CN101377507A - Chemiluminescence reagent kit for detecting herpes simplex virus I-type IgM antibody and preparing method thereof - Google Patents
Chemiluminescence reagent kit for detecting herpes simplex virus I-type IgM antibody and preparing method thereof Download PDFInfo
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Abstract
The invention discloses a chemiluminescent kit for detecting a herpes simplex virus I-type IgM antibody, which comprises a negative control, a positive control, a solid-phase vector which is coated by an anti-human Mu-chain monoclonal antibody, a neutralizing antigen, a herpes simplex virus I-type IgM antibody marked by enzyme, a chemiluminescent substrate solution and a concentrated washing solution; wherein, the enzyme is alkaline phosphatase, the chemiluminescent substrate solution contains 1, 2-diarylethane derivatives. With the kit of the invention to detect the herpes simplex virus I-type IgM antibody, the operation is simple and convenient, the sensitivity is high, the specificity is good, and the detection result coincides with those of other detection methods. The invention also discloses a preparation method of the chemiluminescent kit for detecting a herpes simplex virus I-type IgM antibody.
Description
Technical field
The present invention relates to the immunoassay medical domain, more particularly, relate to a kind of chemical luminescence reagent kit that detects herpes simplex virus I-type IgM antibody and preparation method thereof.
Background technology
Herpes simplex virus (HSV) belongs to a kind of of herpesviral.This viral center is DNA, forms capsid by 162 shell particulates outward, is 20 bodies, and the coating that contains lipid is arranged on every side, and the virion size is 150-200nm.According to serology, epidemiology characteristics, herpes simplex virus is divided into HSV-I and HSV-II amphitypy.Clinical manifestation is more common with mucocutaneous herpes simplex infections, is representative with herpes simplex labialis, genital herpes especially.
After the primary infection, immunoglobulin M (IgM) appears in body at first, occurs immunoglobulin A (IgA) and immunoglobulin G (IgG) subsequently.Antibody capable prevents viral spread, but can not stop recurrence.Detect the specific IgM positive or paired sera specific IgG antibodies and tire rising more than four times or four times, can point out the HSV recent infection.
HSV mainly causes herpetic stomatitis, herpetic keratoconjunctivitis, herpetic meningitis, herpetic vulvovaginitis, eczema bleb, neonate's bleb etc.The HSV at position infects and how to cause (accounting for 95%) by the HSV-I type beyond the reproductive organs, and the HSV of reproductive organs infects, and mainly causes (accounting for 78%) by the HSV-II type.The prompting of IgM antibody positive has HSV to infect in the recent period.IgG antibody positive (no change of tiring) illustrates the previous infection mistake, and certain immunity is arranged.
Pregnant early infection HSV person can cause the miscarriage, in the gestation, late period the infected, can cause fetus and neonate the morbidity.
Herpes simplex virus detection of antibodies method mainly contains radio immunoassay, fluoroimmunoassay and enzyme-linked immunosorbent assay etc.Though radiommunoassay is highly sensitive, the height is special, there is term of validity weak point, has alpha-contamination shortcoming.Though fluoroimmunoassay high specificity, susceptibility height, speed are fast, the unspecific staining problem solves as yet fully, the objectivity deficiency that the result judges, and program is more complicated also.Though enzyme-linked immuno assay is highly sensitive, high specificity, have that endogenous enzyme disturbs, substrate is most of poisonous or be shortcomings such as carcinogen.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) being worldwide to develop very fast on-radiation immunoassay over past ten years, is a kind of supersensitive microdetermination technology that grows up after radiommunoassay and EIA enzyme immunoassay.Chemiluminescence immune assay combines with the high specific immune response of antibody antigen with high-sensitive chemiluminescence detection technology, with chemiluminescent substance labelled antigen or antibody, carry out immune association reaction again, can carry out qualitative, quantitative measurement.Advantages such as this method has high sensitivity, sensing range is wide, easy and simple to handle fast, label is stable, pollution-free, instrument simple economy.It is radioimmunoassay and the desirable substituent of normal enzyme immunoassay, is that present immune quantitative is analyzed optimal method.
The principle that detects herpes simplex virus I-type IgM antibody with chemiluminescence immune analysis method is, testing sample is joined in the White-opalescent microwell plate that is coated with anti-people μ chain monoclonal antibody, in adding subsequently and antigen combine with IgM antibody specificity in the testing sample, add again enzyme labeling in and antigen-specific antibody form the anti-people μ chain monoclonal antibody of solid phase-IgM antibody-antigen-hrp-antibody complex, unreacted free composition is removed in washing.Then, add the luminous substrate liquid of above-mentioned enzyme, the free energy that utilizes chemical reaction to discharge excites intermediate luminous, thus testing molecule concentration in the test sample.
The heavy chain of human immunoglobulin(HIg) has antigentic specificity, and wherein the heavy chain of IgM is a μ chain hypotype.The rising of specific IgM antibodies can be used as the index of recent infection, so detecting of IgM antibody has very important clinical meaning.Anti-people μ chain monoclonal antibody is used for prize law more and detects IgM antibody, and bag is by on solid phase carrier, in conjunction with all IgM in the sample.
The general glutaraldehyde coupling method that adopts is with marker enzyme and antibody coupling.Combine with amino covalence on enzyme and the immunoglobulin (Ig) respectively by its aldehyde radical.Antibody to be marked to fully dialysis of phosphate buffer (PBS), adds equal-volume glycerine with glutaraldehyde coupling method and alkaline phosphatase coupling, preserves standby below-20 ℃.
General adopt the square formation method determine in and the concentration of antigen and enzyme labelled antibody.The component of concentration to be determined joins in the solid-phase coating carrier with different dilutabilitys by square formation, selects suitable concentration according to relative luminous intensity (RLU).
In the kit reagent, antiseptic commonly used has proclin 300, NaN
3, thimerosal etc.
The kit that detects herpes simplex virus I-type IgM antibody on the existing market mostly is the enzyme-linked immuno assay kit, reaction terminating liquid is a sulfuric acid, has corrosivity, have also simultaneously that endogenous enzyme disturbs, substrate is most of poisonous or be the shortcoming of carcinogen, the detection of absorbance also is vulnerable to the influence of multiple external factors such as cut.
Summary of the invention
Be subjected in order to overcome enzyme-linked immuno assay that multiple external factor is disturbed, detection sensitivity is low, the most of poisonous deficiency of substrate, the invention provides a kind of chemical luminescence reagent kit that detects herpes simplex virus I-type IgM antibody, comprise feminine gender, positive reference substance, the solid phase carrier of anti-people μ chain monoclonal antibody bag quilt, in and antigen, the herpes simplex virus I-type antibody of enzyme labeling, chemical luminous substrate liquid and concentrated cleaning solution.Wherein, described enzyme is an alkaline phosphatase, and described chemical luminous substrate liquid contains 1,2-two oxidative ethane analog derivatives.
Use this kit to detect herpes simplex virus I-type IgM antibody, easy and simple to handle, highly sensitive, specificity is good, is consistent with other detection method assays.
On the basis of technique scheme, the present invention can also do following improvement:
Described chemical luminous substrate liquid contains (diamantane)-1,2-two oxidative ethanes, 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 '-phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), 3-(2 '-(5 ＂-chlorine) spiral diamantane)-4-methoxyl-4-(3 ＂-phosphorus acyloxy) benzene-1,2-dioxetane (CSPD) or 3-(2 '-(5 ＂-chlorine) spiral diamantane)-4-methoxyl-4-(3 ＂-phosphorus acyloxy-5 ＂-chlorine) benzene-1,2-dioxetane (CDP-Star).
On the basis of technique scheme, the present invention can also do following improvement:
Described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD, CSPD or CDP-Star 1.5～4.0% (v/v),
Its pH is 7.0～7.5
On the basis of technique scheme, the present invention can also do following improvement:
Described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD 1.5～4％(v/v)，
Its pH is 7.0～7.5
On the basis of technique scheme, the preferred preferred version of the present invention is:
Described chemical luminous substrate liquid contains:
Tris-HCl 0.2mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Proclin 300 (production of SUPELCO company) 0.1% (v/v)
AMPPD 2％(v/v)，
Its pH is 7.2
On the basis of technique scheme, the present invention can also do following improvement:
Described concentrated cleaning solution contains:
NaCl 16％(w/v)
Tween-20 1％(v/v)
Tris-HCl damping fluid 0.2mol/L,
Its pH is 7.0～7.5
The present invention also provides a kind of preparation method who detects the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody, may further comprise the steps:
The preparation of A, feminine gender, positive reference substance:
Negative control product: the negative human serum of HSV-I IgM, 56 ℃ of deactivations in 1 hour, aseptic filtration, 2-8 ℃ of preservation.
Positive reference substance: fixed high titre HSV-I IgM positive human serum, after 56 ℃ of deactivations in 1 hour, with containing 10% (v/v) NBCS, 0.1% (v/v) antiseptic, the 0.02mol/LTris-HCl damping fluid dilution of pH7.2, dilution ratio is 1:150,2-8 ℃ of preservation.
The preparation of the herpes simplex virus I-type antibody of B, enzyme labeling:
The preparation of the herpes simplex virus I-type antibody of enzyme labeling: herpes simplex virus I-type antibody is fully dialysed to the 0.01mol/L phosphate buffer with glutaraldehyde coupling method and alkaline phosphatase coupling, adds equal-volume glycerine, preserves standby below-20 ℃; With enzyme labelled antibody with the enzyme labeling thing with containing the diluted of 0.1mol/LTris-HCl damping fluid, 0.5% (w/v) bovine serum albumin(BSA) (BSA), 0.1% (v/v) proclin 300, pH 7.2～7.6, adopting the square formation method to select the dilutability of enzyme labelled antibody is 1:4000-1:6000.
C, in and the preparation of antigen:
Dilutability with antigen during employing square formation method is selected is 1:2000-1:4000.Dilution adopts the dilution prescription in the above-mentioned B step.
The preparation of the solid phase carrier of D, anti-people μ chain monoclonal antibody bag quilt: will resist people μ chain monoclonal antibody to join in the coating buffer that contains 0.015mol/L natrium carbonicum calcinatum, 0.035mol/L sodium bicarbonate, pH 9.2～9.8 to 2 μ g/mL, add in each hole of microwell plate, every hole 110 μ L placed 24 hours for 4 ℃; Get rid of coating buffer, every hole adds the confining liquid 300 μ L that contain 0.01mol/L phosphate buffer, 1% (w/v) bovine serum albumin(BSA) (BSA), 0.1% (v/v) proclin 300, pH7.0～7.5 respectively, room temperature was placed 3 hours, get rid of confining liquid, pat dry, room temperature removal moisture drying 24 hours, envelope is put 2～8 ℃ of preservations;
The preparation of E, chemical luminous substrate liquid:
Chemical luminous substrate liquid contains:
Tris-HCl 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD, CSPD or CDP-Star 1.5～4.0% (v/v),
Its pH is 7.0～7.5;
The preparation of F, concentrated cleaning solution
Concentrated cleaning solution contains:
NaCl 16％(w/v)
Tween-20 1％(v/v)
Tris-HCl damping fluid 0.2mol/L,
Its pH is 7.0～7.5
This method is convenient to produce, be easy to monitoring industrial processes, the stability between having guaranteed batch.
On the basis of technique scheme, preferred version of the present invention is:
Described chemical luminous substrate liquid contains:
Tris-HCl 0.2mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD 2％(v/v)，
Its pH is 7.2.
Description of drawings
Embodiment
Following institute gives an actual example and only is used to explain the present invention, is not to be used to limit scope of the present invention.
Embodiment 1
The preparation of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody:
Negative control product: 56 ℃ of deactivations in 1 hour of the negative human serum of HSV-I IgM, aseptic filtration, 2-8 ℃ of preservation.
Positive reference substance: fixed high titre HSV-IgM positive human serum, after 56 ℃ of deactivations in 1 hour, with containing 10% (v/v) NBCS, 0.1% (v/v) proclin300, positive reference substance is made in the 0.02mol/LTris-HCl damping fluid dilution of pH 7.2, dilution ratio is 1:150,2-8 ℃ of preservation.
The preparation of the herpes simplex virus I-type antibody of enzyme labeling: herpes simplex virus I-type antibody is fully dialysed to the 0.01mol/L phosphate buffer with glutaraldehyde coupling method and alkaline phosphatase coupling, adds equal-volume glycerine, preserves standby below-20 ℃.Take by weighing Tris 12.120g, bovine serum albumin(BSA) (BSA) 5g, proclin 300 1mL are settled to 1000mL with distilled water, regulate pH to 7.2～7.6, prepared and diluted liquid with HCl.With enzyme labelled antibody enzyme labeling thing diluted, adopting the square formation method to select the dilutability of enzyme labelled antibody is 1:4000.
In and the preparation of antigen: adopt the square formation method select in and the dilutability of antigen be 1:2000.Dilution during dilution use previous step is rapid.
The preparation of the solid phase carrier of anti-people μ chain monoclonal antibody bag quilt: take by weighing natrium carbonicum calcinatum 0.795g, sodium bicarbonate 1.47g, be dissolved in distilled water and constant volume to 500mL, behind the mixing, adjust pH to 9.6, add anti-people μ chain monoclonal antibody to 2 μ g/mL mixing, add then in each hole of microwell plate, every hole 110 μ L place 24h for 4 ℃.Get rid of coating buffer, get NaH
2PO
42H
2O 0.2g, Na
2HPO
412H
2O2.9g, BSA 10g, proclin 300 1mL are settled to 1000mL with distilled water, and the pH value is 7.0～7.5, is mixed with confining liquid.Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.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.
The preparation of chemical luminous substrate liquid.Adopt following prescription: 0.2mol/L Tris-HCl damping fluid, 16% (w/v) NaCl, 0.4% (w/v) KCl, 0.1% (v/v) proclin300 (production of SUPELCO company), 2% (v/v) AMPPD.Get Tris 24g, NaCl 160g, KCl 4g, proclin 300 1mL, AMPPD 20mL after the dissolving of adding distilled water, is settled to 1000mL, regulates pH to 7.2 with HCl.
The preparation of concentrated cleaning solution.Adopt following prescription: 0.2mol/L Tris-HCl damping fluid, 1% (v/v) Tween-20,16% (w/v) NaCl.Get Tris 24g, NaCl 160g, Tween-20 10mL is settled to 1000mL with deionized water, regulates pH to 7.0～7.5 with HCl.Use 20 times of distilled water dilutings during use.
Embodiment 2
The preparation of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody:
The preparation of chemical luminous substrate liquid.Adopt following prescription: 0.25mol/L Tris-HCl damping fluid, 16% (w/v) NaCl, 0.4% (w/v) KCl, 0.1% (v/v) proclin 300,1.6% (v/v) CSPD.Get Tris 30g, NaCl 160g, KCl 4g, proclin300 1mL, CSPD 16mL after the dissolving of adding distilled water, is settled to 1000mL, regulates pH to 7.4 with HCl.
The preparation of the herpes simplex virus I-type antibody of enzyme labeling: with enzyme labelled antibody enzyme labeling thing diluted, adopting the square formation method to select the dilutability of enzyme labelled antibody is 1:6000.
In and the preparation of antigen: adopt the square formation method select in and the dilutability of antigen be 1:4000.
All the other are identical with embodiment 1.
Embodiment 3
The preparation of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody:
The preparation of chemical luminous substrate liquid.Adopt following prescription: 0.15mol/L Tris-HCl damping fluid, 16% (w/v) NaCl, 0.4% (w/v) KCl, 0.1% (v/v) NaN
3, 3.2% (v/v) CPD-Star.Get Tris18g, NaCl 160g, KCl 4g, NaN
31mL, CPD-Star 32mL after the dissolving of adding distilled water, is settled to 1000mL, regulates pH to 7.0～7.5 with HCl.
All the other are identical with embodiment 1.
Embodiment 4
The preparation of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody:
The preparation of chemical luminous substrate liquid.Adopt following prescription: 0.15mol/L Tris-HCl damping fluid, 16% (w/v) NaCl, 0.4% (w/v) KCl, 0.1% (v/v) NaN
3, 4.0% (v/v) AMPPD.Get Tris 18g, NaCl 160g, KCl 4g, NaN
31mL, AMPPD 40mL after the dissolving of adding distilled water, is settled to 1000mL, regulates pH to 7.0～7.5 with HCl.
All the other are identical with embodiment 1.
Embodiment 5
The preparation of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody:
The preparation of chemical luminous substrate liquid.Adopt following prescription: 0.15mol/L Tris-HCl damping fluid, 16% (w/v) NaCl, 0.4% (w/v) KCl, 0.1% (v/v) proclin 300 (production of SUPELCO company), 1.5% (v/v) AMPPD.Get Tris 18g, NaCl 160g, KCl 4g, proclin3001mL, AMPPD 15mL after the dissolving of adding distilled water, is settled to 1000mL, regulates pH to 7.0～7.5 with HCl.
All the other are identical with embodiment 1.
Embodiment 6
The using method of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody.
In 4 ℃ of refrigerators, take out the chemical luminescence reagent kit of embodiment 1 made herpes simplex virus I-type IgM antibody, equilibrium at room temperature 15 minutes; Sample to be tested is diluted with physiological saline 1:100; If blank 1 hole, negative control 2 holes, positive control 2 holes, each hole adds negative control product, positive reference substance or the sample 50 μ L after the dilution except that blank well, with the micro-oscillator mixing that fully vibrates, 37 ℃ of incubations 30 minutes; Get rid of dereaction liquid, 20 times of cleansing solutions after the dilution are filled it up with in every hole, wash plate 5 times, buckle on clean thieving paper at last and do; Except that blank well each hole add enzyme labelled antibody and in and each 50 μ L of antigen, with the micro-oscillator mixing that fully vibrates, 37 ℃ of incubations 30 minutes; Get rid of dereaction liquid, 20 times of cleansing solutions after the dilution are filled it up with in every hole, wash plate 5 times, buckle on clean thieving paper at last and do; Each hole adds chemical luminous substrate liquid 50 μ L, and fully vibrating with micro-oscillator mixes, room temperature (20～25 ℃) lucifuge reaction 30 minutes; On the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, 1 second/hole of Measuring Time; The result judges: the RLU of sample 〉=2.1 * negative control product RLU value mean value, be judged as the positive, otherwise negative.
Embodiment 7
Adopt the chemical luminescence reagent kit of prepared herpes simplex virus I-type IgM antibody among the embodiment 1 to press embodiment 6 described methods to a positive sample, carry out replication 3 times, the luminous intensity RLU coefficient of variation is calculated in each 10 holes, and data are as follows:
Table 1 luminous intensity RLU coefficient of variation experimental result
| Numbering | RLU | CV％ |
| 1 | 420537±38269 | 9.10 |
| 2 | 448971±37220 | 8.29 |
| 3 | 409865±23116 | 5.64 |
The result as seen, CV% illustrates that all less than 10% kit accuracy of the present invention is good, meets the requirements.
Embodiment 8
Adopt the chemical luminescence reagent kit of prepared herpes simplex virus I-type IgM antibody among the embodiment 2 to detect HSV-I IgM positive sample 47 examples simultaneously by embodiment 6 described methods and enzyme-linked immunologic detecting kit (Ke Run of Shenzhen reaches bio-engineering corporation and produces), negative sample 33 examples, relatively the two testing result.The result is as follows:
Two kinds of kit testing results of table 2 table of comparisons
As shown in Table 2, the result of kit of the present invention and herpes simplex virus I-type IgM enzyme-linked immunologic detecting kit is in full accord.
Embodiment 9
The positive sample of the high titre of portion is pressed the dilution proportion of 1:50,1:100,1:200,1:400,1:800, adopt the chemical luminescence reagent kit of prepared herpes simplex virus I-type IgM antibody among the embodiment 4 to detect the relatively sensitivity of the two simultaneously by embodiment 6 described methods and enzyme-linked immunologic detecting kit (Ke Run of Shenzhen reaches bio-engineering corporation and produces).The result is as follows:
Two kinds of kit testing results of table 3 table of comparisons
| Dilution ratio | Kit of the present invention | Elisa kit |
| 1:50 | + | + |
| 1:100 | + | + |
| 1:200 | + | + |
| 1:400 | + | + |
| 1:800 | - | - |
As seen from the above table, when high titre positive sample 1:50-1:400 dilutes, but two kinds of equal test positive of kit, when high titre positive sample 1:800 diluted, two kinds of kit testing results were all negative.Illustrate that kit of the present invention is suitable with enzyme-linked immunologic detecting kit sensitivity.
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 (10)
1. chemical luminescence reagent kit that detects herpes simplex virus I-type IgM antibody, comprise feminine gender, positive reference substance, the solid phase carrier of anti-people μ chain monoclonal antibody bag quilt, in and antigen, the herpes simplex virus I-type antibody of enzyme labeling, chemical luminous substrate liquid and concentrated cleaning solution, it is characterized in that, described enzyme is an alkaline phosphatase, and described chemical luminous substrate liquid contains 1,2-two oxidative ethane analog derivatives.
2. chemical luminescence reagent kit according to claim 1, it is characterized in that, described chemical luminous substrate liquid contains (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), 3-(2 '-(5 ＂-chlorine) spiral diamantane)-4-methoxyl-4-(3 ＂-phosphorus acyloxy) benzene-1,2-dioxetane (CSPD) or 3-(2 '-(5 ＂-chlorine) spiral diamantane)-4-methoxyl-4-(3 ＂-phosphorus acyloxy-5 ＂-chlorine) benzene-1,2-dioxetane (CDP-Star).
3. chemical luminescence reagent kit according to claim 1 is characterized in that, described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD, CSPD or CDP-Star 1.5～4.0% (v/v),
Its pH is 7.0～7.5.
4. chemical luminescence reagent kit according to claim 1 is characterized in that, described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD 1.5～4％(v/v)，
Its pH is 7.0～7.5.
5. chemical luminescence reagent kit according to claim 1 is characterized in that, described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.20mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
proclin300 0.1％(v/v)
AMPPD 2％(v/v)，
Its pH is 7.2.
6. according to any described chemical luminescence reagent kit of claim 1-5, it is characterized in that concentrated cleaning solution wherein contains:
NaCl 16％(w/v)
Tween-20 1％(v/v)
Tris-HCl damping fluid 0.2mol/L,
Its pH is 7.0～7.5.
7. detect the preparation method of the chemical luminescence reagent kit of herpes simplex virus I-type IgM antibody, it is characterized in that, may further comprise the steps:
The preparation of A, feminine gender, positive reference substance: negative control product, 56 ℃ of deactivations in 1 hour of the negative human serum of HSV-I IgM, aseptic filtration, freezing preservation; Positive reference substance, fixed high titre HSV-IIgM positive human serum is after 56 ℃ of deactivations in 1 hour, with containing 10% (v/v) NBCS, 0.1% (v/v) antiseptic, the 0.02mol/L Tris-HCl damping fluid dilution of pH7.2, dilution ratio is 1:150, freezing preservation;
The preparation of the herpes simplex virus I-type antibody of B, enzyme labeling: herpes simplex virus I-type antibody glutaraldehyde coupling method and alkaline phosphatase coupling, phosphate buffer to 0.01mol/L pH 7.4 is fully dialysed, add equal-volume glycerine, preserve standby below-20 ℃; With enzyme labelled antibody with the enzyme labeling thing with containing the diluted of 0.1mol/L Tris-HCl damping fluid, 0.5% (w/v) bovine serum albumin(BSA) (BSA), 0.1% (v/v) proclin 300, pH7.2～7.6, adopting the square formation method to select the dilutability of enzyme labelled antibody is 1:4000-1:6000;
C, in and the preparation of antigen: adopt the square formation method select in and the dilutability of antigen be 1:2000-1:4000, dilution adopts the dilution in the above-mentioned B step;
The preparation of the solid phase carrier of D, anti-people μ chain monoclonal antibody bag quilt: will resist people μ chain monoclonal antibody to join in the coating buffer that contains 0.015mol/L natrium carbonicum calcinatum, 0.035mol/L sodium bicarbonate, pH9.2～9.8 to 2 μ g/mL, add in each hole of microwell plate, every hole 110 μ L placed 24 hours for 4 ℃; Get rid of coating buffer, every hole adds the confining liquid 300 μ L that contain 0.01mol/L phosphate buffer, 1% (w/v) bovine serum albumin(BSA) (BSA), 0.1% (v/v) proclin 300, pH7.0～7.5 respectively, room temperature was placed 3 hours, get rid of confining liquid, pat dry, room temperature removal moisture drying 24 hours, envelope is put 2～8 ℃ of preservations;
The preparation of E, chemical luminous substrate liquid:
Chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.15～0.25mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD, CSPD or CDP-Star 1.5～4.0% (v/v),
Its pH is 7.0～7.5;
The preparation of F, concentrated cleaning solution: concentrated cleaning solution contains
NaCl 16％(w/v)
Tween-20 1％(v/v)
Tris-HCl damping fluid 0.2mol/L,
Its pH is 7.0～7.5.
8. the preparation method of the chemical luminescence reagent kit of detection herpes simplex virus I-type IgM antibody according to claim 7 is characterized in that, described chemical luminous substrate liquid contains:
Tris-HCl damping fluid 0.2mol/L
NaCl 16％(w/v)
KCl 0.4％(w/v)
Antiseptic 0.1% (v/v or w/v)
AMPPD 2％(v/v)，
Its pH is 7.0～7.5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353777A (en) * | 2011-07-05 | 2012-02-15 | 深圳市卫武光明生物制品有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN109283334A (en) * | 2018-09-30 | 2019-01-29 | 潍坊市康华生物技术有限公司 | A kind of recombinant antigen composition and its kit detecting herpes simplex virus type II IgG antibody |
| CN112213483A (en) * | 2020-09-18 | 2021-01-12 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and preparation method thereof |
-
2008
- 2008-04-30 CN CN 200810105741 patent/CN101377507A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353777A (en) * | 2011-07-05 | 2012-02-15 | 深圳市卫武光明生物制品有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN102353777B (en) * | 2011-07-05 | 2014-07-02 | 深圳市卫光生物制品股份有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN109283334A (en) * | 2018-09-30 | 2019-01-29 | 潍坊市康华生物技术有限公司 | A kind of recombinant antigen composition and its kit detecting herpes simplex virus type II IgG antibody |
| CN109283334B (en) * | 2018-09-30 | 2021-09-07 | 山东硕景生物科技有限公司 | Recombinant antigen composition for detecting herpes simplex virus II type IgG antibody and kit thereof |
| CN112213483A (en) * | 2020-09-18 | 2021-01-12 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and preparation method thereof |
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