CN101377504A - Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody - Google Patents
Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody Download PDFInfo
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Abstract
The invention discloses a toxoplasma gondii IgM antibody detection kit combined with the FITC-anti-FITC indirect coating technology and the chemiluminescent immunoassay technology, and a preparation method thereof. The kit of the invention is composed of a negative control, a positive control, solid-phase vectors for anti-FITC antibodies, anti-human Mu-chain monoclonal antibodies of FITC markers, toxoplasma gondii antigens which are marked by horse radish peroxidase, chemiluminescent substrates and concentrated washing solutions. The kit of the invention can be used as the aided detection index for prenatal prepotency diagnosis, and has vital significances for improving the birth population quality and doing the family planning and the prepotency well.
Description
Technical field
The present invention relates to Medical Immunology and vitro diagnostic techniques field, particularly, the invention provides the anti-FITC antibody indirect of a kind of FITC-coating technique and measure Toxoplasma Gondi IgM antibody mensuration kit and preparation method thereof in conjunction with chemiluminescence immunoassay technology.
Background technology
Arc worm (Toxoplasma gondii) is the enteron aisle coccidia of cats, find in the spleen monocyte of just combing toe mouse (Ctenodactylus gondii) by French scholar Nicolle and Manceaux, it is arc that polypide is, so the called after toxoplasma gondii.Toxoplasmosis (toxoplasmosis) claim toxoplasmosis again, is by the caused a kind of infecting both domestic animals and human parasitic disease of arc worm.According to the literature, arc worm can infect multiple mammal, and the animal infection rate can reach 10-50%.The whole world has 1,000,000,000 people to infect arc worm approximately, and the average infection rate of population of China is 5.16%.Arc worm is that pregnant woman's intrauterine infection causes one of five big pathogen of embryo's deformity, and the congenital toxoplasmosis patient can cause intelligence development obstacle, nervous system lesion such as brain paralysis, epilepsy, meningitis, symptom such as retarded.China has 90,000 neonates damaged by toxoplasmosis every year approximately, and expensive every year more than one hundred million units have brought white elephant for society and family, have also seriously influenced the health of newborns of China simultaneously.
The antibody that occurs the earliest behind the arch insect infection is IgM antibody, then prompt for acute arch insect infection if in serum, detect IgM antibody, the IgG antibody of Chu Xianing peaked after infection in 2～5 months gradually subsequently, and the sustainable long period exists, the IgG antibody positive illustrates toxoplasma gondii infection once, generally mostly is chronic infection.At present, arc worm teratogenesis has been caused generally understanding, to unusual product history, unusual contact history or there is clinical symptoms person to monitor, formulate the effectively preventing measure to strengthen detection effectively to women of child-bearing age's arch insect infection, for improving the overall quality of newborns, do family planning and bearing and rearing better children well and be extremely important.
At present, the laboratory diagnostic method of arc worm mainly comprises aetology test and serological test.The aetology test has the meaning of making a definite diagnosis, but has shortcomings such as operation is difficult, sensitivity is low.Thereby serological test is the important auxiliary diagnosis means of present widespread use.Serological diagnostic method commonly used mainly comprises stain test (Sabin-Feldman DT), indirect hemagglutination test (IHA), radiommunoassay (RIA) and enzyme-linked immuno assay (EIA) etc.Wherein there is the shortcoming that needs worm operation alive in stain test, though indirect hemagglutination test is with DT coincidence rate height but poor repeatability, sensitized erythrocyte instability, though radiommunoassay is highly sensitive, high special but have that the term of validity is short, alpha-contamination shortcoming, though the enzyme-linked immuno assay term of validity is long, high specificity exists substrate most of poisonous or be shortcoming such as carcinogenic.
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.Have that high sensitivity, sensing range are wide, easy and simple to handle fast, advantages such as label good stability, 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.
Fluorescein isothiocynate (FITC) is a kind of very stable fluorescence element, very easily is combined on the protein molecular, and not only mark is simple, and label is very stable.FITC is attached to as haptens has very strong immunogenicity on the protein carrier, FITC reaction on anti-FITC monoclonal antibody and the protein that is marked at, and not only specificity height, and affinity also is higher than the combination of antigen-antibody.The present invention is with anti-FITC antibody sandwich solid phase carrier, wrap indirectly by the albumen of FITC mark by the anti-FITC antibody forming system of FITC-, not only be convenient to amplify and produce, be easy to monitoring industrial processes, and shortcoming such as the loss of activity of having avoided the direct coated solid phase to exist is big, process optimization is loaded down with trivial details, the stability between having guaranteed simultaneously batch.
The invention solves at present clinically aetology test operation inconvenience, sensitivity is low, put be excused from an examination that the agent term of validity is short, radioactive waste contamination hazard and enzyme are excused from an examination that agent is subjected to that multiple factor is disturbed, the most of deficiency such as poisonous of substrate.Toxoplasma Gondi IgM antibody chemical luminescent detecting kit of the present invention, through clinical trial certificate, easy and simple to handle, highly sensitive, specificity is good, and is high with aetology assay degree of conformity, and a kind of serodiagnosis technology of efficient, sensitive, stable, special arc worm acute infection is provided.
Summary of the invention
The purpose of this invention is to provide the anti-FITC antibody indirect of a kind of FITC-coating technique in conjunction with chemical luminescent detecting kit of chemiluminscence immunoassay Toxoplasma Gondi IgM antibody and preparation method thereof.
Kit of the present invention is made up of the solid phase carrier of Toxoplasma Gondi IgM antibody negative control, positive control, anti-FITC antibody sandwich, the anti-people μ chain monoclonal antibody of FITC mark, toxoplasma antigen, chemical luminous substrate and the concentrated cleaning solution of horseradish peroxidase-labeled.
Another object of the present invention is for providing the preparation method of mentioned reagent box.
(1) preparation of negative control
Collection detects the negative normal human serum of Toxoplasma Gondi IgM antibody, HIV (1+2) antibody, HCV antibody, TP antibody, HBsAg more than 6 parts through the ELISA method, filtration sterilization, packing, low-temperature storage.
(2) preparation of positive control
Collection detects the Toxoplasma Gondi IgM antibody positive through the ELISA method, and HIV (1+2) antibody, HCV antibody, TP antibody, HBsAg negative patients serum is more than 6 parts, filtration sterilization, packing, low-temperature storage.
(3) preparation of anti-FITC antibody sandwich solid phase carrier
With 0.020M pH value is the anti-FITC antibody sandwich liquid that 7.4 phosphate buffer is mixed with desired concn, and coating buffer is carried on the solid phase carrier, after the 0.02M pH7.4 phosphate buffer sealing that contains 20% NBCS, 5%L-fucose, the dehumidifier drying, aluminium foil bag is airtight, 2-8 ℃ of preservation.Wherein, described bag is microwell plate or magnetic-particle by the solid phase carrier of anti-FITC antibody.
(4) preparation of FITC labelled antibody
After the dialysis of antibody usefulness pH9.60.05M carbonate buffer solution, with the DMSO solution lucifuge reaction of FITC, through 50mM NH
4After the Cl cessation reaction, the dialysis of 0.02M pH7.4 phosphate buffer, low temperature, keep in Dark Place.
(5) preparation of enzyme mark toxoplasma antigen
With easy sodium periodate oxidizing process with horseradish peroxidase and toxoplasma antigen coupling.
(6) preparation of chemical luminous substrate
Based on the described chemical luminous substrate A of 1000mL liquid, comprise 1.7716g luminol, 0.051g 4-xenol, 0.012g 4-iodobenzene boric acid, 11.4g boric acid, 4.9g borax, its pH value is 8.0～10.0;
Based on the described chemical luminous substrate B of 1000mL liquid, comprise 0.329g urea peroxide, 1ml Tween20,51.58g Na
2HPO
412H
2O, 8.74g NaH
2PO42H
2O, its pH value is 7.0～7.6.
(7) preparation of concentrated cleaning solution
The 0.2M pH7.4Tris-HCl damping fluid that preparation contains 16% NaCl, 0.4% KCl, 0.1% polysorbas20,0.1% Proclin300 uses by 20 times of dilutions during use as concentrated cleaning solution.
Kit of the present invention adopts the anti-FITC antibody indirect of FITC-coating technique to combine with chemiluminescence, avoided direct physical absorption to have shortcomings such as the immunocompetence loss is big, sterically hindered, accuracy difference, and simplified process optimization, be convenient to monitoring industrial processes.
Kit of the present invention utilizes the anti-FITC antibody forming system of FITC-to wrap indirectly by anti-people μ chain monoclonal antibody, anti-people μ chain monoclonal antibody compound with the anti-FITC antibody of Toxoplasma Gondi IgM antibody reaction formation solid phase-FITC mark in the sample to be tested, form the toxoplasma antigen sandwich complex of the anti-people μ chain monoclonal antibody-horseradish peroxidase-labeled of the anti-FITC antibody of solid phase-FITC mark again with the toxoplasma antigen reaction of horseradish peroxidase-labeled, catalytic substrate chemiluminescence then, thereby detect the content of Toxoplasma Gondi IgM antibody in the serum by the chromogenic substrate in the light signal replacement EIA enzyme immunoassay that detects the luminous substrate generation, improved detection sensitivity greatly, the most of shortcoming such as poisonous or carcinogenic of avoided enzyme to be excused from an examination substrate that agent exists, and it is fast to have reaction velocity, cost is low, advantages such as easy storage.Kit of the present invention can detect the Toxoplasma Gondi IgM antibody content in the human body behind the arc worm of acute infection very single-mindedly.
Embodiment
Embodiment 1 preparation Toxoplasma Gondi IgM antibody chemical luminescence immune assay determination reagent kit of the present invention
One, the preparation of negative control, positive control
1, the preparation of negative control
The normal human serum that collection detects Toxoplasma Gondi IgM antibody, HIV (1+2) antibody, HCV antibody, TP antibody, HBsAg feminine gender through the ELISA method is more than 6 parts, filtration sterilization, packing, low-temperature storage.
2, the preparation of positive control
Collection detects the Toxoplasma Gondi IgM antibody positive through the ELISA method, and HIV (1+2) antibody, HCV antibody, TP antibody, HBsAg negative patients serum is more than 6 parts, filtration sterilization, packing, low-temperature storage.
Two, anti-FITC bag is by the preparation of microwell plate
1, bag quilt
The preparation of coating buffer: with 5.8g Na
2HPO
412H
2O and 0.59g NaH
2PO
42H
2O is fixed molten to 1000ml with distilled water.
To add an amount of anti-FITC in the coating buffer, wrap by microwell plate with every hole 100 μ l behind the mixing, and place 2-8 ℃ of bag by 18-24h.
2, sealing
The preparation of confining liquid: with 5.8g Na
2HPO
412H
2O, 0.59g NaH
2PO
42H
2O, 200ml NBCS, 50g L-fucose, 1ml Proclin300 add in the clean container, dissolving mixing, fixed molten to 1000ml with distilled water at last.
Every hole adds confining liquid 120 μ l respectively, places 2-8 ℃ of sealing 18-24h.Get rid of confining liquid, on thieving paper, pat dry.28 ℃ of dehumidifying, after dry 24 hours, use the aluminium foil bag envelope, 2～8 ℃ of preservations of labeling postposition.
Three, the preparation of the anti-people μ of FITC mark chain antibody
1, FITC marking process
(1), preparation pH9.60.05M carbonate buffer solution: with 2.94g NaHCO
3, 1.59g Na
2CO
3Add in the clean container, add distilled water and be settled to 1000ml.
(2), 2mg/ml antibody is dialysed three times under 2-8 ℃ of condition with the carbonate buffer solution of pH9.6.
(3), FITC is dissolved among the DMSO, concentration is 1mg/ml.
(4), in protein: the ratio of FITC=1mg:150 μ g slowly is incorporated in FITC in the antibody-solutions, and the limit edged rocks gently itself and antibody are mixed, and lucifuge 2-8 ℃ was reacted 8 hours.
(5), the NH that adds 5M
4Cl is to final concentration 50mM, 2-8 ℃ of cessation reaction 2 hours.
(6), label is dialysed in PBS more than four times, limpid to dislysate.
(7), the evaluation of label
Protein concentration (mg/ml)=[A
280-0.31 * A
495]/1.4=1.5mg/ml
F/P ratio: 3.1 * A
495/ [A
280-0.31 * A
495]=4, this value should be between 2.5-6.5.
(8), the FITC labelled protein should place the phosphate buffer of pH7.4, adds 0.1%NaN
3, 1%BSA, 2-8 ℃ keeps in Dark Place.
2, the preparation of FITC labelled antibody dilution
With 0.2g NaH
2PO
42H
2O, 2.9g Na
2HPO
412H
2O, 10g PEG20000 and 1ml Proclin300 add in the clean container, and be fixed molten to 1000ml with distilled water.
3, the preparation of FITC labelled antibody working fluid
The FITC labelled antibody of preparation is diluted to variable concentrations respectively with dilution, uses the chessboard titrimetry to select best dilutability and be 1:5000.
Four, the preparation of horseradish peroxidase-labeled toxoplasma antigen
1, the marking process of toxoplasma antigen
Adopt the EDC method with horseradish peroxidase and toxoplasma antigen coupling, add 50% glycerine, place-20 ℃ frozen.
2, the preparation of toxoplasma antigen label dilution
With 0.2g NaH
2PO
42H
2O, 2.9g Na
2HPO
412H
2O, 10g PEG20000 and 1ml Proclin300 add in the clean container, and be fixed molten to 1000ml with distilled water.
3, the preparation of toxoplasma antigen label working fluid
The toxoplasma antigen label of preparation is diluted to variable concentrations respectively with dilution, uses the chessboard titrimetry to select best dilutability and be 1:4000.
Five, chemical luminous substrate A liquid
Luminol 1.7716g
4-xenol 0.051g
4-iodobenzene boric acid 0.012g
Boric acid 11.4g
Borax 4.9g
Distilled water is fixed molten to 1000mL
pH 8.0～10.0
Six, chemical luminous substrate B liquid
Urea peroxide 0.329g
Tween20 1ml
Na
2HPO
4·12H
2O 51.58g
NaH
2PO
4·2H
2O 8.74g
Distilled water is fixed molten to 1000mL
pH 7.0～7.6
A liquid mixes with B liquid equal proportion during use.
Seven, concentrated cleaning solution
160g NaCl, 4g KCl, 24.2g trishydroxymethylaminomethane (Tris), 1ml polysorbas20,1ml Proclin 300 are dissolved in the 900ml distilled water, adjust pH to 7.4 with HCl, fixed molten with distilled water to 1000ml.
Dilute 20 times with distilled water during use.
The present inventor has carried out the optimization of system to the reaction pattern and the reaction conditions of kit, and the influence of test findings is tested with regard to the different reaction time, determines the optimal reaction time.By the influence test to measured value shows to the mensuration of luminous duration of chemical luminous substrate liquid and different fluorescent lifetime: be measured as the best between 5-30 minute after adding chemical luminous substrate liquid, its result is also comparatively accurate.
Embodiment 2 preparations Toxoplasma Gondi IgM antibody chemical luminescence immune assay determination reagent kit of the present invention
As solid phase carrier, will resist FITC to be coupled to magnetic-particle surface outside with glutaraldehyde method divided by magnetic-particle, all the other all prepare the Toxoplasma Gondi IgM antibody chemical luminescence immune assay determination reagent kit with the method identical with embodiment 1.
The using method of embodiment 3 kits of the present invention
The concrete operations of the Toxoplasma Gondi IgM antibody chemical luminescence immune assay determination reagent kit of above embodiment 1 preparation are as follows:
1) in 2-8 ℃ of refrigerator, takes out Toxoplasma Gondi IgM and measure kit (chemoluminescence method), equilibrium at room temperature 30 minutes;
2) sample to be tested is diluted by 1:1000 in advance with physiological saline;
3) TOX IgM negative control 3 holes, positive control 2 holes are established in each experiment.Add negative control, positive control and prediluted sample to be tested 50 μ l respectively, every then hole adds 50 μ l FITC labels respectively, vibration mixing, 37 ℃ of incubations 30 minutes;
4) wash 5 times with the cleansing solution after the dilution, cleansing solution is filled it up with in every hole, soaks 10 seconds at every turn, pats dry on clean thieving paper at last;
5) the enzyme-added label 100 μ l in each hole, vibration mixing, 37 ℃ of incubations 30 minutes;
6) wash 5 times with the cleansing solution after the dilution, cleansing solution is filled it up with in every hole, soaks 10 seconds at every turn, pats dry on clean thieving paper at last;
5) each hole adds each 50 μ l of chemical luminous substrate liquid A, B, room temperature (20～25 ℃) lucifuge reaction 5 minutes.
6) must measure in the 5th～30 minute after adding chemical luminous substrate liquid, on the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, 1 second/hole of Measuring Time.
7) Cutoff value=2.1NC, if the RLU value 〉=Cutoff value of sample to be tested then sample be judged to be positive sample; RLU value＜Cutoff value then sample is judged to be negative sample.
Embodiment 4 kit clinical testings of the present invention are estimated
Kit with embodiment 1 preparation carries out parallel detection to 81 routine normal person's samples with arc worm acute infection patient sample 45 examples of making a definite diagnosis, and trace routine and result's judgement are carried out in strict accordance with the using method that embodiment 3 provides.
Testing result is as shown in table 1:
Table 1 kit clinical testing of the present invention evaluation result
The described data of table 1 show, kit specificity of the present invention, susceptibility are all good, and kit positive predictive value of the present invention, negative predictive value are 100%.Kit of the present invention can be used as the supplementary means that antenatal prenatal and postnatal care is diagnosed, and for improving the overall quality of newborns, does family planning and bearing and rearing better children well and is extremely important.
Claims (10)
1, a kind of Toxoplasma Gondi IgM antibody chemical luminescence immune assay determination reagent kit, it is characterized in that described kit is made up of the solid phase carrier of negative control, positive control, anti-FITC antibody, the anti-people μ chain monoclonal antibody of FITC mark, toxoplasma antigen, chemical luminous substrate and the concentrated cleaning solution of horseradish peroxidase-labeled.
2, kit as claimed in claim 1 is characterized in that, described solid phase carrier is microwell plate or magnetic-particle.
3, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is luminol or different luminol.
4, kit as claimed in claim 1, it is characterized in that, described concentrated cleaning solution is: contain the 0.2M pH7.4 Tris-HCl damping fluid of 16%NaCl, 0.4% KCl, 0.1% polysorbas20,0.1% Proclin300, use with 20 times of dilutions of distilled water during use.
5, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
Preparation negative control, positive control, anti-FITC antibody sandwich solid phase carrier, mark monoclonal antibody, antigen, preparation luminous substrate and concentrated cleaning solution, the above-mentioned component of packing also is assembled into finished product.
6, method as claimed in claim 5 is characterized in that, described negative control is to detect the negative normal human serum potpourri of Toxoplasma Gondi IgM antibody through ELISA.
7, method as claimed in claim 5 is characterized in that, described positive control is the potpourri that detects Toxoplasma Gondi IgM antibody positive patient serum through ELISA.
8, method as claimed in claim 5, it is characterized in that, describedly adopted following method preparation with anti-FITC bag by solid phase carrier: with 0.020M pH value is the anti-FITC coating buffer that 7.4 phosphate buffer is mixed with desired concn, and coating buffer is carried on the solid phase carrier, after the 0.02MpH7.4 phosphate buffer sealing that contains 20% NBCS, 5%L-fucose, the dehumidifier drying, aluminium foil bag is airtight, 2-8 ℃ of preservation.
9, method as claimed in claim 5, it is characterized in that, the compound method of described chemical luminous substrate is: based on the described chemical luminous substrate A of 1000mL liquid, comprise 1.7716g luminol, 0.051g 4-xenol, 0.012g 4-iodobenzene boric acid, 11.4g boric acid, 4.9g borax, its pH value is 8.0～10.0.
Based on the described chemical luminous substrate B of 1000mL liquid, comprise 0.329g urea peroxide, 1ml Tween20,51.58g Na
2HPO
412H
2O, 8.74g NaH
2PO
42H
2O, its pH value is 7.0～7.6.
10, method as claimed in claim 5, it is characterized in that, the prescription of described concentrated cleaning solution is: contain the 0.2M pH7.4 Tris-HCl damping fluid of 16% NaCl, 0.4% KCl, 0.1% polysorbas20,0.1% Proclin300, use with 20 times of dilutions of distilled water during use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810104140 CN101377504A (en) | 2008-04-16 | 2008-04-16 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810104140 CN101377504A (en) | 2008-04-16 | 2008-04-16 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101377504A true CN101377504A (en) | 2009-03-04 |
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ID=40421148
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|---|---|---|---|
| CN 200810104140 Pending CN101377504A (en) | 2008-04-16 | 2008-04-16 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
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| CN102368071A (en) * | 2011-06-30 | 2012-03-07 | 同昕生物技术（北京）有限公司 | Chemiluminescent immunoassay kit for detecting mycoplasma pneumoniae IgM antibody |
| CN102368068B (en) * | 2011-06-30 | 2014-01-22 | 同昕生物技术（北京）有限公司 | Kit for detecting chlamydia pneumoniae IgM antibody |
| CN102368071B (en) * | 2011-06-30 | 2014-01-22 | 同昕生物技术（北京）有限公司 | Chemiluminescent immunoassay kit for detecting mycoplasma pneumoniae IgM antibody |
| CN102818892A (en) * | 2012-08-16 | 2012-12-12 | 北京恩济和生物科技有限公司 | Detection kit for prostate specific antigen and preparation method thereof |
| CN102914645A (en) * | 2012-11-12 | 2013-02-06 | 武汉伊艾博科技有限公司 | Method for fixing antibody in solid phase carrier in immune adsorption reaction process |
| CN106226539A (en) * | 2016-07-08 | 2016-12-14 | 广州东林生物科技有限公司 | Electrochemiluminescence cleaning buffer solution |
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| CN111999494A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
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