CN101975859A - Magnetic microparticle separation chemiluminescent immunoassay detection method for hepatitis B virus surface antigen - Google Patents
Magnetic microparticle separation chemiluminescent immunoassay detection method for hepatitis B virus surface antigen Download PDFInfo
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Abstract
The invention provides an in-vitro detection method for a hepatitis B virus surface antigen. The method adopts magnetic microparticle separation chemiluminescent immunoassay technology which is the result of the combination of enzyme labeling technology, magnetic microparticle separation technology and chemiluminescent immunoassay technology and has the characteristics of high sensitivity, high specificity, high repeatability and the like. The method can be used for assaying the hepatitis B surface antigen content of human serum and blood plasma. Clinically, the index is generally used for determining infection with hepatitis B viruses and mainly used for monitoring the illness condition, treatment effect and prognosis of the hepatitis B .
Description
Technical field
The invention belongs to the immune detection analysis technical field, relate to a hepatitis b virus infected diagnosis index hepatitis b virus s antigen of clinical judgement, a kind of magnetic separating chemiluminescence immunologic detection method of hepatitis b virus s antigen is provided, has been applicable to the detection by quantitative of human serum, blood plasma hepatitis b virus s antigen.
Background technology
Hepatitis B is called serum hepatitis, virus B hepatitis (abbreviation hepatitis B) again, is by hepatitis type B virus (Hepatitis B Virus, the infectious disease that HBV) causes.By blood and body fluid communication, has chronic carrier state.Hepatitis B clinical manifestation variation easily develops into chronic hepatitis and cirrhosis, and some patients can be changed primary carcinoma of liver into.
Hepatitis b virus s antigen (HBs-Ag) is one of serologic marker thing of hepatitis B, this albumen is by the S gene code, multiple antigenic determinants such as " a ", " b ", " y ", " r ", " w " are arranged, wherein " a " is common antigenic determinant, and " d ", " y " and " r ", " w " are main hypotype determinant.HBs-Ag mainly contains adr, adw, and ayr reaches 4 kinds of hypotypes such as ayw.The serologic marker that occurs the earliest when HBs-Ag is acute infection.Usually 1-6 is all before symptom occurs just can detect for this antigen, and the normal body of immunity can produce surface antibody, and this antibody virus that can neutralize is a kind of protection antibody.It is the active sign of virus that HBs-Ag detects the positive, means that usually the patient has infectiousness; Negative situation or be that patient never infects, or be rehabilitation or invisible infection after infecting.Symptom occurs back 4 months, and the immunity normal individual can be removed HBs-Ag, and body begins rehabilitation, HBs-Ag shows as feminine gender at convalescence, and still, some people is the individuality (as the AIDS patient) of infant or immunity difference especially, can form chronic hbv-infection, HBs-Ag can lasting masculin.
Usually to detect HBs-Ag as standard, during acute infection, HBs-Ag can detect about 4 weeks after the infection, also usually with certain clinical symptoms in the popularity monitoring of hepatitis B; Chronic infection is diagnostic criteria more than 6 months with the HBs-Ag positive usually then.As a kind of serologic marker thing, HBs-Ag not only is used in the diagnosis, also is used for the blood that examination is contributed.
Detection to hepatitis b virus s antigen mainly is to adopt immunological method clinically.Clinical immunoassay technology is a technology of utilizing antigen-antibody reaction principle detection of biological substance in vivo, the introducing of this technology makes clinical chemistry test that revolutionary variation take place: test item constantly increases, the sensitivity of method is higher, specificity is stronger, and automaticity is higher.After the eighties in last century, the immunology detection technology develops rapidly along with the maturation of monoclonal antibody, artificial synthetic polypeptide, gene engineering expression antigen and various labelling techniques, radiommunoassay (RIA) and enzyme immunoassay (EIA) (EIA) replace traditional immunoprecipitation and immunity cohesion gradually, susceptibility, the specificity of detection are all improved greatly, and the application of chemiluminescence subsequently is further improved the susceptibility of immunology detection and the range of linearity.The detection by quantitative that be applied as disease, observation of curative effect and the CORD PARALYSIS of these technology in clinical examination provides more direct and objective data.
Summary of the invention
The object of the present invention is to provide a kind of magnetic microparticle separating chemiluminescence immunologic detection method with highly sensitive, that specificity good, applicability is wide detection hepatitis b virus s antigen.
The kit of the hepatitis b virus s antigen magnetic microparticle separating chemiluminescence immunity that the present invention generates is formed and is comprised: 1. calibration solution (contain certain density hepatitis b virus s antigen, be used for determining the CutOff value); 2. hepatitis b virus s antigen yin, yang reference substance (positive control contains certain density hepatitis b virus s antigen, the result's who is used to check quality control); 3. the anti-reagent of hepatitis b virus s antigen No. 1, No. 2 (coupling have the antibody of hepatitis b virus s antigen of biology enzyme and the antibody of the hepatitis b virus s antigen that coupling has FITC); 4. magnetic separation agent (in conjunction with the magnetic particle suspension of anti-FITC); 5. cleaning fluid concentrate (being used to prepare cleaning fluid); 6. substrate solution (luminous substrate of biological enzyme).
The detection step of described hepatitis b virus s antigen magnetic microparticle separating chemiluminescence immunologic detection method is as follows:
(1) application of sample and immune response: in test tube, add sample, calibration solution and reference substance that 5-100 μ L handles well, add the anti-reagent of 10-500 μ L hepatitis b virus s antigen then respectively No. 1 and No. 2, mixing, 37 ℃ incubation 5-60 minute;
(2) immune complex combines with magnetic particle: add 20-100 μ L magnetic separation agent in test tube, mixing, 37 ℃ incubation 0-30 minute (also not incubation);
(3) magnetic separates and washing: make magnetic particle sedimentation in magnetic field, remove supernatant; Add 100-600 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then makes magnetic particle sedimentation in magnetic field, removes supernatant; Can add the washing lotion cleaning more repeatedly (also can save);
(4) add substrate solution and read concentration of specimens: every pipe adds the luminous substrate of 50-300 μ L biological enzyme, and the enzymatic substrate is luminous, according to the S/CO value that the luminous value and the Cut Off value of sample are read hepatitis B surface antigen in the sample, and judgement sample yin and yang attribute.
The principle of work of described hepatitis b virus s antigen magnetic microparticle separating chemiluminescence immunologic detection method is: at first, sample to be checked combines the sandwich complex of formation " sandwich " structure with enzymic-labelled antibody, FITC labelled antibody specificity, sandwich complex combines (combining with anti-FITC on the magnetic particle by the FITC of coupling on the antibody) again with the magnetic particle specificity; Through washing, bond is left by externally-applied magnetic field, and unconjugated antigen, antibody and other materials are removed; Add substrate, detect its luminous value with chemiluminescent analyzer.In the finite concentration scope, the hepatitis b virus s antigen concentration in the sample to be tested is directly proportional with luminous value, and the S/CO value of reading hepatitis B surface antigen in the sample according to the luminous value and the Cut Off value of sample is judged the sample yin and yang attribute at last.
Technical solution of the present invention is as follows:
(1) preparation of magnetic separation agent: FITC antibody is connected the magnetic particle surface.Described magnetic particle diameter has superparamagnetism between 0.01-5.0 μ m, amino (NH2-) or carboxyl (COOH-) reactive group are contained in the surface.Described FITC antibody and magnetic particle can pass through chemical cross-linking agent, as glutaraldehyde, 1-ethyl-3-[3-dimethylaminopropyl] carbo diimide hydrochloride (EDC) etc. is with the form of covalent bond, also can carry out coupling by the form of physisorption (electrostatic interaction, ionic link or hydrophobic effect etc.).
(2) preparation of enzyme labelled antibody in the anti-reagent of hepatitis b virus s antigen: with the antibody coupling of biology enzyme and hepatitis b virus s antigen, described biology enzyme can be horseradish peroxidase (HRP), also can be alkaline phosphatase (ALP).Coupling method can be several different methods such as sodium periodate oxidizing process, glutaraldehyde method or Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 2-IminothiolaneHCl (2IT) be crosslinked.
(3) substrate solution: described substrate solution can be the luminous substrate luminol (Luminol) of HRP catalysis, also can be the luminous substrate AMPPD of ALP catalysis, CSPD and CSPD-Star etc.
(4) calibration solution and reference substance: choose human serum, it is mixed back the dilution by a certain percentage with dilution form through deactivation.
Description of drawings
Fig. 1 is the consistance of kit of the present invention with the external import HBsAg of renowned company chemical illuminating reagent clinical sample test.
Embodiment
Embodiment 1: anti-FITC antigen and the coupling of 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] ethane sulfonic acid), pH 4.5-5 solution 10ml washing 3 times.Magnetic particle is resuspended with this solution 1ml, adds the anti-FITC antibody of 2mg, mixes.Add 100 μ l 10mg/ml EDC solution, mixed the back room temperature reaction 2 hours.Contain 0.01M phosphate buffer (PBS) the pH7.4 washing magnetic bead 3 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: the coupling of the antibody of alkaline phosphatase (ALP) and hepatitis b virus s antigen
Get the antibody of 5mg hepatitis b virus s 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 15 minutes, places 5 minutes under the room temperature.Use PG10 pillar desalination, collect eluted protein.
Get 7.5mg ALP solution, Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) the solution 50 μ l that add 6.69mg/mL, room temperature was placed 30 minutes, added glycocoll and stopped priming reaction, and room temperature was placed 5 minutes.Use PG10 pillar desalination, collect eluted protein.
The bladder chalone C antibody of collecting is mixed with the ALP of collection, and 4 ℃ were reacted 20 hours, used Supperdex200 gel chromatography column separating purification then, collected second peak and the 3rd peak is a conjugate, and it is stored in 4 ℃.
Embodiment 3: detect hepatitis b virus s antigen in the serum
With this kit 680 routine clinical serum serum are detected, determined the computing method of CutOff value.
Material and instrument
(1) hepatitis b virus s antigen kit;
(2) chemical illumination immunity analysis instrument MAGLIA60: the Beijing Bei Ai Kang Biotechnology Co., Ltd produces;
(3) magnetic separator: the Beijing Bei Ai Kang Biotechnology Co., Ltd produces;
(4) water bath (being used for 37 ℃ of temperature bathes).
The detection step is as follows:
(1) application of sample and immune response: in test tube, add sample, calibration solution and reference substance that 100 μ L handle well, add the anti-reagent of 30 μ L hepatitis b virus s antigens then respectively No. 1 and No. 2, mixing, 37 ℃ of incubations 30 minutes;
(2) immune complex combines with magnetic particle: add 60 μ L magnetic separation agents in test tube, mixing, 37 ℃ of incubations 6 minutes;
(3) magnetic separates and washing: make magnetic particle sedimentation in magnetic field, remove supernatant; Add 300 μ L cleaning fluids, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then makes magnetic particle sedimentation in magnetic field, removes supernatant.Repeat twice adding, 300 μ L cleaning fluids again, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then makes magnetic particle sedimentation in magnetic field, removes supernatant;
(4) add substrate solution and read concentration: every pipe adds 200 μ L substrate solutions, and the enzymatic substrate is luminous, according to the S/CO value that the luminous value and the Cut Off value of sample are read hepatitis B surface antigen in the sample, judges the sample yin and yang attribute.
Testing result:
When the CutOff value is the luminous mean value of calibration solution divided by 6 the time, the yin and yang attribute of sample is distinguished better, and coincidence rate is greater than 97%.
Embodiment 4: the performance evaluation of this kit
According to the characteristics of external diagnosis reagent, detect sensitivity, positive and negative coincidence rate, precision and the stability of this kit as usual.Material and instrument, detection step are with embodiment 3.
Testing result:
(1) sensitivity: the sensitivity reference material to three kinds of hypotypes of national reference material detects result such as following table:
| Ay2.0? | ?+? |
| Ay1.0? | ?+? |
| Ay0.5? | ?-? |
| Adw2.0? | ?+? |
| Adw1.0? | ?+? |
| Adw0.5? | ?+? |
| Adr1.0? | ?+? |
| Adr0.5? | ?+? |
| Adr0.2? | ?-? |
(2) positive and negative coincidence rate: 3 positive reference materials and 20 negative reference materials to national reference material detect, and the result all meets;
(3) precision: the internal control material of three kinds of concentration is carried out precision detect, the coefficient of variation of the S/CO value that records is calculated in every kind of material replicate determination 10 times, and the result shows that the coefficient of variation is between 1.2%～7.5%.
(4) stability: kit was placed 37 ℃ after 8 days, and test internal control product are basic, normal, high, and the result is still in the Quality Control scope; 2～8 ℃ of placements of kit after 12 months, can be shown that kit has good stability by national reference material standard, meet the clinical practice needs.
Claims (8)
1. a hepatitis b virus s antigen magnetic microparticle separating chemiluminescence immune analysis detection method is characterized in that combining enzyme labeling technology, magnetic particle isolation technics and chemiluminescence detection technology, and has generated detection kit.
2. enzyme labeling technology according to claim 1 is characterized in that the method for antibody of biology enzyme marking hepatitis B table antigen is as follows:
Described biology enzyme can be horseradish peroxidase (HRP), also can be alkaline phosphatase (ALP).Coupling method can be 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. magnetic separation technique according to claim 1 is characterized in that the preparation method of magnetic separation agent is as follows:
FITC antibody is connected the magnetic particle surface.Described magnetic particle diameter has superparamagnetism between 0.01-5.0 μ m, amino (NH2-) or carboxyl (COOH-) reactive group are contained in the surface.Described FITC antibody and magnetic particle can pass through chemical cross-linking agent, as glutaraldehyde, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) etc. is with the form of covalent bond, also can carry out coupling by the form of physisorption (electrostatic interaction, ionic link or hydrophobic effect etc.).
4. chemiluminescence luminescent detection techniques according to claim 1, it is characterized in that the used substrate solution of chemiluminescence can be the luminous substrate luminol (Luminol) of HRP catalysis, also can be the luminous substrate AMPPD of ALP catalysis, CSPD and CSPD-Star etc.
5. kit according to claim 1 is characterized in that reference substance and the calibration solution in the kit is the human serum of choosing through deactivation, its mixing back is diluted by a certain percentage with dilution form.
6. kit according to claim 1 is characterized in that the anti-reagent of hepatitis B surface antigen in the kit comprises coupling the antibody of hepatitis B surface antigen of biology enzyme and the antibody of the hepatitis B surface antigen that coupling has FITC are arranged.
7. the antibody that is used for coupling FITC according to claim 6 can be monoclonal antibody, also can be polyclonal antibody.
8. detection method according to claim 1, it is as follows to it is characterized in that detecting step:
(1) application of sample and immune response: in test tube, add sample, calibration solution and reference substance that 5-100 μ L handles well, add the anti-reagent of 10-500 μ L hepatitis B surface antigen then, mixing, 37 ℃ incubation 5-60 minute;
(2) immune complex combines with magnetic particle: add 20-100 μ L in test tube, 0.1-10mg/mL magnetic separation agent, mixing, 37 ℃ incubation 0-30 minute (also not incubation);
(3) magnetic separates and washing: make magnetic particle sedimentation in magnetic field, remove supernatant; Add 100-600 μ L cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then makes magnetic particle sedimentation in magnetic field, removes supernatant; Can add washing lotion once more and clean twice (also can save);
(4) add substrate solution and read concentration of specimens: every pipe adds the luminous substrate of 50-300 μ L biological enzyme, and the enzymatic substrate is luminous, according to the S/CO value that the luminous value and the Cut Off value of sample are read hepatitis B surface antigen in the sample, and judgement sample yin and yang attribute.
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