CN103472233A - Detection kit for hepatitis C virus and applications thereof - Google Patents
Detection kit for hepatitis C virus and applications thereof Download PDFInfo
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- CN103472233A CN103472233A CN2013102525635A CN201310252563A CN103472233A CN 103472233 A CN103472233 A CN 103472233A CN 2013102525635 A CN2013102525635 A CN 2013102525635A CN 201310252563 A CN201310252563 A CN 201310252563A CN 103472233 A CN103472233 A CN 103472233A
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Abstract
The invention discloses a detection kit for hepatitis C virus (HCV) and applications thereof. The detection kit comprises HCV mosaic antigens, HCV mosaic antigen monoclonal antibodies, a decontaminating agent buffer solution, HRP labeled mouse anti-human IgG, HRP labeled anti-HCV monoclonal antibodies, and a color developing solution. The detection kit is capable of shortening the time of window phase greatly by one-time combined diagnosis on HCV antigens and antibodies, reducing false negatives rate significantly and avoiding defects of HCV RNA detection. The structure of full-length core antigens is modified, mosaicism of non-structural regions is realized, and expression of the mosaic antigens is realized, so as to detect the HCV antibodies in serum as soon as possible, realize fusion of the mosaic antigens and coated monoclonal antibodies, and avoid combination.
Description
Technical field
The invention belongs to field of biological pharmacy, in particular to a kind of hepatitis C detection kit and the application in the hepatitis C early detection thereof.
Background technology
Hepatitis virus (hepatitis C virus, HCV) is the NANB hepatitis virus of finding in 1989.Main by the propagation of the approach such as blood transfusion, intravenous injection.According to estimates, there are 1.7 hundred million hepatitis c virus infection persons in the whole world at present, and the third liver infection rate of China is approximately 3.0%, has at present 4000～6,000 ten thousand third hepatopaths at least.In the infected, 60%-80% can develop into chronic hepatitis or sustainable development becomes cirrhosis and liver cancer, and case fatality rate is higher.The clear and definite medicine that also is of no curative effect is in the market treated, and does not also have vaccine to be prevented to prevent its further propagation.Therefore the early diagnosis of HCV is of great importance for the infection sources, guiding clinical treatment and the prognosis judgement of examination HCV.It is mainly at present the detection of anti-HCV and HCV RNA in serum.But after HCV infects to " window phase " (average out to 6～12 weeks) of growing that has of anti-HCV, even the patient by hyperinfection, the antibody test technology can not detect the existence of HCV.So early diagnosis index that anti-HCV can not infect as HCV.Characteristics such as although the detection of HCV RNA have in early days, responsive and special, the method is had relatively high expectations on technology and equipment, time-consuming, and recall rate is low, is difficult to promote in routine work or basic hospital laboratory.This research carrys out the infection of the HCV of detection window phase by catch HCV-cAg and antibody simultaneously, by the joint-detection to HCV antigen/antibody combination and HCV-cAg, make " window phase " that HCV infects foreshorten to 2 weeks, even shorter, thereby reach accurately, fast, testing goal easily.
Existing detection method comprises anti-HCV enzyme immunoassay (ELA), and the ELA technology is applicable to people at highest risk's examination, also can be used for HCV the infected's primary dcreening operation.But whether anti-HCV turns out cloudy can not be as the performance assessment criteria of Anti-viral Treatment.The ELA technology of utilizing recombinant HCV antigen to develop, developed into the third generation.This technology can detect in sample the mixed antibody for different HCV antigens, and that detects at present is special up to more than 90%.Owing to lacking more responsive goldstandard, the susceptibility of anti-HCV-ELA technology also is difficult to determine.After infecting, HCV also had one period long term of approximately 40～70 days (average 66 days) before HCV antigen/antibody combination produces.Now, the blood donor is infected and have infectivity, and applying current third generation ELA detection reagent can not detect, and this stage is called infects the front window phase of rear seropositive conversion.The existence of window phase is one of important threat of transfusion safety, the receptor is still had through inputting anti-HCV and screen negative blood and the danger of HCV infection.
In addition, prior art has also reported that the discovery of the nuclease system relevant with reparation with nucleic acid replication has been applied to molecular detection technology.For example, round pcr is considered to one and can detects highly delicately and the method for the target nucleic acid sequence that increases.The high affinity of nucleic acid hybridization and specificity make the possibility that develops into of nucleic acid detection technique, and this sensitivity is far beyond the sensitivity based on the antibody test technology.Therefore, these detection techniques, after appropriate trace routine is combined, are used further to detect the effect that the HCV RNA in the patient blood sample produces satisfactory.After HCV infects, 6～15 days (average 11 days) HCV RNA just occur in the infected's blood, and reached a higher level before seropositive conversion, use the NAT detection technique that susceptibility is higher to carry out the routine screening to the blood donor, can greatly reduce the danger that window phase infects.Although detecting successfully to have filtered out to infect in developed country and area, NAT belongs to the blood donor, but because the expensive accurate instrument of NAT Technology Need, the higher more expensive and easy cross pollution of experiment skill, reagents ratio cause false positive higher, make it be difficult to be generalized to single blood donor, also limited significantly in the application of developing country.
Summary of the invention
The objective of the invention is logical a kind of hepatitis C detection kit; In another aspect of this invention, relate to a kind of kit detected for the hepatitis C window phase.
One aspect of the present invention relates to a kind of hepatitis C detection kit, and described kit comprises the mouse-anti human IgG of HCV chimeric antigen, HCV chimeric antigen monoclonal antibody, detergent damping fluid, HRP mark, the anti-HCV monoclonal antibody of HRP mark, nitrite ion.
In a preferred embodiment of the present invention, described HCV chimeric antigen obtains by the following method: synthetic three couples of primer: Primer1:5 '-CGGGCACGTTGTAGGCATC-3 '
Primer2：5’-AACGGACGGCTTTAGGACGA-3’
Primer3：5’-TGGGTACCTTATGGCAAGTTCCTCGCCGG-3’
Primer4：5’-TAGAATTCTACCAGTCCTACCTCGCCGCTG-3’
Primer5：5’-TCTAGACAATGTCTTACTCTTGGACAGGC-3’
Primer6：5’-GGATCCCGGGGCCGGGCATGAGACACGCTG-3’】
Use above-mentioned primer, take the HCV viral RNA as masterplate, divide the fragment amplification by PCR, obtain NS-3, NS-5 and Core genetic fragment, mode by double digestion is inserted in the pRSET plasmid vector and builds recombinant vector, and recombinant vector is transformed to the Escherichia coli induction expression protein, immobilization metallic ion (Ni2 for expression product
+) affinity chromatogra carries out purifying, obtains the HCV chimeric antigen, HCV chimeric antigen molecular weight is the 24kDa left and right.
In another preferred embodiment of the present invention, described HCV chimeric antigen monoclonal antibody is by adopting above-mentioned HCV chimeric antigen immunity BALB/c mouse to obtain.
The present invention also relates to the application of mentioned reagent box on the other hand, and described kit is for detection of hepatitis C.
In another aspect of this invention, also relate to the application of mentioned reagent box for detection of the window phase hepatitis C.
Kit of the present invention, by HCV antigen and antibody are carried out to the disposable diagnosis of combining, shortens HCV " window phase " time, the defect that also greatly reduces loss simultaneously and avoid HCV RNA to detect greatly.By the total length cAg is changed to structure, chimeric non-structural area, and realize the expression of chimeric antigen, and in order to detect the HCV antibody in serum as far as possible, and compatible with coated monoclonal antibody, avoid mutually combining.
The accompanying drawing explanation
Fig. 1: the construction method schematic diagram of recombinant plasmid.
Embodiment
Embodiment 1
(1) preparation of antigen and Purification:
Synthetic three couples of primer: Primer1:5 '-CGGGCACGTTGTAGGCATC-3 '
Primer2：5’-AACGGACGGCTTTAGGACGA-3’
Primer3：5’-TGGGTACCTTATGGCAAGTTCCTCGCCGG-3’
Primer4：5’-TAGAATTCTACCAGTCCTACCTCGCCGCTG-3’
Primer5：5’-TCTAGACAATGTCTTACTCTTGGACAGGC-3’
Primer6：5’-GGATCCCGGGGCCGGGCATGAGACACGCTG-3’】
Use above-mentioned primer, take the HCV viral RNA as masterplate, by PCR, divide the fragment amplification, obtain NS-3, NS-5 and Core genetic fragment, be inserted in the pRSET plasmid vector and build recombinant vector (concrete inserted mode is referring to Fig. 1).It is transformed to Escherichia coli (BL21) induction expression protein, immobilization metallic ion (Ni2 for expression product
+) affinity chromatogra carries out purifying, obtains target antigen albumen, molecular weight of albumen is the 24kDa left and right.
(2) foundation of hybridoma and monoclonal antibody preparation
With the antigen protein immunity BALB/c mouse of the preparation of purifying, through HAT, HT, select to cultivate and after limiting dilution assay carries out cloning, with indirect elisa method and Dot blot, it is screened and CHARACTERISTICS IDENTIFICATION.Set up the anti-HCV-core of secretion, NS3, the hybridoma cell strain of NS5 monoclonal antibody.Hybridoma cell strain is inoculated into the Syngenic mice of histocompatbility, and hybridoma is bred and made mouse produce tumour.The monoclonal antibody that contains a large amount of hybridoma secretions in the mouse ascites of generation tumour cell and serum.Concrete operations are as follows:
A. material
Through autoclaved norphytane, 5～8 week age Balb/c mouse, the hybridoma cell strain cell in the growth period of taking the logarithm, RPMI-1640 nutrient solution, NBCS (FCS), 1. 20mM pH7.8-7.9Tris-HCl damping fluid/20mM NaCl, 20mM pH7.8-7.9Tris-HCl damping fluid/40mM NaCl, 20mM pH7.8-7.9Tris-HCl damping fluid/80mM NaCl, 20mM pH7.8-7.9Tris-HCl damping fluid/saturated ammonium sulfate liquid, DEAE-cellulose column.
B. method of operating
1. in mouse peritoneal injection 0.5ml norphytane, repopulating cell in 1-9 week after injection.
2. collect the hybridoma of exponential phase, with the washing of the RPMI-1640 liquid containing 5%FCS once, the centrifugal 10min of 1000r/min.Sampling, expect blue dyeing with platform, counts viable count, again with the RPMI-1640 liquid of 5%FCS, is made into the suspension of 1.0 * 107 cells/ml.
3. give the small white mouse inoculation of having injected norphytane hybridoma, every lumbar injection 1ml (containing 1.0 * 107 cell/ml).
Inoculate latter 10 days left and right temporal lesion volume maximums, now by abdominal cavity, extract ascites, got 1 time desirable 10 times every 1～3 day.Serum can be by separating after oxter artery or heart blood sampling.
4. mouse ascites, with after 4 times of cold PBS liquid dilutions, in the centrifugal 30min of 1.0 * 105rpm, goes precipitation.
5. slowly drip saturated ammonium sulfate liquid in supernatant at 4 ℃, the limit edged stirs, and making solution is finally 50% ammonium sulfate concentrations.
6. this solution is put 30-60min in ice, and then 5, the centrifugal 10min of 000rpm, remove supernatant.
7. precipitation is dissolved in (solution may be muddy) in Tris-HCl damping fluid (40mM NaCl).
8. the bag filter of packing into the desalination of dialysing in Tris-HCl damping fluid (20mM NaCl), the centrifugal precipitation of going.
9. after solution dilution (1: 100 or more dilution for many times), in 280nm, survey protein content, estimate protein content.In general every ml ascites, contain the about 25.0-36.0mg of total protein.
10. cross the DEAE-cellulose column: the high 40cm of cellulose column, with 20mM NaCl Tris damping fluid balance.The dialysis sample dilutes with Tris damping fluid equivalent.It is 1.0ml-2.0ml/min that sample enters post bed speed, with NaCl linear gradient wash-out.Most of monoclonal IgG, in 40mM and 80mM NaCl wash-out, also has the monoclonal antibody of few exception in 120-150mM NaCl wash-out.Survey OD
280nmcollect protein peak, monoclonal IgG-80 ℃ saves backup.
(3) preparation of antigen-antibody combined detection kit
HCV chimeric antigen (one or more) is coated from the monoclonal antibody of different epi-positions, and the damping fluid washing micropore with containing detergent, add sample in micropore, incubation, washing.Add the anti-HCV monoclonal antibody of horseradish peroxidase (HRP) mark and the anti-human IgG in mouse source of HRP mark, incubation, washing, colour developing, detect at 495nm.
As shown in Table 1 and Table 2, the initial stage that kit of the present invention can be to greatest extent infects at HCV detect HCV to the testing result of kit of the present invention, and recall rate and accuracy rate will be far above coherent detection reagent in the market.
Table 1
The relevant disease intercrossing is estimated
Table 2
Hepatitis C virus in the series plasma sample
The detection time of cAg/Ab, RNA and antibody
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. a hepatitis C detection kit, described kit comprises the mouse-anti human IgG of HCV chimeric antigen, HCV chimeric antigen monoclonal antibody, detergent damping fluid, HRP mark, the anti-HCV monoclonal antibody of HRP mark, nitrite ion.
2. hepatitis C detection kit according to claim 1, described HCV chimeric antigen obtains by the following method: synthetic three couples of primer: Primer1:5 '-CGGGCACGTTGTAGGCATC-3 '
Primer2：5’-AACGGACGGCTTTAGGACGA-3’
Primer3：5’-TGGGTACCTTATGGCAAGTTCCTCGCCGG-3’
Primer4：5’-TAGAATTCTACCAGTCCTACCTCGCCGCTG-3’
Primer5：5’-TCTAGACAATGTCTTACTCTTGGACAGGC-3’
Primer6：5’-GGATCCCGGGGCCGGGCATGAGACACGCTG-3’；
Use above-mentioned primer, take the HCV viral RNA as masterplate, divide the fragment amplification by PCR, obtain NS-3, NS-5 and Core genetic fragment, mode by double digestion is inserted in the pRSET plasmid vector and builds recombinant vector, and recombinant vector is transformed to the Escherichia coli induction expression protein, immobilization metallic ion (Ni2 for expression product
+) affinity chromatogra carries out purifying, obtains the HCV chimeric antigen, HCV chimeric antigen molecular weight is the 24kDa left and right.
3. hepatitis C detection kit according to claim 1 and 2, described HCV chimeric antigen monoclonal antibody is by adopting above-mentioned HCV chimeric antigen immunity BALB/c mouse to obtain.
4. the application of claim 1-3 any one kit, described kit is for detection of hepatitis C.
5. the application of claim 1-3 any one kit, for detection of the application of window phase hepatitis C.
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| CN1690206A (en) * | 2004-04-21 | 2005-11-02 | 吉林大学第一医院 | Chimeric antigen gene of hepatitis B and hepatitis C and its nucleic acid vaccine |
| CN1755365A (en) * | 2004-09-29 | 2006-04-05 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of method that detects antibody of HCV |
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| US20120070435A1 (en) * | 2002-06-20 | 2012-03-22 | Paladin Labs, Inc. | Chimeric antigens for eliciting an immune response |
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| JP3665371B2 (en) * | 1994-08-31 | 2005-06-29 | 株式会社先端生命科学研究所 | Epitope chimera antigen peptide for hepatitis C virus infection or group determination, production method thereof, and infection or group determination method using the same |
| CN1309664A (en) * | 1998-07-21 | 2001-08-22 | 康耐克斯研究发展优化有限公司 | Anti hepatitis C virus antibody and uses thereof |
| US20120070435A1 (en) * | 2002-06-20 | 2012-03-22 | Paladin Labs, Inc. | Chimeric antigens for eliciting an immune response |
| CN1690206A (en) * | 2004-04-21 | 2005-11-02 | 吉林大学第一医院 | Chimeric antigen gene of hepatitis B and hepatitis C and its nucleic acid vaccine |
| CN1755365A (en) * | 2004-09-29 | 2006-04-05 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of method that detects antibody of HCV |
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Non-Patent Citations (4)
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Application publication date: 20131225 |