obviously is better than the former, therefore suggestion with adopt can be simultaneously be good at the polyclonal antibody of multiple antigens such as worm source property and worm's ovum source property, they all can be prepared by common immunization method, form with fluorescein-labelled according to a conventional method after purified again.Experimental result shows that during with fluorescein (F) the anti-circulating antigen antibody of mark (P), every milliliter of grammol ratio that is labeled the F/P in the antibody is that 1-2 is good, and ratio is excessive may to have a negative impact on the contrary because of the reason such as sterically hindered that is prone to a plurality of antibody.The same with general immunity test, each component is because of reasons such as the raw material source of its preparation and/or methods in the detectable, and difference to some extent on the result of use is even different to detect effect also inequality with batch mixed ratio of each component of preparing when mixing.Therefore, except that compositions such as all requisite damping fluid, inorganic salts and surfactant in common reagent, component A and B component best working concentration separately is or/and the suitableeest mixed ratio all can be selected definite by the best titre of the checkerboard type square formation of routine immunization test.
On the foregoing basis, through repetition test and relatively discovery, if can change used solid phase carrier into have the appropriate format that can contain liquid dimpled by common surface plate form, for example have one or forms such as several columns or hemispherical recess, can further improve effect and the level of detecting, and operation is also very convenient.Especially when adopting the combined type solid phase carrier that the lining rete is arranged, compound rete only overlayed on the basal surface of recess to compare effect close with all surfaces that comprises the recess sidewall all being covered with rete, but it be preferable for the former to say at least economically.
Be not difficult to find out that by foregoing it is exactly to have utilized the multistage amplification and the principle of the fluorescein-anti-fluorescein antibody system that is better than the BAS system in the detectable that the present invention detects one of important feature of medicine box.Though utilization has special affinity very high between the micromolecule fluorescein of haptens character and macromolecular anti-fluorescein antibody and can produce multistage amplification report is arranged recently, but before foregoing of the present invention proposes, to parasitic disease, especially do not used effectively but seeing in the detection of snail fever circulating antigen.In addition to the improvement of solid phase carrier form and the selection of optimised form, make testing tool of the present invention no matter in the simplification of method of operating, the length of detection time with environmental baseline required degree and extensive adaptability, still detect effect and level, especially all be far superior to the whole bag of tricks in the aforementioned documents on the detection level to low infectiosity.For example on method of operating, requisite wash water can replace being strict with the distilled water and the damping fluid of use with the clean water at tap water or any water source such as well water, river in the detection in spotting method, and also save fully be generally spotting method instrument and equipments such as indispensable constant temperature oven, baking box, shaking table; Examinee's blood sample both can be common used serum, also can be for not adding the whole blood of separation; Detect and consuming timely also can be reduced to by a few hours of spotting method 20-30 minute; Each detection scale requires also can a collection of to reduce to a person-portion a collection of by necessary tens of person-portions up to a hundred of spotting method, and the detection cost also can reduce more than 50%.Wherein some advantage obviously be present the whole bag of tricks can't realize fully, they all have the Practical significance and the value of particular importance undoubtedly, for in the outpatient service fast detecting, provide a great convenience condition in particular for carrying out in environment and the extremely simple and crude remote districts of condition and popularizing to detect on the spot, and make it to be achieved.Detecting on the effect, the sensitivity that detects the snail fever circulating antigen with medicine box of the present invention can reach 10
-9The level of g has improved 30 times than present spot test (Dot-ELISA) method.Light to picking up from, in, the serum of the schistosomiasis endemic district excrement inspection positive patient of heavy different infectiosities detects positive rate and is respectively 92.9～100%, the positive rate that serum of acute schistosomicide patient is detected is 100%, and normal person or the false positive rate of suffering from other parasitic disease human serum are no more than 0.11%, shown the snail fever circulating antigen, especially the detection to low-grade infection serum has desirable susceptibility and specificity, this also be present various testing tool and method can't be obtained.The comparing data that adopts the present invention to detect the parties concerned of the detection technique that medicine box and method and aforementioned documents report sees Table 1, the detection method of bibliographical information is the spot test method, wherein the instrument of " contrast 1 " usefulness is that the people reported tight self-service grade, the instrument of " contrast 2 " usefulness be Qiu Lishu etc. the people reported, the instrument of " contrast 3 " usefulness is people's report such as Zhou Rui.
What below introduce is example as content of the present invention, but scope of the present invention is not limited in following example.
Example 1
It is supporting substrate that solid phase carrier in this routine medicine box adopts the PVC sheet material that has some cylindric recesses, and is coated with the cellulose nitrate rete on the baseplane in each recess.
Detectable in this routine medicine box is made up of A, B two parts, mixes with inorganic salts, damping fluid, surfactant and distilled water etc. routinely.Prepare the anti-FITC of rabbit (fluorescein isothiocynate) immune serum of efficient valency with the inoculation method, with the bond of sodium periodate method mark preparation with HRP, obtain component A behind ammonium sulfate precipitation, wherein each antibody molecule is on average in conjunction with 1.5 HRP molecules.By tiring (10 at the intestines related antigen of imago of blood fluke release and the height of multiple antigen preparation such as pellicle antigen and worm's ovum source property antigen
6) the anti-circulating antigen antibody of polyclone through ammonium sulfate saltout and column chromatography purification after, use the FITC mark, make 1.75 FITC molecules of the average mark of each antibody molecule, obtain B component.Will be by 1: the dilution B component of 25-1000 with by 1: 100 dilution with amount antigen after doing the selection of square formation titre on the checkerboard type solid phase carrier, the best dilute concentration of determining B component is 1: 200.With this B component of tiring to use by 1: 25～1000 different dilution component A with quadrat method determined the optimum dilution degree of component A be 1: 100 and in detectable the suitableeest blending ratio of A and B two components be 2: 1.With this routine detectable four extinction values (OD value) testing result of standard female and positive serum is respectively 0.95,0.98,0.89 and 0.92, standard deviation 0.039, the coefficient of variation 4.12%.
Example 2
Solid phase carrier and the method for preparing detectable are with example 1.Average mark 1.7 molecule HRP of each antibody molecule of component A in the detectable wherein, the average mark 1.9 molecule FITC of each antibody molecule in the B component, its best dilute concentration is all with example 1, and the suitableeest blending ratio of A and B is 1: 1.This routine reagent is respectively 1.10,1.05 to four extinction values that standard serum detects, and 1.15 and 0.97, standard deviation 0.082, the coefficient of variation 7.66%.
Example 3
Solid phase carrier and the method for preparing detectable are with example 1.Average mark 1.95 molecule HRP of the every antibody molecule of component A in the detectable wherein, the average mark 1.5 molecule FITC of the every antibody molecule of B component, its best dilute concentration is all with example 1, and the suitableeest blending ratio of A and B is 1.5: 1.This routine reagent is respectively 1.30,1.20 to four extinction value testing results of standard serum, and 1.26 and 1.15, standard deviation 0.066, the coefficient of variation 5.13%.
More than three routine detectable actual measurement tiring all of infecting that serum determine more than 1: 4096, meet the requirement of actual measurement fully.
Method of operating when the above-mentioned detection medicine box of the present invention uses is very simple.At first diluted test serum (available in case of necessity whole blood, but note not haemolysis) is paved with a certain recess bottom surface in the solid phase carrier, room temperature is placed 10 minutes (time expand is not limit) for 25 ℃, the serum deprivation that inclines, wash 10 times from the beginning after control do.Add by 1: the detectable of 50-100 working concentration dilution, place hypsokinesis in 10 minutes for 25 ℃ and go, wash from the beginning more than 10 times, preferably again with the distillation washing once, control is done at last.Add and contain H
2O
2Tmb substrate solution reaction 5-10 minute, promptly available range estimation or spectrophotometer are done colorimetric analysis, judged result.There is no the requirement of other more instrument and equipment and operating conditions aspect in addition.
Table 1
| Recall rate % | Detect consuming time | The operation difficulty or ease | Need view device equipment | Everyone part testing cost | The on-the-spot applicability of blood fluke | |
| The present invention | 92.9-100 | The 20-30 branch | Very easy | Do not have | 0.3 unit | Fine |
| Contrast 1 | 82.5-92.1 | 5 hours | Difficult | Baking oven, shaking table | 0.6 unit | Relatively poor |
| Contrast 2 | 81.3 | ＞5 hours | Difficult | Constant temperature oven, shaking table | / | Relatively poor |
| Contrast 3 | 80.9 | ＞5 hours | Difficult | Do not have | / | Relatively poor |
Claims (7)
1, a kind of medicine box that is used to detect the snail fever circulating antigen, solid phase carrier and detectable two parts that comprising cooperatively interacts uses are formed, wherein solid phase carrier employing pvc material is a supporting substrate, the anti-fluorescein antibody of detectable usefulness horseradish peroxidase-labeled and potpourri with fluorescein-labeled anti-schistosome circulating antigen antibody, in using the anti-fluorescein antibody of horseradish peroxidase-labeled, combined marker enzyme molecule is 1.5-2 on each antibody molecule, and in every milliliter of fluorescein-labeled anti-schistosome circulating antigen antibody, the grammol ratio of fluorescein and anti-schistosome circulating antigen antibody is 1-2.
2, medicine box as claimed in claim 1, it is characterized in that the anti-schistosomiasis circulating antigen antibody that is labeled in the said detectable for can be simultaneously at the polyclonal antibody of blood fluke worm source property and worm's ovum source property antigen.
3, medicine box as claimed in claim 1 or 2 is characterized in that being selected to determine by the checkerboard type square formation titre of immunity test with the anti-fluorescein antibody of enzyme labeling and with the optimization blending ratio between fluorescein-labeled anti-schistosomiasis circulating antigen antibody in the said detectable.
4, medicine box as claimed in claim 1 is characterized in that said solid phase carrier is for having the form of a recess at least.
5,, it is characterized in that said solid phase carrier is for being coated with the complex form of cellulose nitrate rete on the Polyvinylchloride supporting substrate as claim 1 or 4 described medicine boxs.
6, medicine box as claimed in claim 4 is characterized in that said solid phase carrier is for only being coated with the complex form of cellulose nitrate rete on the bottom surface of supporting substrate recess.
7, medicine box as claimed in claim 3 is characterized in that said solid phase carrier is the supporting substrate at the band recess of being made by Polyvinylchloride sheet material, and only is coated with the complex form of cellulose nitrate rete at the base fabric of recess.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92108014A CN1034975C (en) | 1992-01-17 | 1992-01-17 | Medicinal box for high-sensitivity quick detection of schistosomiasis circulating antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92108014A CN1034975C (en) | 1992-01-17 | 1992-01-17 | Medicinal box for high-sensitivity quick detection of schistosomiasis circulating antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1062601A CN1062601A (en) | 1992-07-08 |
| CN1034975C true CN1034975C (en) | 1997-05-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92108014A Expired - Fee Related CN1034975C (en) | 1992-01-17 | 1992-01-17 | Medicinal box for high-sensitivity quick detection of schistosomiasis circulating antigen |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6287783B1 (en) * | 1999-03-18 | 2001-09-11 | Biostar, Inc. | Optical assay device and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4158049A (en) * | 1976-09-27 | 1979-06-12 | Edna Mcconnell Clark Foundation | Antigen fraction of Schistosoma mansoni eggs suitable for testing for schistosomiasis |
| EP0291180A1 (en) * | 1987-04-24 | 1988-11-17 | Takeda Chemical Industries, Ltd. | Assay for anti-pre-S antibody |
| EP0328388A2 (en) * | 1988-02-12 | 1989-08-16 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Wash solution, test kit and method for the determination of an immunological ligand |
-
1992
- 1992-01-17 CN CN92108014A patent/CN1034975C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4158049A (en) * | 1976-09-27 | 1979-06-12 | Edna Mcconnell Clark Foundation | Antigen fraction of Schistosoma mansoni eggs suitable for testing for schistosomiasis |
| EP0291180A1 (en) * | 1987-04-24 | 1988-11-17 | Takeda Chemical Industries, Ltd. | Assay for anti-pre-S antibody |
| EP0328388A2 (en) * | 1988-02-12 | 1989-08-16 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Wash solution, test kit and method for the determination of an immunological ligand |
Non-Patent Citations (1)
| Title |
|---|
| 《中国寄生虫学与寄生虫病杂志》 1990年 第8卷 第4期 1990.1.1 周蕊 * |
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| CN1062601A (en) | 1992-07-08 |
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