CN101368955A - Chemical luminescence immune analysis reagent box for detecting herpes simplex virus type II IgG antibody - Google Patents
Chemical luminescence immune analysis reagent box for detecting herpes simplex virus type II IgG antibody Download PDFInfo
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- CN101368955A CN101368955A CN 200810105411 CN200810105411A CN101368955A CN 101368955 A CN101368955 A CN 101368955A CN 200810105411 CN200810105411 CN 200810105411 CN 200810105411 A CN200810105411 A CN 200810105411A CN 101368955 A CN101368955 A CN 101368955A
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Abstract
The invention discloses a test kit and a preparation method thereof for detecting herpes simplex virus type II IgG antibody, wherein, alkaline phosphatase is adopted to mark the antibodies, and the chemiluminescence zymolyte liquid includes 1,2-butyl dioxide derivatives. The test kit is composed of solid phase envelope carrier, biotinylated antigen, positive and negative reference substance, enzyme-labeled antibody, the chemiluminescence zymolyte liquid and the condensed washing liquid. The device and method belongs to medical detection field, establishes the method of sensitively and quickly detecting herpes simplex virus type II IgG antibody, has better precision, and is a reliable method for detecting herpes simplex virus type II IgG antibody.
Description
Technical field
The present invention relates to the clinical examination field, kit of a kind of employing biotin-avidin coating technique and chemiluminescence immunoassay technology detection herpes simplex virus I I type IgG antibody (HSV-II IgG) and preparation method thereof is provided.
Background technology
The herpes simplex virus diameter is about 120-150 μ m, outwards is made up of coating, body quilt, three kinds of concentric structures of capsid successively, and body is made of cellulosic, and cell membrane contains the fat composition.Can survive the several months at low temperatures, under damp and hot 50 ℃ and dry 90 ℃ of conditions, can eliminate in 30 minutes.The mankind are unique natural reservoir (of bird flu viruses) of herpes simplex virus, and population infection is up to 80%-90%, and 10% is asymptomatic.
Herpes simplex virus is divided into two kinds on I type and II type, and I type herpesviral mainly is by the close contact transmission of respiratory tract, skin and mucous membrane, infects the mucocutaneous and organ of waist with the upper part.II type herpesviral mainly causes genital herpes, and mainly propagates by direct contact focus, and causes cutaneous lesions.The pathogen of genital herpes 90% is an II type herpesviral, and only 10% is I type herpesviral.
In case herpes simplex virus virus enters human body, in first postictal several weeks, the sustainable generation of antibody also is present in the blood in bleb, and is extremely important for the defence naturally of human body.The antibody of herpes simplex mainly contains two kinds, and a kind of is IgM antibody, generally occurs in the high-incidence season of virus breeding, and the duration is shorter; Another kind of antibody is IgG antibody, and this antibody is in case produce existence for a long time.As long as infected herpes simplex, IgG antibody will exist in vivo.Can there be the IgG antibody of herpes simplex virus throughout one's life in most of adults.
Binding constant between the biotin-avidin is up to 10
15(mol/L)
-1, just very stable once combination.Biotin and Avidin both can with multiple material markings such as enzyme, but a series of big molecular biological activity materials such as coupling antibody again, this provides more effective and sensitive means for clinical detection antigen or antibody.Particularly in recent years, the appearance of Streptavidin has overcome traditional biological element-Avidin system and has had the to a certain degree shortcoming of non-specific binding, makes the application of this system increasingly extensive.
Summary of the invention
The purpose of this invention is to provide and a kind of the biotin-avidin coating technique is combined with chemiluminescence immunoassay technology, be used to kit that detects HSV-II type IgG antibody and preparation method thereof.
Technical scheme of the present invention: a kind of HSV-II type IgG antibody chemical luminescence immune assay determination reagent kit, it consists of: the solid phase carrier of Avidin bag quilt; Biotinylated herpes simplex virus I I type antigen; The negative control product; Positive reference substance; The anti-human IgG antibody of alkali phosphatase enzyme mark; Chemical luminous substrate liquid and concentrated cleaning solution that above-mentioned enzyme acted on.
According to kit of the present invention, described solid phase carrier is microwell plate or plastic tube; The luminous agent of the chemical luminous substrate liquid of described alkaline phosphatase is 1,2-two oxidative ethane analog derivatives, comprise 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), CSPD or CDP-Star.
The preparation method of herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of the present invention may further comprise the steps:
1) the Avidin bag is by solid phase carrier:
Avidin added to mixing is mixed with desired concn among the PBS that 0.05mol/L pH is 7.0-7.5, and coating buffer is carried on the solid phase carrier; Behind physiological saline washing above-mentioned solid phase carrier, with containing 1%BSA, 0.1% antiseptic, the pH value is that the 0.01mol/L PBS of 7.0-7.5 seals the above-mentioned solid phase carrier as confining liquid.
In said method, the solid phase carrier of described Avidin is microwell plate or plastic tube;
2) biotinylation herpes simplex virus I I type antigen
Biotin (BNHS) is dissolved in the solution that dimethyl sulfoxide (DMSO) (DMSO) is mixed with 10mg/mL, with 0.1mol/L sodium borate buffer liquid with the herpes simplex virus I I type antigen diluent of purifying to 1-2mg/mL, the two is mixed in the ratio of 1:50, stirring reaction 4h under the room temperature, be 4 ℃ of dialysed overnight among the PBS of 7.0-7.5 at 0.01mol/L pH then, bond adds equal-volume glycerine, preserves standby below-20 ℃.
3) preparation yin and yang attribute reference substance;
After the many parts high HSV-I IgG positive human serum mixing of tiring, 56 ℃ of 1h deactivations, aseptic filtration is suitably made positive reference substance after the dilution, and dilution is 10% NBCS, 0.1% antiseptic, 0.02M Tris-HCl damping fluid, pH7.0-7.5.
The negative human serum of many parts of HSV-I IgG is mixed, and the negative control product are made in aseptic filtration after 56 ℃ of 1h deactivations.
4) the anti-human IgG antibody of preparation alkali phosphatase enzyme mark;
Adopting glutaraldehyde method with alkaline phosphatase and anti-human IgG antibody's coupling, is that the PBS of 7.0-7.5 fully dialyses to 0.01mol/L pH then, adds equal-volume glycerine, and preservation is standby below-20 ℃.
5) preparation chemical luminous substrate liquid;
Chemical luminous substrate liquid consist of 0.15-0.25mol/L Tris-HCl, 16% NaCl, 0.4% KCl, 0.1% antiseptic, 1.5%-4.0% luminous agent, pH are 7.0-7.5.
In said method, the luminous agent of the chemical luminous substrate liquid of described alkaline phosphatase is 1,2-two oxidative ethane analog derivatives, comprise (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
6) preparation concentrated cleaning solution
Concentrated cleaning solution consist of 0.15-0.25mol/L Tris-HCl, 16% NaCl, 1% Tween-20, pH are 7.0-7.5.Dilute 20 times with deionized water during use.
With the above-mentioned semi-manufacture packing for preparing, be assembled into the finished product kit.
Beneficial effect of the present invention: the kit applicating biotin-Avidin coating technique among the present invention, not only can keep being labeled the biologically active of antigen, and because of affinity high between biotin and the Avidin, reduced bag by the non-special absorption in the process, and under the condition of high dilution, still can reach the effect same, thereby save the consumption of coated antibody with direct coated.Applied chemistry luminescence immunoassay technology can avoid in the enzyme-linked immuno assay kit testing process external factor such as cut to result's influence.The combination of two kinds of methods has improved the sensitivity of herpes simplex virus I I type IgG antibody test effectively, is a kind of reliable detection method.
Embodiment
Embodiment 1 preparation herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of the present invention
1) preparation of Avidin solid phase microwell plate
It is that mixing is made into 2.5 μ g/mL in 7.2 the PBS coating buffer that Avidin is added 0.05mol/LpH, joins then in each hole of microwell plate, and every hole 110 μ L place 24h for 4 ℃.Give a baby a bath on the third day after its birth time with physiological saline, every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours, confining liquid be formulated as 1.3mmol/LNaH
2PO
42H
2O, 8.7mmol/L NaH
2PO
412H
2O, 1%BSA and 0.1% biological preservative are settled to 1000ml, and the pH value is 7.2.
Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out envelope immediately, 2～8 ℃ of preservations of labeling postposition.
2) preparation of enzyme labelled antibody
Adopting glutaraldehyde method with alkaline phosphatase and anti-human IgG antibody's coupling, is that 7.2 PBS fully dialyses to 0.05mol/L pH then, adds equal-volume glycerine, and preservation is standby below-20 ℃.
3) preparation of biotinylated antigen
Biotin (BNHS) is dissolved in the solution that dimethyl sulfoxide (DMSO) (DMSO) is mixed with 10mg/mL, with 0.1mol/L sodium borate buffer liquid with the herpes simplex virus I I type antigen diluent of purifying to 1mg/mL; The two is mixed stirring reaction 4h under the room temperature in the ratio of 1:50; The bag filter of packing into is 4 ℃ of dialysed overnight among 7.2 the PBS to 0.05mol/L pH value, and bond adds equal-volume glycerine, and packing is in a small amount preserved standby below-20 ℃.
Adopt the square formation method to select the working concentration of biotinylated antigen and enzyme labelled antibody to be respectively 1:2000 and 1:4000, with biotinylated antigen and enzyme labelled antibody enzyme labelled antibody diluted, its constituent is Tris 12.120g, HCl 10mL, BSA 5g, Proclin 300 1ml, deionized water is settled to 1000mL.
4) preparation of yin and yang attribute reference substance
After the many parts high HSV-II IgG positive human serum mixing of tiring, 56 ℃ of 1h deactivations, aseptic filtration, with containing 10% NBCS, 0.1% antiseptic, the 0.02M Tris-HCl damping fluid of pH7.2 dilute makes positive reference substance, and dilution ratio is 1:500.
The negative human serum of many parts of HSV-II IgG is mixed, and the negative control product are made in aseptic filtration after 56 ℃ of 1h deactivations.The all freezing preservation of yin and yang attribute reference substance.
5) preparation of chemical luminous substrate liquid
Get Tris 24g, HCl 15ml, NaCl 160g, KCl 4g, proclin 300 1mL, AMPPD 20mL behind the adding deionized water dissolving, is settled to 1000mL.
6) preparation of concentrated cleaning solution
Get Tris 24g, HCl 15mL, NaCl 160g, Tween-20 10mL is settled to 1000mL with deionized water.Dilute 20 times with deionized water during use.
With the above-mentioned semi-manufacture packing for preparing, be assembled into the finished product kit.
Embodiment 2 preparations herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of the present invention
As outside the solid phase carrier, all the other all prepare herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit with the method identical with embodiment 1 divided by plastic tube.
Embodiment 3 preparations herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of the present invention
The preparation of chemical luminous substrate liquid: get Tris 27g, HCl 16.88mL, NaCl 160g, KCl 4g, proclin300 1mL, CPD-Star 32mL after the dissolving of adding distilled water, is settled to 1000mL.
All the other are identical with embodiment 1.
Embodiment 4 preparations herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of the present invention
The preparation of chemical luminous substrate liquid: get Tris 21g, HCl 13.13mL, NaCl 160g, KCl 4g, NaN
31mL, CSPD 16mL after the dissolving of adding distilled water, is settled to 1000mL.
All the other are identical with embodiment 1.
The using method of embodiment 5 kits of the present invention
The concrete operations of the herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit of above embodiment 1 preparation are as follows:
1) in 4 ℃ of refrigerators, takes out kit, equilibrium at room temperature 15 minutes; Take out coated slab, insert on the grillage.
2) blank 1 hole is established in each test, and each two hole of negative control hole and positive control hole are except that blank well, each hole adds biotinylated antigen 50 μ L, add the yin and yang attribute reference substance again and with the sample 100 μ L of physiological saline 1:100 dilution, with the micro-oscillator mixing that fully vibrates, 37 ℃ of incubations 30 minutes;
3) 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;
4) every hole adds enzyme labeling thing 100 μ L, with the micro-oscillator mixing that fully vibrates, and 37 ℃ of incubations 30 minutes;
5) 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;
6) every hole adds 50 μ L chemical luminous substrate liquid, and fully vibrating with micro-oscillator mixes, and room temperature was educated 30 minutes, measured the luminous intensity (RLU) in each hole in regular turn on the chemiluminescence measuring instrument, 1 second/hole of Measuring Time;
7) CUT OFF=2.1 * negative control product RLU, RLU is positive greater than Cut off, otherwise negative.
The methodology calibrating of embodiment 6 kits of the present invention
1) precision
To with a positive sample, carry out replication 3 times according to embodiment 5 described methods of operating, each 10 holes, the precision of investigation kit of the present invention.As calculated, the CV% of three mensuration RLU is respectively 8.6%, 7.7% and 8.9%, all less than 10%, illustrates that kit accuracy of the present invention is good, meets the requirements.
2) specificity
Detect through the herpes simplex virus I I of clinical definite type infected person anteserum sample 105 examples according to embodiment 5 described methods of operating with kit of the present invention, healthy human serum sample 92 examples are investigated coincidence rate.As shown in Table 1, kit of the present invention detects 2 parts of feminine genders in herpes simplex infections person serum sample, coincidence rate 98.1%, and the testing result of healthy human serum sample conforms to fully with the clinical detection result, overall coincidence rate is 99.0%.
Table 1 kit specific detection of the present invention result
| Test item | The example number | Positive test symbol |
| Positive sample | 105 | 105 |
| Negative sample | 92 | 0 |
3) sensitivity
With the high titre positive sample of portion with 1:40,1:80,1:160,1:320, the dilution proportion of 1:640 is investigated the sensitivity (seeing Table 2) of kit of the present invention.
Table 2 kit sensitivity of the present invention testing result
| Test item | Testing result |
| 1:40 | + |
| 1:80 | + |
| 1:160 | + |
| 1:320 | + |
| 1:640 | - |
As shown in Table 2, but when high titre positive sample 1:40-1:320 dilution kit of the present invention test positive still, when 1:640 diluted, testing result was negative, illustrated that the sensitivity of kit of the present invention is higher.
The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit is characterized in that described kit comprises: the solid phase carrier of Avidin bag quilt; Biotinylated herpes simplex virus I I type antigen; The negative control product; Positive reference substance; The anti-human IgG antibody of alkali phosphatase enzyme mark; Chemical luminous substrate liquid and concentrated cleaning solution
2. kit as claimed in claim 1 is characterized in that, the solid phase carrier of described Avidinization is microwell plate or plastic tube.
3. kit as claimed in claim 1, it is characterized in that, the luminous agent of described alkaline phosphatase chemical luminous substrate liquid is 1,2-two oxidative ethane analog derivatives, comprise (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
4. kit as claimed in claim 1, it is characterized in that, described chemical luminous substrate liquid consist of 0.20mol/L Tris-HCl, 16% NaCl, 0.4% KCl, 2% 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 " phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, 1% Proclin 300.
5. the preparation method of a herpes simplex virus I I type IgG antibody chemical luminescence immune assay determination reagent kit is characterized in that:
1) the Avidin bag is by solid phase carrier
It is that mixing is mixed with desired concn in the 0.05mol/L phosphate buffer (PBS) of 7.0-7.5 that Avidin is added to pH, and coating buffer is carried on the solid phase carrier; Behind physiological saline washing above-mentioned solid phase carrier, with containing 1%BSA, 0.1% antiseptic, pH are that the 0.01mol/L PBS of 7.0-7.5 seals the above-mentioned solid phase carrier as confining liquid.
2) biotinylation herpes simplex virus I I type antigen
Biotin (BNHS) is dissolved in the solution that dimethyl sulfoxide (DMSO) (DMSO) is mixed with 10mg/mL, with 0.1mol/L sodium borate buffer liquid with the herpes simplex virus I I type antigen diluent of purifying to 1-2mg/mL, with the two in stirring reaction 4h under the ratio mixed room temperature of 1:50, be 4 ℃ of dialysed overnight among the PBS of 7.0-7.5 at 0.01mol/L pH then, bond adds equal-volume glycerine, preserves standby below-20 ℃.
3) preparation yin and yang attribute reference substance;
After the many parts high HSV-II IgG positive human serum mixing of tiring, 56 ℃ of 1h deactivations, aseptic filtration is suitably made positive reference substance after the dilution, and dilution is 10% NBCS, 0.1% antiseptic, 0.02mol/LTris-HCl damping fluid, pH 7.0-7.5.
The negative human serum of many parts of HSV-II IgG is mixed, and the negative control product are made in aseptic filtration after 56 ℃ of 1h deactivations.
4) the anti-human IgG antibody of alkali phosphatase enzyme mark;
Adopting glutaraldehyde method with alkaline phosphatase and anti-human IgG antibody's coupling, is that the PBS of 7.0-7.5 fully dialyses to 0.01mol/L pH then, adds equal-volume glycerine, and preservation is standby below one 20 ℃.
5) preparation chemical luminous substrate liquid
Chemical luminous substrate liquid consist of 0.15-0.25mol/L Tris-HCl, 16% NaCl, 0.4% KCl, 0.1% antiseptic, 1.5%-4.0% luminous agent, pH are 7.0-7.5.
6) preparation concentrated cleaning solution
Concentrated cleaning solution consist of 0.15-0.25mol/L Tris-HCl, 16% NaCl, 1% Tween-20, pH are 7.0-7.5.Dilute 20 times with the distillation deionized water during use.
With the above-mentioned semi-manufacture packing for preparing, be assembled into the finished product kit.
6. method as claimed in claim 5 is characterized in that, the solid phase carrier of described Avidin bag quilt is microwell plate or plastic tube.
7. method as claimed in claim 5, it is characterized in that, the luminous agent of described alkaline phosphatase chemical luminous substrate liquid is 1,2-two oxidative ethane analog derivatives, comprise (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
8. method as claimed in claim 5, it is characterized in that, described chemical luminous substrate liquid consist of 0.20mol/L Tris-HCl, 16% NaCl, 0.4% KCl, 2% 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 " phosphorus acyloxy) phenyl-1; 2-two oxidative ethanes, 1% Proclin 300, pH is 7.2.
9. method as claimed in claim 5 is characterized in that, described concentrated cleaning solution consist of 0.20mol/L Tris-HCl, 16% NaCl, 1% Tween-20, pH are 7.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810105411 CN101368955A (en) | 2008-04-29 | 2008-04-29 | Chemical luminescence immune analysis reagent box for detecting herpes simplex virus type II IgG antibody |
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|---|---|---|---|
| CN 200810105411 CN101368955A (en) | 2008-04-29 | 2008-04-29 | Chemical luminescence immune analysis reagent box for detecting herpes simplex virus type II IgG antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091500A (en) * | 2013-02-28 | 2013-05-08 | 苏州和锐医药科技有限公司 | Antibody detection kit (OmpC-IgA) |
| CN111007249A (en) * | 2019-11-27 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Herpes simplex virus antibody IgG chemiluminescence immunoassay kit and preparation method thereof |
-
2008
- 2008-04-29 CN CN 200810105411 patent/CN101368955A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091500A (en) * | 2013-02-28 | 2013-05-08 | 苏州和锐医药科技有限公司 | Antibody detection kit (OmpC-IgA) |
| CN111007249A (en) * | 2019-11-27 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Herpes simplex virus antibody IgG chemiluminescence immunoassay kit and preparation method thereof |
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Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111028 |
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Application publication date: 20090218 |