CN101533011A - A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method - Google Patents
A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method Download PDFInfo
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Abstract
The invention provides a Japanese blood fluke ovum antibody magnetic particle EILSA detecting method, which belongs to verminosis immunodiagnosis technology. The invention adopts the immunodetection theory of antigen sandwich assay method, which uses the surface of magnetic microsphere which is connected with the Japanese blood fluke ovum antigen and has the diameter of 0.1 to 5.0 micrometer as the solid phase reagent; the Japanese blood fluke dissoluble ovum antigen labeled by biological enzyme is used as enzyme reagent. The magnetic separation reagent and the enzyme reagent are respectively combined with the Japanese blood fluke ovum antibody in the sample to form the solid phase- antigen- antibody- enzyme antigen sandwiched immunocomposite. The invention is characterized in that first, magnetic particles are used for replacing traditional enzyme plate ELISA as the solid phase carriers so that the immune reaction is carried on in the condition of liquid phase and the reaction if more abundant and quick; compared with the traditional ELISA method, the method is characterized by the quick detecting speed, the high specificity and the good repetitiveness; second, due to the adoption of bi-antigen sandwich method theory, one kit can be used for the detection of both people and animal Japanese blood flukes.
Description
Technical field
The invention belongs to the immune detection analysis technical field, the immune detection analytical technology that relates to a kind of infectious diseases common to human beings and animals, a kind of magnetic particle separation enzyme-linked immunoassay of fast detecting Japanese blood fluke ovum antibody is provided especially, has been applicable to the qualitative detection of people and animals' Japanese blood fluke ovum antibody.
Background technology
At present, area, China middle and lower reach of Yangtze River snail fever is still seriously popular, existing 810,000 blood fluke patients, 60,000 snail fever oxen.6,000 ten thousand people are exposed among the risk of infection, and acute infection constantly takes place.Although China utilizes measure such as the synchronous chemotherapy of people and animals to obtain very big success on the control snail fever, snail fever remains an important public health problem in area, China middle and lower reach of Yangtze River at present
[1]
Traditionally, with the aetology method detection snail fever of schistosoma japonice ovum in patient's ight soil, but this method is time-consuming, effort, the loss height, and can not make early diagnosis to this disease.Since the eighties in 20th century, the method for immune detection is used to the diagnosis of snail fever gradually, and has obtained good effect.
In the immune detection of snail fever, parasitic egg antibody is the most frequently used detection material, if detect antibody of Schistosoma japonicum in people and animals' blood, then the deducibility people and animals suffer from or once suffered from snail fever.At present, immunological detection method more advanced and commonly used is indirect enzyme-linked immunosorbent assay (indirect ELISA).This method is with Schistosoma japonicum soluble egg antigen (Schistosomajaponicum Soluble Egg Antigen, Sj-SEA) ELISA Plate of bag quilt is the solid phase carrier of separating, with the anti-people of biology enzyme mark or raise two anti-be enzyme marking reagent, the Japanese blood fluke ovum antibody in qualitative detection people and animals' serum or the blood plasma.This method is simple to operate, highly sensitive, accuracy is good, therefore obtains increasingly extensive application.But also there are some shortcomings in this method; 1. this method only can detect the antibody of single subclass.For example, the kit that detects IgG antibody just can not detect antibody such as IgM, IgA.Because may only there be IgM antibody in the snail fever early infection stage, so there is certain phenomenon of failing to pinpoint a disease in diagnosis; 2. since the anti-people of the biology enzyme mark that adopts or he raises two anti-can only with the antibody specific bond of people or poultry, so every kind of kit can only be at people or poultry, can not be general.3. the test serum sample often needs to dilute in advance, has increased the complicacy of operation.
Magnetic particle separation enzyme-linked immunoassay technology be a kind of be the solid phase carrier of separating with the magnetic particle, immune magnetic particle isolation technics is combined with elisa technique and a kind of novel immunologic detection method set up.In the traditional E LISA method; the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface; and in magnetic particle separation enzyme-linked immune detection; magnetic particle is suspended in the liquid phase; the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase; thereby reaction is fast, thoroughly, compare with traditional E LISA have highly sensitive, the advantage that detection time is short.
Dual-antigen sandwich method is a kind of method that is used to detect antibody, and its detection principle is; Antibody to be measured at first combines with the antigen that is connected solid phase surface, adds the antigen of biology enzyme mark then.Because antibody has two antigen binding sites, can combine with solid phase antigen and biology enzyme labelled antigen respectively, colour developing or luminescence-producing reaction by biological enzyme can reach the purpose that detects antibody.At present, this method successfully has been used for the detection such as hepatitis B surface antibody etc.
Summary of the invention
The object of the present invention is to provide a kind of have highly sensitive, specificity good, applicability is wide, easy and simple to handle and save the magnetic particle separation enzyme-linked immunoassay of the detection Japanese blood fluke ovum antibody of detection time.
The reagent of described Japanese blood fluke ovum antibody magnetic particle separation enzyme-linked immunoassay is formed and is comprised: 1. magnetic separation agent (being combined with the magnetic particle suspension of Sj-SEA); 2. enzyme marking reagent (coupling has the Sj-SEA solution of biology enzyme); 3. cleaning fluid; 4. positive quality control product (serum that contains a certain amount of anti-Sj-SEA antibody); 5. negative quality-control product (serum that does not contain anti-Sj-SEA antibody); 6. substrate solution (colour developing of biological enzyme or luminous substrate).
The detecting operation method of described Japanese blood fluke ovum antibody magnetic particle separation enzyme-linked immunoassay is as follows:
(1) application of sample and immune response: in test tube, add 5-100 μ L serum sample, positive quality control product or negative quality-control product, add 20-100 μ L then, the magnetic separation agent of 0.1-10mg/mL, behind the mixing, 37 ℃ incubation 5-30 minute;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add 100-500 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then makes magnetic particle sedimentation in magnetic field, removes supernatant.So repeat 2-4 time;
(3) add enzyme marking reagent: in each test tube, add the SEA working solution 30-120 μ L of biology enzyme mark, behind the mixing, 37 ℃ incubation 5-30 minute;
(4) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add 100-500 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then makes magnetic particle sedimentation in magnetic field, removes supernatant.So repeat 2-4 time;
(5) add substrate solution: every pipe adds the colour developing or the luminous substrate of 50-300 μ L biological enzyme, behind the mixing, 37 ℃ incubation 5-30 minute;
(6) color development stopping: every pipe adds 300 μ L stop buffers, and is placed on the magnetic separator and separated 10 minutes;
(7) value of reading: on spectrophotometer or luminous intensity detector, read absorbance or luminous intensity values;
The principle of work of described Japanese blood fluke ovum antibody magnetic particle separation enzyme-linked immunoassay is: at first, and test sample, positive quality control product and magnetic separation agent mixing incubation.An antigen binding site of the anti-Sj-SEA antibody in the sample combines with the Sj-SEA on magnetic particle surface.Through washing, remove unconjugated antibody and other impurity.Add enzyme marking reagent and incubation, the Sj-SEA of biology enzyme mark combines formation solid phase antigen-antibody-enzyme-labelled antigen sandwich complex with another antigen binding site of anti-SEA antibody on being combined in magnetic bead.Unconjugated enzyme mark Sj-SEA and other impurity are removed in washing, add enzymatic colour developing or luminous substrate at last.In the finite concentration scope, the anti-Sj-SEA antibody content in the sample to be tested is directly proportional with colour developing or luminous degree, can make the judgement that has or not Sj-SEA antibody in the sample to be tested in view of the above.
Technical solution of the present invention is as follows:
(1) preparation of magnetic separation agent: Sj-SEA is connected the magnetic particle surface.Described magnetic particle diameter has superparamagnetism and magnetic field responsiveness between 0.1-5.0 μ m, amino (NH2-) reactive group or carboxyl (COOH-) reactive group are contained in the surface.Described Sj-SEA can pass through chemical cross-linking agent with being connected of magnetic particle, as glutaraldehyde, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) etc. is with the form of covalent bond, and also can be by the form of physisorption (electrostatic interaction, ionic link or hydrophobic effect etc.).
(2) preparation of enzyme marking reagent: biology enzyme and Sj-SEA is covalently bound.Described biology enzyme can be horseradish peroxidase (HRP), also can be alkaline phosphatase (ALP).Covalently bound method can be passed through several different methods such as sodium periodate oxidizing process, glutaraldehyde method or Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 2-IminothiolaneHCl (2IT) be crosslinked.
(3) substrate solution: described substrate solution can be the chromogenic substrate methyl biphenyl amine (Tetrabenzidine of HRP catalysis, TMB) or luminous substrate luminol (Luminol), also can be the chromogenic substrate list phosphoric acid phenolphthalein (PMP) or the luminous substrate AMPPD of ALP catalysis, CSPD and CSPD-Star etc.
(4) positive quality control serum: described positive quality control serum is the popular epidemic-stricken area acute infection of Japanese schistosomiasis patients serum, and this patient has epidemic disease water contact history, can find schistosoma japonice ovum in the ight soil, has typical clinical symptoms and sign.
(5) negative quality controlled serum: described negative quality controlled serum in the life of non-schistosomiasis endemic epidemic-stricken area, does not have the healthy population serum of epidemic disease water contact history for always.
The present invention has the following advantages:
(1) serum need not dilute, and can be directly used in detection;
(2) can detect IgG antibody, also can detect each subclass antibody such as IgM, IgA, can be used for the early diagnosis of snail fever;
(3) can detect the interior anti-Sj-SEA antibody of human serum, also can detect the anti-Sj-SEA antibody in the various animal blood serums.Can be used for people and various animal, as the detection of snail fever such as farm cattle, horse, dog;
(4) highly sensitive, the good reproducibility, easy and simple to handle of this method is saved detection time.
Description of drawings
Fig. 1 detects Japanese blood fluke ovum antibody immune response synoptic diagram.
Embodiment
The coupling of embodiment 1:Sj-SEA and surface amino groups (COOH-) magnetic particle, preparation magnetic separation agent
Get magnetic particle that the 100mg surface contains carboxyl (COOH-) reactive group with 0.1M MES (2-[N-morpholino] ethanesulfonic acid), pH 4.5-5 solution 10ml washing 3 times.Magnetic particle is resuspended with this solution 1ml, adds 2mg Sj-SEA, mixes.Add 100 μ l 10mg/ml EDC solution, mix back room temperature reaction 2h.Contain the 0.01M PBS pH7.4 solution washing magnetic bead three times of 1% bovine serum albumin(BSA) (BSA) with 10ml after, be mixed with the magnetic separation agent working fluid of 2.5mg/ml with this solution.
Embodiment 2:Sj-SEA coupling alkaline phosphatase (ALP), the preparation enzyme marking reagent
Get 5mg SJ-SEA antigen, be concentrated into 5mg/ml, add activator 2-IminothiolaneHCl (2IT) the solution 10 μ l of 13.76mg/mL, room temperature is placed and is added glycocoll termination priming reaction after 20 minutes, places 5 minutes under the room temperature.Use Sephadex G25 pillar desalination, collect the albumen eluting peak.
Get 5mg ALP solution, add Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) the solution 50 μ l of 6.69mg/mL, room temperature was placed 30 minutes, added glycocoll and stopped priming reaction, and room temperature was placed 5 minutes.Use Sephadex G25 pillar desalination, collect the albumen eluting peak.
The Sj-SEA of activation is mixed with the ALP of activation, and room temperature was placed 10 hours, used Supperdex200 gel chromatography column separating purification then, collected second peak and the 3rd peak, removed the free Sj-SEA and the ALP that do not connect, and connector is stored in 4 ℃.
Liquid making method is preserved in the enzyme marking reagent dilution: Tris 12.11g, and sodium chloride 8.77g, Sodium azide 1g adds purified water to 600mL, proofreaies and correct pH value to 7.5.Add TWEEN-20 2mL, bovine serum albumin(BSA) 10g adds purified water and is settled to 1000mL.Filter with 0.22 μ m filtrator, in 4 ℃ of preservations.
Above-mentioned Sj-SEA-ALP connector is diluted to 5 μ g/mL with above-mentioned dilution preservation liquid is mixed with the enzyme marking reagent working fluid.
Embodiment 3: Japanese blood fluke ovum antibody magnetic particle separation enzyme immune detection
Material and instrument
(1) magnetic separation agent: see the preparation of above-mentioned magnetic separation agent;
(2) enzyme marking reagent: see the preparation of above-mentioned enzyme marking reagent;
(3) cleansing solution: 500mL (Beijing Bei Ai Kang Biotechnology Co., Ltd's production);
(4) the molten thing of substrate (PMP solution): 300mL (Beijing Bei Ai Kang Biotechnology Co., Ltd's production);
(5) stop bath: 600mL (Beijing Bei Ai Kang Biotechnology Co., Ltd's production);
(6) Schistosoma japonicum country reference material: ((N1-N15), 1 part of sensitivity reference material (P16), 1 part of precision reference material (P17) are bought from Nat'l Pharmaceutical ﹠ Biological Products Control Institute for P1-P15), 15 parts of negative reference materials for 15 parts of positive reference materials.
(7) serum sample:
20 parts of Japanese schistosomiasis acute patient's serum, 55 parts of chronic patients serum, 45 parts of patients with terminal serum, 378 parts of Pest-or disease-free area crowd serum, 13 parts of liver rot patients serums, 16 parts of paragonimiasis patients serums, 15 parts of trichinosis patients serums, 10 parts of cysticercosis patients serums;
(8) the separation enzyme-linked immunoassays instrument of magnetic I type machine: the Beijing Bei Ai Kang Biotechnology Co., Ltd produces;
(9) magnetic separator; The Beijing Bei Ai Kang Biotechnology Co., Ltd produces;
(10) water bath (being used for 37 ℃ of temperature bathes).
The detection step is as follows:
(1) application of sample and immune response: in flat based tubes, add 20 μ L serum samples, positive quality control product and negative quality-control product, add 100 μ L magnetic separation agents, behind the mixing, 37 ℃ of incubations 10 minutes;
(2) wash: flat based tubes is placed on the magnetic separator separated 2 minutes, remove supernatant, every pipe adds cleaning fluid 300 μ L, fully behind the mixing, again flat based tubes is placed on the magnetic separator and separated abandoning supernatant 2 minutes, bounce separation vessel to remove wall built-up liquid, repeat twice;
(3) add enzyme marking reagent: in each test tube, add enzyme marking reagent 60 μ L, behind the mixing, 37 ℃ of incubations 10 minutes;
(4) wash: flat based tubes is placed on the magnetic separator separated 2 minutes, remove supernatant, every pipe adds cleaning fluid 300 μ L, fully behind the mixing, again flat based tubes is placed on the magnetic separator and separated abandoning supernatant 2 minutes, bounce separation vessel to remove wall built-up liquid, repeat twice;
(5) add substrate solution: every pipe adds 100 μ L substrate solutions, behind the mixing, and 37 ℃ of incubations 15 minutes;
(6) color development stopping: every pipe adds 300 μ L stop buffers, and is placed on the magnetic separator and separated 10 minutes;
(7) read absorbance: exempt from analyzer I (Beijing Bei Ai Kang Biotechnology Co., Ltd's production) with spectrophotometer or magnetic enzyme and read absorbance, wavelength is 492nm/550nm.
Testing result:
(1) positive coincidence rate and negative match-rate:
Detect the positive national reference material of Schistosoma japonicum that Nat'l Pharmaceutical ﹠ Biological Products Control Institute buys (P1-P15), negative national reference material (N1-N15).Positive coincidence rate is 100%, and negative match-rate is 100%.
(2) sensitivity:
Detect the sensitivity reference material P16 of Schistosoma japonicum country, carry out doubling dilution from 1:50 to 1:25600, be decided to be sensitivity with the positive extension rate of previous extension rate of highly diluted pipe, sensitivity is 1:1600;
(3) precision:
Detect the precision reference material P17 of Schistosoma japonicum country, do 10 pipes simultaneously, calculate its coefficient of variation, the coefficient of variation＜10%;
(4) serum sample testing result:
1) laboratory detection result: 20 parts of acute schistosomiasis patients serums all detect, none routine omission; 55 parts of chronic schistosomiasis patients serum testing results are all positive; 45 parts of advanced schistosomiasis patients have four routine omissions; 30 parts of Pest-or disease-free area crowd serum are all negative; 13 parts of clonorchiasis patients serums, 16 parts of paragonimiasis patients serums, 15 parts of trichinosis patients serums, 10 parts of testing results of cysticercosis patients serum are all negative.
2) rig-site utilization result:
Detected Xiong Kou town, Qianjiang City, Hubei Province, Lao Xin town, Long Wan town (the popular district of snail fever severe) 1987 parts of crowd (nature person) serum samples in 2007, wherein 469 routine excrement are examined positive snail fever human serum, it is positive to detect 465 examples, and sensitivity is 99.15% (465/469).Detect 112 routine Pest-or disease-free area health the same period and go into serum sample, 2 examples are positive, and specificity is 98.21% (110/112).This method shows higher sensitivity and specificity preferably in actual applications.
Detected in Caidian District San Dian village, Wuhan City, Hubei Province, Sanyo village, friendly village (the popular district of snail fever moderate) 866 parts of crowd (nature person) screening serums in 2007, wherein 77 routine excrement are examined positive snail fever human serum, detect 76 examples, sensitivity is 98.70% (76/77).
Detect the hot peace of Wuhan City, Hubei Province Dongxihu District in 2007 and cross Red Star group, Bai Quan farm East Lake group (the low popular district of snail fever) crowd's screening serum, detect 267 routine patients serums, the positive snail fever human serum of 10 routine excrement inspections, the result is positive entirely, and sensitivity is 100% (10/10).
Detected 248 parts of examinations of Long Gang town, Yangxin County, Hubei Province bridge shop village's (the popular district of snail fever severe) nature person's serum sample in 2008, wherein excrement is examined positive snail fever human serum totally 84 examples, and the result is positive entirely, and sensitivity is 100% (84/84).
Detected Jingzhou City, Hubei Province Jianglin County province seed multiplication farm (the popular district of snail fever severe) nature person's serum sample 289 examples in 2008 and carry out examination, wherein excrement is examined positive snail fever human serum totally 101 examples, detects 99 examples, and sensitivity is 98.02% (99/101)
List of references
1.Ross?AGP，Li?YS，Sleigh?AC，McManus?DP.Schistosomiasis?control?in?the?People’s?republic?ofChina.Parasitol?Today，1997，13：152-156.
Claims (3)
1, the magnetic particle separation enzyme-linked immunization method of a kind of Japanese blood fluke ovum antibody is characterized in that detecting step and is:
(1) application of sample and immune response: in test tube, add 5-100 μ L serum sample, positive quality control product or negative quality-control product, add 20-100 μ L then, the magnetic separation agent of 0.1-10mg/mL, behind the mixing, 37 ℃ incubation 5-30 minute;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add 100-500 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then makes magnetic particle sedimentation in magnetic field, removes supernatant.So repeat 2-4 time.
(3) add enzyme marking reagent: Schistosoma japonicum soluble egg antigen (the Schistosoma japonicum soluble egg antigen that in each test tube, adds the biology enzyme mark, Sj-SEA) working solution 30-120 μ L, behind the mixing, 37 ℃ incubation 5-30 minute;
(4) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add 100-500 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then makes magnetic particle sedimentation in magnetic field, removes supernatant.So repeat 2-4 time.
(5) add substrate solution: every pipe adds the colour developing or the luminous substrate of 50-300 μ L biological enzyme, behind the mixing, 37 ℃ incubation 5-30 minute.
(6) color development stopping (being only applicable to development process): every pipe adds 100-300 μ L stop buffer, and is placed on the magnetic separator and separated 10 minutes;
(7) value of reading: on spectrophotometer or luminous intensity detector, read absorbance or luminous intensity values.
2, magnetic separation agent according to claim 1 is characterized in that: be the connector of Schistosoma japonicum soluble egg antigen (Sj-SEA) and magnetic particle, and further be suspended in the suspension in the damping fluid.Described magnetic particle diameter has superparamagnetism and magnetic field responsiveness between 0.1-5.0 μ m, amino (NH2-) or carboxyl (COOH-) reactive group are contained in the surface.Described Sj-SEA can pass through chemical cross-linking agent with being connected of magnetic particle, as glutaraldehyde, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) etc. is with the form of covalent bond, and also can be by the form of physisorption (electrostatic interaction, ionic link or hydrophobic effect etc.).Its working concentration is 0.25-8mg/ml.
3, enzyme marking reagent according to claim 1 is characterized in that: be the connector of Sj-SEA and biology enzyme, and be dissolved in the solution in the damping fluid.Described biology enzyme can be horseradish peroxidase (HRP), also can be alkaline phosphatase (ALP).The method of attachment of biology enzyme and Sj-SEA can be passed through several different methods such as sodium periodate oxidizing process, glutaraldehyde method or Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 2-IminothiolaneHCl (2IT) cross-linking method.Its working concentration is 0.5-5 μ g/mL.
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