CN103033612A - IgM antibody detection kit for five TORCH tests and preparation of IgM antibody detection kit - Google Patents
IgM antibody detection kit for five TORCH tests and preparation of IgM antibody detection kit Download PDFInfo
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Abstract
The invention relates to a pathogenic microorganism detection kit, in particular to an IgM antibody detection kit for five TORCH tests and preparation of the IgM antibody detection kit. The kit is used for detecting IgG-class antibodies in a detection sample in five TORCH tests, namely TOX, rubella virus (RV), cytomegalovirus (CMV) and herpes simplex I/II virus (HSV-I/II).
Description
Technical field:
The present invention relates to a kind of detection reagent of pathogenic microorganism, particularly five IgM antibody of a kind of TORCH (band Western blot) detection kit and preparation method thereof.This kit is for detection of five of TORCH in the sample, i.e. the antibody of the IgG class of Infection of Toxoplasma Gondii (TOX), rubella virus (RV), cytomegalovirus (CMV) and herpes simplex 1 type and 2 types viruses (HSV-I/II).
Background technology:
TORCH refers to cause congenital intrauterine infection and perinatal infection, thereby cause the English name abbreviation of the one group of pathogenic microorganism that encloses newborn baby's deformity, T(Toxopasma wherein) be Infection of Toxoplasma Gondii, R (Rubella.Virus) is rubella virus, C (Cytomegalo.Virus) is cytomegalovirus, and H (Herpes.Virus) namely is herpes simplex I/II type.This group infected by microbes of TORCH has common feature, all can cause the mother and baby to infect.The pregnant woman is because primary infection easily occurs in endocrine alteration and immunity degradation, and the interior potential virus of pregnant woman's body of previous infection also is activated easily and recurrent infection occurs.When torch infection occured the pregnant woman, virus can be propagated by placenta or birth canal and infect fetus, causes premature labor, miscarriage, stillborn foetus or monster etc., and the infringement that causes a plurality of systems of neonate, a plurality of organs, causes the symptoms such as dysnoesia in various degree.The infection of TORCH and prenatal and postnatal care have important and close relationship.
Infection of Toxoplasma Gondii (TOX): the fetal anomaly that pregnant early stage arch insect infection causes mainly comprises: hydrocephalus, little deformity of brain, choroidoretinitis and brain calcification.Hematogenous infection can cause the infringement of many organ necrosis of fetus property, such as hepatosplenomegaly, myocarditis and thrombopenia etc.
Rubella virus (RV): mainly pass through respiratory infectious, can make the fetus teratogenesis behind the infection of pregnant women, virus forms congenital infection by the placental infection fetus, be called congenital rubella syndrome (CRS), mainly be congenital cataract, congenital heart disease and nerve deafness, 20 all postoperative infection persons are almost without impact.Rubella-infection occurs in the pregnancy period more early, and the teratogenesis of fetus is also more serious.
Cytomegalovirus (CMV): can cause intrauterine fetal growth retardation, little capitiform, encephalitis, retina arteries and veins film inflammation, jaundice, hepatosplenomegaly, hemolytic anemia etc. after the infection, infant mortality rate is higher, and the cmv infection rate due to the perinatal period breast milk toxin expelling is 63%.
Herpes simplex virus (HSV I/II type): usually hide at neuromere, the physiological change of parent makes the HSV activation during gestation, and pregnant early infection can destroy the plumule face and cause miscarriage, though pregnant middle and advanced stage is sent out monster less, can cause the fetus an d neonate morbidity.
Therefore, for the birth rate that reduces invalid youngster and improve the overall quality of newborns, the clinical position person should further strengthen the communication and education to the pregnant woman, actively carries out the serological screening of torch infection in order to find early bad gestation and in time process; Also answer routine to carry out TORCH to the neonate and detect, understand the neonatal TORCH infection situation, so as early to intervene, early treatment.The examination of torch infection has important practical significance to prenatal and postnatal care.
The IgG antibody-like is the important indicator of TORCH previous infection or infection period among five of the TORCH; IgM is the early infection index, and the detection of specific IgM is the reliable basis of diagnosing fetal intrauterine infection in the placenta; And the joint-detection of IgG antibody and IgM antibody among five of the TORCH is the important method of judging TORCH early infection or previous infection.
China's ELISA Enzyme-multiplied immune technique commonly used or gold mark Fast Detection Technique detect respectively five IgM of TOCRH or IgG antibody-like, but the testing result of the method is vulnerable to the interference of the aspects such as rheumatoid factor, lacks degree of accuracy.
Western blot is that protein transduction is moved on on the film, then utilizes antibody to detect.To known expressing protein, available corresponding antibodies detects as primary antibodie, to the expression product of new gene, and can be by merging the antibody test of part.Its principle is the protein example that separates through PAGE, transfers on the solid phase carrier (for example cellulose nitrate film), and solid phase carrier is with non-covalent bond form adsorbed proteins, and can keep polypeptide type and the biologic activity thereof of electrophoretic separation constant.With the protein on the solid phase carrier or polypeptide as antigen, play immune response with corresponding antibody, react with enzyme or isotope-labeled second antibody, the colour developing of process substrate or radioautograph are with the protein ingredient of the specific destination gene expression of detection electrophoretic separation again.This technology also is widely used in detecting the expression of protein level.
The method of using at present band immunoblot assay five IgM of joint-detection TOCRH or IgG antibody-like has no report, and the present invention is through research, with band five IgM of western blot determination TOCRH or IgG antibody-like, degree of accuracy is high, and is highly sensitive, simple to operate, noiseless, low price.
Summary of the invention:
The invention provides a kind of high sensitivity, high specific, steady quality, operating aspect, low price also can satisfy the preparation method of five IgM antibody-likes of TORCH detection kit.
Five IgM antibody of TORCH of the present invention (band Western blot) detection kit comprises following reagent:
1) be coated with the detection reagent strip of total schizolysis antigen of five IgM antibody-likes of TORCH,
2) sample dilution,
3) be marked with the enzyme working fluid of horseradish peroxidase,
4) developer A,
5) developer B,
6) 20 times of concentrated washing lotions.
Wherein 1) described detection reagent strip, material therefor comprises: carrier board, such as material class carrier boards such as PVC, nitrocellulose filter (NC film) or pvdf membrane, Infection of Toxoplasma Gondii (TOX) schizolysis antigen, rubella virus (RV) schizolysis antigen, cytomegalovirus (CMV) schizolysis antigen, herpes simplex 1 type and the TORCH raw materials such as 2 types virus (HSV-I/II) schizolysis antigen, anti-human IgM are mixed with 5 coated lines that coating buffer is made.
The preparation method of wherein said detection reagent strip comprises:
With the phosphate buffer of 0.01M pH7.2-7.4 as coated dilution, Infection of Toxoplasma Gondii schizolysis antigen, rubella virus schizolysis antigen, cytomegalovirus schizolysis antigen, herpes simplex 1 type and 2 type virolysis antigens, anti-human IgM are diluted to 0.5-5mg/ml, then line is coated successively on nitrocellulose filter or pvdf membrane: with the coated film freeze-drying or dry, then stick on the PVC material class carrier board and make coated film, then be cut into the wide detection reagent strip of 3-5mm, coated line speed 1-2ul/cm.
Its preparation method comprises:
1) coated raw material is selected:
Select suitable concn and IgG class Infection of Toxoplasma Gondii (TOX) schizolysis antigen of tiring and specificity recombinant antigen, rubella virus (RV) schizolysis antigen and specificity recombinant antigen, cytomegalovirus (CMV) schizolysis antigen and specificity recombinant antigen, herpes simplex 1 type and 2 types virus (HSV-I/II) schizolysis antigen and the raw materials such as specificity recombinant antigen, human IgG antibody.
2) coated film preparation:
With the phosphate buffer (PBS solution) of 0.01M pH7.2-7.4 as coated dilution, the coated raw material of 5 TORCH is diluted to suitable working concentration, then line is coated successively on nitrocellulose filter (NC film) or pvdf membrane: the anti-human IgM of Quality Control detection line (C line), detection line Infection of Toxoplasma Gondii (TOX) schizolysis antigen, rubella virus (RV) schizolysis antigen, cytomegalovirus (CMV) schizolysis antigen, herpes simplex 1 type and 2 types virus (HSV-I/II) schizolysis antigen be totally 5 coated lines, with the coated film freeze-drying or dry, then stick on the material class carrier boards such as PVC and make coated film, then be cut into the wide detection reagent strip of 3-5mm, namely be prepared into the detection reagent strip.Auspiciously see accompanying drawing 1:
Described coating buffer working concentration can be 0.5-5mg/ml, coated line speed 1-2ul/cm.
Wherein 2) its prescription of described sample dilution is composed as follows:
Adjust pH is 7.2-7.4
Wherein 3) described enzyme Working solution prescription is composed as follows:
Select specific anti-anti-human IgM(such as anti-human IgM specific fragment μ chain) monoclonal antibody, with sodium periodate method mark horseradish peroxidase (HRP), make protoenzyme, ELISA tires 〉=1:5000.
Preparation enzyme working fluid dilution:
Adjust pH is 7.2-7.4
Preparation enzyme working fluid: protoenzyme is namely got the enzyme working fluid with enzyme working fluid diluted to suitable concentration.
Be the enzyme working fluid of 1:2000 ~ 1:4000(v:v) such as final concentration concentration.
Wherein 4) prescription of described developer A is composed as follows:
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
H2O2(30%) 1ml
Purified water adds to 1000ml
Wherein 5) prescription of described developer B is composed as follows:
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
TMB·2HCL 0.80g
Purified water adds to 1000ml
Wherein 6) prescription of described 20 times of concentrated washing lotions is composed as follows:
Sodium dihydrogen phosphate 11.8g
Sodium hydrogen phosphate 116g
Sodium chloride 170g
Purified water adds to 1000ml
Above reagent is splendid attire proportionally, select to detect 1-3 time consumption, and each reagent can be distinguished splendid attire, together is packaged in the same packing box again, and the low temperature preservation operates according to the method for describing in the instructions during use and gets final product.The consumption of relevant reagent is advisable with people's 1-3 time consumption.
The present invention comprises that also five IgM antibody of TORCH of the present invention (band Western blot) detection kit using method: this using method may further comprise the steps:
1) five IgM antibody of TORCH (band Western blot) detection kit is taken out from 2-8 ℃ of refrigerator, balance is to room temperature.
2) it is for subsequent use 20 times of concentrated washing lotions to be diluted to 1 times of washing lotion.
3) take out five IgM antibody of TORCH (band Western blot) and detect reagent strip, put into reactive tank, add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
4) sample is diluted with sample dilution 1:50 ~ 1:100, add the rear sample of 1.5ml dilution, and numbering, under the room temperature condition (18-25 ℃), hatch 30-60min with waving shaking table, discard the sample dilution.
5) add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
6) add 1.5ml enzyme working fluid, under the room temperature condition (18-25 ℃), hatch 30-60min with waving shaking table, discard the enzyme working fluid.
7) add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
8) add respectively 1ml developer A and developer B, hatch 10-20min with waving shaking table, discard nitrite ion.
9) wash film 3 times, judged result with purified water or distilled water.After film dried, but result's long preservation.
10) result judges:
A) only have Quality Control detection line (C line) colour developing, TORCH detects negative;
B) Quality Control detection line (C line) colour developing, TORCH(TOX, RV, CMV, HSV) colour developing of any one schizolysis antigen, it is positive to show that then this project IgM antibody-like detects.
C) Quality Control detection line (C line) does not develop the color, and it is invalid that this time detects.
The sample that the present invention can detect comprises, people's whole blood, serum, blood plasma, cerebrospinal fluid etc.
Detection method of the present invention finds that through experiment the term of validity is 1 year under the 2-8 ℃ of condition.The joint-detection that is used for whole blood, serum, blood plasma, five IgM antibody-likes of cerebrospinal fluid equal samples TORCH.The method sample demand is little, does not need specific installation, the visual inspection result, easy fast, specificity is good, highly sensitive, and cost is low, is easy to extensive popularization.And cooperate five IgG antibody of patented invention TORCH (band Western blot) detection kit to use simultaneously previous infection and the recent infection that can distinguish five of TORCH, provide strong foundation for TORCH detects.
Description of drawings:
Five IgM antibody of Fig. 1: TORCH (band Western blot) detection kit detects the reagent strip legend:
Embodiment:
Further specify by the following examples the present invention, mainly be to help the reader better to understand the present invention, not as limitation of the present invention.
Embodiment 1,
1, detect the preparation of reagent strip:
Show such as Fig. 1: detect reagent strip in five IgM antibody of TORCH (band Western blot) detection kit by the PVC carrier board, pvdf membrane, Infection of Toxoplasma Gondii (TOX) schizolysis antigen, rubella virus (RV) schizolysis antigen, cytomegalovirus (CMV) schizolysis antigen, herpes simplex 1 type and the TORCH raw materials such as 2 types virus (HSV-I/II) schizolysis antigen, anti-human IgM are mixed with 9 coated lines compositions that coating buffer is made.
With the phosphate buffer (PBS solution) of 0.01M pH7.2-7.4 as coated dilution, the coated raw material of 5 TORCH is diluted to suitable working concentration, then line is coated successively on pvdf membrane: the anti-human IgM of Quality Control detection line (C line), detection line Infection of Toxoplasma Gondii (TOX) IgM class schizolysis antigen, rubella virus (RV) IgM class schizolysis antigen, cytomegalovirus (CMV) IgM class schizolysis antigen, herpes simplex 1 type and 2 types virus (HSV-I/II) IgM class schizolysis antigen be totally 5 coated lines, coated film is dried, then stick on the material class carrier boards such as PVC and make coated film, then be cut into the wide detection reagent strip of 4mm, namely be prepared into the detection reagent strip.Auspiciously see accompanying drawing 1:
Described coating buffer working concentration can be 1.5mg/ml, coated line speed 1-2ul/cm.
2, sample dilution preparation:
Adjust pH is 7.2-7.4
3, enzyme working fluid preparation
Select specific anti-anti-human IgM(such as anti-human IgM specific fragment μ chain) monoclonal antibody, with sodium periodate method mark horseradish peroxidase (HRP), make protoenzyme, ELISA tires 〉=1:5000.
Preparation enzyme working fluid dilution:
Adjust pH is 7.2-7.4
Preparation enzyme working fluid: protoenzyme is namely got the enzyme working fluid with enzyme working fluid diluted to 1:3000 concentration.
4,20 times of concentrated washing lotion preparations:
Sodium dihydrogen phosphate 11.8g
Sodium hydrogen phosphate 116g
Sodium chloride 170g
Purified water adds to 1000ml
5, developer A preparation
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
H2O2(30%) 1ml
Purified water adds to 1000ml
6, developer B preparation
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
TMB·2HCL 0.80g
Purified water adds to 1000ml
7, five IgM antibody of TORCH (band Western blot) detection kit preparation:
TORCH is detected reagent strip, sample dilution, enzyme working fluid, 20 times of concentrated washing lotions, developer A, developer B be assembled into the kit kit that gets product by usage ratio.
Embodiment 2:
1, five IgM antibody of TORCH (band Western blot) detection kit using method:
1) five IgM antibody of TORCH (band Western blot) detection kit is taken out from 2-8 ℃ of refrigerator, balance is to room temperature.
2) it is for subsequent use 20 times of concentrated washing lotion 1:20 doubly to be diluted to 1 times of washing lotion.
3) take out five IgM antibody of TORCH (band Western blot) and detect reagent strip, put into reactive tank, add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
4) sample is diluted with sample dilution 1:50 ~ 1:100, add the rear sample of 1.5ml dilution, and numbering, under the room temperature condition (18-25 ℃), hatch 30min with waving shaking table, discard the sample dilution.
5) add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
6) add 1.5ml enzyme working fluid, under the room temperature condition (18-25 ℃), hatch 60min with waving shaking table, discard the enzyme working fluid.
7) add 1 times of washing lotion of 1.5ml, under the room temperature condition (18-25 ℃), wash film 3 times with waving shaking table, each 5min.
8) add respectively 1ml developer A and developer B, hatch 10min with waving shaking table, discard nitrite ion.
9) wash film 3 times, judged result with purified water or distilled water.After film dried, but result's long preservation.
2, the result judges:
1) only have Quality Control detection line (C line) colour developing, TORCH detects negative;
2) Quality Control detection line (C line) colour developing, TORCH(TOX, RV, CMV, HSV) colour developing of any one IgM schizolysis antigen, it is positive to show that then this project IgM antibody-like detects.
3) Quality Control detection line (C line) does not develop the color, and it is invalid that this time detects.
3, individual event enzyme linked immunological (ELISA) detection kit of five of five IgM antibody of TORCH (band Western blot) detection kit and TORCH detects every clinical sample 50 examples simultaneously, the positive coincidence rate 100% of result, negative match-rate 100%, and haemolysis, piarhemia, jaundice equal samples on the result without impact.
4, use the same sample of five IgM antibody of TORCH (band Western blot) detection kit Parallel testing 10 times, colour developing is homogeneous as a result.
Claims (10)
1. five IgM antibody assay kits of a TORCH comprise following reagent:
1) be coated with the detection reagent strip of total schizolysis antigen of five IgM antibody-likes of TORCH,
2) sample dilution,
3) be marked with the enzyme working fluid of horseradish peroxidase,
4) developer A,
5) developer B,
6) 20 times of concentrated washing lotions.
2. according to claim 1 detection kit, wherein said detection reagent strip, material therefor comprises: carrier board, nitrocellulose filter or pvdf membrane, Infection of Toxoplasma Gondii schizolysis antigen, rubella virus schizolysis antigen, cytomegalovirus schizolysis antigen, herpes simplex 1 type and 2 type virolysis antigens, anti-human IgM.
3. according to claim 1 detection kit, the preparation method of wherein said detection reagent strip comprises:
With the phosphate buffer of 0.01M pH7.2-7.4 as coated dilution, Infection of Toxoplasma Gondii schizolysis antigen, rubella virus schizolysis antigen, cytomegalovirus schizolysis antigen, herpes simplex 1 type and 2 type virolysis antigens, anti-human IgM are diluted to 0.5-5mg/ml, then line is coated successively on nitrocellulose filter or pvdf membrane: with the coated film freeze-drying or dry, then stick on the PVC material class carrier board and make coated film, then be cut into the wide detection reagent strip of 3-5mm, coated line speed 1-2ul/cm.
4. according to claim 1 detection kit, wherein 2) its prescription of described sample dilution is composed as follows:
Adjust pH is 7.2-7.4.
5. according to claim 1 detection kit, wherein 3) described enzyme Working solution prescription is composed as follows:
Select the monoclonal antibody of specific anti-human IgM, with sodium periodate method mark horseradish peroxidase, make protoenzyme, ELISA tires 〉=1:5000,
Preparation enzyme working fluid dilution:
Adjust pH is 7.2-7.4
Protoenzyme is namely got the enzyme working fluid with enzyme working fluid diluted to suitable concentration.
6. according to claim 1 detection kit,
Wherein 4) prescription of described developer A is composed as follows:
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
H2O2(30%) 1ml
Purified water adds to 1000ml
Wherein 5) prescription of described developer B is composed as follows:
Citric acid 5.00g
Na2HPO4·12H2O 17.45g
TMB·2HCL 0.80g
Purified water adds to 1000ml.
7. according to claim 1 detection kit, wherein 6) prescription of described 20 times of concentrated washing lotions is composed as follows:
Sodium dihydrogen phosphate 11.8g
Sodium hydrogen phosphate 116g
Sodium chloride 170g
Purified water adds to 1000ml.
8. according to claim 1 detection kit, each reagent is splendid attire in accordance with the appropriate ratio, selects to detect 1-3 time consumption, and each reagent can be distinguished splendid attire, together is packaged in the same packing box again.
9. the using method of the detection kit of claim 1 may further comprise the steps:
1) five IgM antibody assay kits of TORCH are taken out from 2-8 ℃ of refrigerator, balance is to room temperature,
2) it is for subsequent use 20 times of concentrated washing lotions to be diluted to 1 times of washing lotion,
3) take out five IgM antibody tests of TORCH reagent strip, put into reactive tank, add 1 times of washing lotion of 1.5ml, under the room temperature condition, wash film 3 times with waving shaking table, each 5min,
4) sample is diluted with sample dilution 1:50 ~ 1:100, add the rear sample of 1.5ml dilution, and numbering, under the room temperature condition, hatch 30-60min with waving shaking table, discard the sample dilution,
5) add 1 times of washing lotion of 1.5ml, under the room temperature condition, wash film 3 times with waving shaking table, each 5min,
6) add 1.5ml enzyme working fluid, under the room temperature condition, hatch 30-60min with waving shaking table, discard the enzyme working fluid,
7) add 1 times of washing lotion of 1.5ml, under the room temperature condition, wash film 3 times with waving shaking table, each 5min,
8) add respectively 1ml developer A and developer B, hatch 10-20min with waving shaking table, discard nitrite ion,
9) wash film 3 times, judged result with purified water or distilled water.After film dried, but result's long preservation,
10) result judges:
A) only have the colour developing of Quality Control detection line, TORCH detects negative,
B) Quality Control detection line colour developing, the colour developing of TORCH any one schizolysis antigen, it is positive to show that then this project IgM antibody-like detects,
C) the Quality Control detection line does not develop the color, and it is invalid that this time detects.
10. according to claim 1 detection kit, the sample that can detect comprises, people's whole blood, serum, blood plasma, cerebrospinal fluid.
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| LI JIANG ET AL: "Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections", 《BIOSENSORS AND BIOELECTRONICS》 * |
| MIKROGEN DIAGNOSTIK: "recomLine TORCH Screening IgM test kit", 《INSTRUCTIONS FOR USE》 * |
| 程茂良等: "温岭市孕妇TORCH 五项感染检测结果分析", 《临床和实验医学杂志》 * |
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| CN103454415A (en) * | 2013-09-23 | 2013-12-18 | 青岛中仁药业有限公司 | Preparing method of enzyme linked immunoblotting family-planning four-item combined kit |
| CN106053365A (en) * | 2016-05-26 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for measuring myeloperoxidase and preparation method of kit |
| CN106501528A (en) * | 2016-10-27 | 2017-03-15 | 杭州量康科技有限公司 | Based on the method that Dried blood spots detect ToRCH10 item antibody |
| CN109270279A (en) * | 2018-10-18 | 2019-01-25 | 郑州安图生物工程股份有限公司 | TORCH-IgG antibody mixing quality-control product |
| WO2021134304A1 (en) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | Kit, method and immunoassay analyzer for screening torch infection |
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