CN1700010A - Hepatitis B virus large protein pre-S1 antigen detection reagent kit - Google Patents
Hepatitis B virus large protein pre-S1 antigen detection reagent kit Download PDFInfo
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- CN1700010A CN1700010A CN 200510077451 CN200510077451A CN1700010A CN 1700010 A CN1700010 A CN 1700010A CN 200510077451 CN200510077451 CN 200510077451 CN 200510077451 A CN200510077451 A CN 200510077451A CN 1700010 A CN1700010 A CN 1700010A
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Abstract
The invention provides a monoclonal antibody which is obtained by using antigen immune animal that has fabric epitope B hepatitis virus surface antigen big protein fore S zone by sifting hybridomas colon. The invention also provides monoclonal antibody medium box and it's preparing method and application. The medium box can be used as index of early stage diagnosis B hepatitis virus infection to quote weather individual has infectious after it has virus infection, it can also ascertain the patient's estimation and the probability of cirrhosis and liver cancer aroused by it according to monitoring B hepatitis big protein expression vival.
Description
Technical field
The invention belongs to biological technical field, more particularly, the invention provides monoclonal antibody, the kit that contains this monoclonal antibody, its preparation method and the application thereof that large protein pre-S surface antigen for hepatitis B virus district antigen preparation that a kind of use has the conformation type epi-position goes out.Utilize this kit can diagnose the situation of duplicating of hepatitis type B virus in the human body quickly and accurately.
Background technology
Hepatitis B is the global infectious disease that is caused by hepatitis type B virus (HBV), and according to estimates, the whole world is nearly 300,000,000 hepatitis carriers at present, and this numeral is also increasing.Wherein, China is the high popular district of hepatitis B, according to China's end of the seventies and the beginning of the nineties twice national hepatitis the data presentation of epidemiology survey, the people that our country infected hepatitis B has 6.9 hundred million, infection rate is 57.6%, and the people who carries hepatitis B for a long time has 1.2 hundred million, the carrying rate 9.75% of hepatitis B virus surface antigen, accumulate over the years existing chronic hepatitis B patient about more than 2,000 ten thousand.HBV belongs to and to have a liking for hepatovirus family, can cause oxyhepatitis, chronic hepatitis and cirrhosis, and with the confidential relation that has of liver cancer.Still do not have the chronic infection of effective antiviral method treatment hepatitis type B virus at present, therefore the prevention and the detection of virus are exactly the important means of avoiding infection, and also can provide important information for development, the prognosis of hepatitis B to the dynamic monitoring of virus infections.
Hepatitis B virus surface antigen (HBsAg) is the outer membrane protein of HBV, and is coded by the genomic S of HBV district, and S divides into 3 sections of preS1, preS2 and S genes, and it is identical that their read frame phase place, and arranged in series continuously can shared 1 termination codon.These 3 genes three kinds of surface proteins of encoding respectively: (1) main surface protein: be called for short main albumen, by the S gene code, be the principal ingredient of peplos and subunit's particle, because of its molecular weight little, therefore be referred to as small protein, HBsAg often refers to the albumen by the S gene code traditionally; (2) medium molecule S albumen (M albumen) is coded by S and Pre-S2 gene regions; (3) big molecule S albumen (L albumen) is coded by S, Pre-S1 and Pre-S2 gene regions.(Large protein LP) is molecular weight maximum in three kinds of albumen to large protein.The preceding S1 of large protein is in the upstream in preceding S2 district, preceding S1 and preceding S2 section form the preceding S district albumen of conformation type, composition as large protein, be positioned at the virion surface, at virus infections, duplicate, stimulate body to produce aspect the immune response and diagnosis, treatment and the prognosis of hepatitis B patient all had the important references meaning.
The virion that 3 kinds of different shapes are arranged among the HBV infected person anteserum: pellet shapes particle (diameter 22nm), tubular particle (diameter 22nm, length 100-1000nm), Dane particle.The situation difference that these 3 kinds of albumen of S, Pre-S1 and Pre-S2 exist on the variable grain surface, the Dane particle is identical with the envelope protein composition of tubular particle, and each virus contains the S albumen of 300-400 molecule and the M albumen and the L albumen of 40-80 molecule.Whether the envelope protein of pellet shapes albumen is formed and then duplicated with virus and different: in viral duplicator was arranged, roughly the same on M albumen on the pellet shapes particle coating and S protein content and the Dane particle have L albumen, but ratio was lower than 1/20; In virus-free duplicator, the envelope protein of pellet shapes particle almost all is a S albumen, and no L albumen only has 1% M albumen.
Among the hepatitis B infected person, the distribution of outer membrane granule in blood flow: great majority are the small spherical particles of 22nm in patient's blood flow of the infected, only by large protein or 5% the middle albumen of having an appointment simultaneously form; Virus replication phase patient has simultaneously that minority is different in size, the cast particle of diameter 22nm, is made up of albumen among main albumen, the 2%-5% and 5%-10% large protein.At the non-replicative phase of virus, large protein is very low and nothing in serum, is trapped in the liver cell in a large number.Therefore can judge whether the HBV virus in virus carrier's body is duplicating according to detecting having or not of large protein in the blood.
The enzyme linked immunological adsorption technology is the technology that has situation of hepatitis B surface antibody in the at present the most frequently used detection patient serum, utilizes monoclonal antibody to detect HBsAg usually.Monoclonal antibody has very high specific owing to be antibody at an epitope, can the specific recognition corresponding antigens.Utilize the monoclonal antibody of solid-phase coating that the combination specifically of the HBsAg in the serum is got off, the anti-HBsAg polyclonal antibody of adsorptive enzyme mark again, to the substrate reactions colour developing, the colour developing result just can reflect the content of HBsAg in serum as quantizating index, embodies the infection level of hepatitis type B virus.
In the immune detection of serum large protein, all utilize the distinctive section preS1 of large protein as immunogene at present, the preparation monoclonal antibody makes up diagnostic reagent.It is that the higher structure that folding back, dependent protein space forms occurs that but a very important part is arranged in the immune epitope of albumen, when making monoclonal antibody, if the antigen that obtains is low structure, good more in theory sequence, also can be when reality form higher structure, may be folded, curl and lose the chance that exposes epi-position; Some the peptide section that constitutes epi-position is lacking under the folding assisted situation of other peptide sections, the topological structure of formation may with physiological status under different.The monoclonal antibody that such antigen is made must cause omission in actual applications, resembles present pre S 1 antigen diagnostic kit, and clinical practice finds that it is negative that the preceding S1 of the sample kit of a large amount of DNA positives detects.S1 section before the individualism not on the hepatitis B large protein, but the preceding S district of the conformation type that exists preceding S1 and preceding S2 district to form, preceding S district in the LP large protein in the hepatitis B is a conformation type albumen, has complicated topological structure, only, could really capture the existence of large protein at the monoclonal antibody specific of this labyrinth.
Purpose of the present invention is just in order to solve the deficiency that is used as hepatitis B large protein index detection means at present clinically with anti-preS1 monoclonal antibody, utilization is at the levels of replication of the monoclonal antibody diagnosis of hepatitis b virus of large protein pre-S district conformation type epi-position, and detection kit and assay method easy and simple to handle, accurate sensitivity are provided, thereby can improve the recall rate of hepatitis B virus large protein, so that it is clinical from protein level observation virus replication level, can take effective comprehensive therapeutic plan as early as possible, the generation of prevention cirrhosis and liver cancer.
Summary of the invention
The invention provides diagnostic kit and preparation method thereof, can diagnose out the infection conditions of duplicating of hepatitis type B virus by this kit exactly.Owing to used special monoclonal antibody at conformation type hepatitis B large protein, can reflect the level that exists of large protein in the serum truly, there is not cross reactivity, specificity is good, is convenient to quality control, also demonstrates high sensitivity.
On the one hand, the invention provides a kind of monoclonal antibody, it is characterized in that, it is to obtain by hepatitis b virus s antigen large protein antigen immune mouse, screening hybridoma clone that use has a conformation type epi-position.Preferably, described monoclonal antibody is prepared by following method: with the pure product of hepatitis b virus s antigen large protein with conformation type epi-position that have the large protein pre-S surface antigen for hepatitis B virus district recombinant antigen of conformation type epi-position or extract from serum the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal, collect ascites, monoclonal antibody purification.
On the other hand, the present invention also provides above-mentioned monoclonal antibody to be used for detecting the application of the kit of hepatitis B replication situation in preparation.
On the other hand, the invention provides a kind of preparation monoclonal antibody method, it comprises: with the pure product of hepatitis b virus s antigen large protein with conformation type epi-position that have the large protein pre-S surface antigen for hepatitis B virus district recombinant antigen of conformation type epi-position or extract from serum the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal, collect ascites, monoclonal antibody purification.
On the other hand, the invention provides hepatitis b virus s antigen large protein ELISA measuring reagent kit, it contains monoclonal antibody, it is characterized in that described monoclonal antibody obtains animal immune, screening hybridoma clone by using conformation type hepatitis B virus surface antigen large protein.Preferably, described monoclonal antibody is prepared by following method: with the pure product of hepatitis b virus s antigen large protein with conformation type epi-position that have the large protein pre-S surface antigen for hepatitis B virus district recombinant antigen of conformation type epi-position or extract from serum the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal, collect ascites, monoclonal antibody purification.
Above-mentioned kit of the present invention can also contain the LP positive, negative control, ELISA Plate, horseradish peroxidase (HRP) mark polyclonal antibody, auxiliary reagent, and wherein, described monoclonal antibody is to be coated on the ELISA Plate.
On the other hand, the present invention also provides the method for making of mentioned reagent box, and it comprises the preparation of the LP positive, negative control, the preparation that the monoclonal antibody bag is marked Polyclonal Antibody Preparation and auxiliary reagent by the making of plate, enzyme.
The LP positive in the kit of the present invention, negative control prepare from human serum.A kind of preparation process comprises: collect hepatitis B acute stage patients serum and healthy normal human serum, and high-temperature inactivation, filtration sterilization, packing promptly obtain the LP positive, negative control.
Monoclonal antibody bag in the kit of the present invention is made of the mouse-anti human monoclonal antibodies coated elisa plate that the pure product of HBV-LP albumen obtain mouse immune by plate.ELISA Plate can be selected homemade plate or import plate for use; Specification can be 96 holes flat board or 12 * 8, the removable batten of 12X4.A kind of making step of monoclonal antibody coated elisa plate is as follows:
1) preparation monoclonal antibody: with the pure product of hepatitis b virus s antigen large protein that extract in large protein pre-S surface antigen for hepatitis B virus district recombinant antigen or the serum, after routinely the Balb/c mouse being carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal is collected ascites and promptly obtain anti-LP monoclonal antibody behind reorganization proteinG prepackage chromatographic column purifying.
2) bag quilt: said monoclonal antibody is added each hole of ELISA Plate with 0.05M carbonic acid buffer dilution back, every hole 100 μ l, absorption is spent the night, wash plate with the tween phosphate buffer, spend the night with the tween phosphate buffer sealing that contains bovine serum albumin(BSA) again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.
Enzyme mark polyclonal antibody in the kit of the present invention prepares with horseradish peroxidase (HRP) mark HBsAg polyclonal antibody.A kind of enzyme mark Polyclonal Antibody Preparation step is as follows:
A) with NaIO
4-glycol method is carried out the oxidation of HRP, reaches final concentration 10mg/ml.
B) polyclonal antibody and HRP dialysed 6 hours in the alkaline carbonic acid salt buffer, realized the mark of HRP to polyclonal antibody, used NaBH after reaction finishes
4The solution cessation reaction is again to the PBS dialysed overnight.
C) use saturated ammonium sulphate, obtain the HRP enzyme mark anti-HBsAg polyclonal antibody of purifying.
Auxiliary reagent in the kit of the present invention comprises integrated enzyme reaction substrate solution, colour developing liquid, reaction terminating liquid and cleaning buffer solution, and a kind of method of preparing auxiliary reagent is as follows:
A) substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (pH5.0) preparation;
B) colour developing liquid B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.1mg/ml;
C) reaction terminating liquid: 2mol/L sulfuric acid;
D) cleaning buffer solution (20 times of concentrates, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation.
On the other hand, the present invention also provides the method that detects the situation of duplicating of hepatitis type B virus, comprising making the contacted step of mentioned reagent box and serum or plasma sample.
More specifically, detection method of the present invention comprises the steps:
A) antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the 50 μ l positives, negative control sera respectively, or the test serum sample, HRP-polyclonal antibody solution is added each hole, every hole 50 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times.
B) chromogenic reaction: every hole adds substrate solution A successively, each 50 μ l of colour developing liquid B, and 37 ℃ of water bath heat preservations 10 minutes, every hole adds 50 μ l reaction terminating liquids again and finishes reaction.
C) colorimetric:, measure OD value and record at 450nm with microplate reader with the light absorption value zeroing in blank hole.
D) result judges:
A. positive quality control serum OD value is more than 2.0, and negative quality controlled serum OD value is 0.05 when following, and this is measured effectively.
B. testing sample OD value is greater than being judged to the hepatitis b virus s antigen large protein positive at 0.105 o'clock.
On the other hand, the invention provides the quantitative ELISA measuring reagent kit of a kind of hepatitis b virus s antigen large protein, it contains LP standard items, monoclonal antibody, ELISA Plate, horseradish peroxidase-labeled polyclonal antibody, auxiliary reagent, it is characterized in that, described monoclonal antibody obtains, and is coated on the described ELISA Plate animal immune, screening hybridoma clone by using conformation type hepatitis B virus surface antigen large protein.Preferably, described monoclonal antibody is prepared by following method: with the pure product of hepatitis b virus s antigen large protein with conformation type epi-position that have the large protein pre-S surface antigen for hepatitis B virus district recombinant antigen of conformation type epi-position or extract from serum the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal, collect ascites, monoclonal antibody purification.
On the other hand, the present invention also provides the method for making of mentioned reagent box, and it comprises the preparation of standard items, the preparation that the monoclonal antibody bag is marked Polyclonal Antibody Preparation and auxiliary reagent by the making of plate, enzyme.
LP standard items in the kit of the present invention are by genetic engineering, molecular biology and the recombinant expressed preparation of biochemical means.A kind of preparation process comprises: with baculovirus expression hepatitis b virus s antigen large protein, utilize the albumen label of amalgamation and expression to do affinity purification, promptly obtain the LP standard items.
Monoclonal antibody bag in the kit of the present invention is made of the mouse-anti human monoclonal antibodies coated elisa plate that the pure product of HBV-LP albumen obtain mouse immune by plate.ELISA Plate can be selected homemade plate or import plate for use; Specification can be 96 holes flat board or 12 * 8, the removable batten of 12X4.A kind of making step of monoclonal antibody coated elisa plate is as follows:
1) prepares monoclonal antibody: after routinely the Balb/c mouse being carried out immunity with the pure product of hepatitis b virus s antigen large protein that extract in large protein pre-S surface antigen for hepatitis B virus district recombinant antigen or the serum, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal is collected ascites and promptly obtain anti-LP monoclonal antibody behind reorganization proteinG prepackage chromatographic column purifying.
2) bag quilt: said monoclonal antibody is added each hole of ELISA Plate with 0.05M carbonic acid buffer dilution back, every hole 100 μ l, absorption is spent the night, wash plate with the tween phosphate buffer, spend the night with the tween phosphate buffer sealing that contains bovine serum albumin(BSA) again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.
Enzyme mark polyclonal antibody in the kit of the present invention prepares with horseradish peroxidase (HRP) mark HBsAg polyclonal antibody.A kind of enzyme mark Polyclonal Antibody Preparation step is as follows:
A) with NaIO
4-glycol method is carried out the oxidation of HRP, reaches final concentration 10mg/ml.
B) polyclonal antibody and HRP dialysed 6 hours in alkaline CB damping fluid, realized the mark of HRP to polyclonal antibody, used NaBH after reaction finishes
4The solution cessation reaction is again to the PBS dialysed overnight.
C) use saturated ammonium sulphate, obtain the HRP enzyme mark anti-HBsAg polyclonal antibody of purifying.
Auxiliary reagent in the kit of the present invention comprises integrated enzyme reaction substrate solution, colour developing liquid, reaction terminating liquid and cleaning buffer solution, and a kind of method of preparing auxiliary reagent is as follows:
A) substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (pH5.0) preparation;
B) colour developing liquid B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.1mg/ml;
C) reaction terminating liquid: 2mol/L sulfuric acid;
D) cleaning buffer solution (20 times of concentrates, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation.
On the other hand, the present invention also provides the method that detects the situation of duplicating of hepatitis type B virus, comprising making the contacted step of mentioned reagent box and serum or plasma sample.
More specifically, detection method of the present invention comprises the steps:
A) antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the standard items that 50 μ l have diluted good variable concentrations respectively, or test serum sample, HRP-polyclonal antibody solution is added each hole, every hole 50 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times.
B) chromogenic reaction: every hole adds substrate solution A successively, each 50 μ l of colour developing liquid B, and 37 ℃ of water bath heat preservations 10 minutes, every hole adds 50 μ l reaction terminating liquids again and finishes reaction.
C) colorimetric:, measure OD value and record at 450nm with microplate reader with the light absorption value zeroing in blank hole.
D) result calculates:
A production standard curve: with standard items concentration is horizontal ordinate, and the OD value that standard items are measured is an ordinate, makes typical curve; Basis of calculation curvilinear regression coefficients R
2, work as R
2This was measured effectively in＞0.98 o'clock;
B calculates the test serum sample concentration: the LP concentration that calculates the test serum sample according to the OD value of testing sample from typical curve.
Detect index p reS1 reagent with existing hepatitis b virus s antigen large protein and compare, kit of the present invention has the following advantages:
A) monoclonal antibody that obtains of the conformation type antigen immune that forms with the preS1+preS2 section of kit of the present invention is as the diagnosis index of hepatitis b virus infected levels of replication, overcome in the past that detectable only obtains the low recall rate that antibody detects with the preS1 immunity, reached identical 96% or more with the PCR testing result.
B) the large protein monoclonal antibody is to the not reaction of S albumen, and the conformation type epi-position of preS is had high degree of specificity, can effectively reflect the level that exists of large protein in the serum, improves the recall rate of the HBV of replication status.
C) the matched standard items of kit can quantitative Analysis go out the content of hepatitis B large protein contained in the blood sample sample, and preceding S1 reagent in the past are qualitative.
Embodiment
Further specify the present invention with embodiment below.It should be understood that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
The making of the qualitative enzyme-linked immunologic detecting kit of embodiment 1:LP
A kind of hepatitis b virus s antigen large protein enzyme-linked immunologic detecting kit (96 person-portion), its composition comprises:
Each 1 bottle of LP feminine gender, positive control;
LP monoclonal antibody bag is by 1 of plate (96 hole);
1 bottle of the HBsAg polyclonal antibody of horseradish peroxidase (HRP) mark, the 6ml/ bottle;
Each 1 bottle of substrate solution A, colour developing liquid B, each 5ml/ bottle;
1 bottle of reaction terminating liquid, the 5ml/ bottle;
1 bottle of cleaning buffer solution (20X concentrates), the 30ml/ bottle.
Concrete operations are as follows:
1. prepare LP feminine gender, positive control:
1) collect serum: secure good health from hospital or blood station normal person, healthy puerpera's placental blood hepatitis B patient serum of cleer and peaceful acute stage, standby in-70 ℃ of preservations;
2) packing:
The LP positive control serum: acute stage hepatitis B patient serum, for the LP positive, 1 hour handle back filtration sterilization through 60 ℃ through calibrating, branch is packed in the 1.5ml eppendorf pipe under aseptic condition, every pipe 0.5ml.Be stored in 4 ℃.
The LP negative control sera: healthy puerpera's placenta serum or other normal human serums are the LP feminine gender through calibrating.Get more than many parts serum and merge in batch, through 60 ℃ handled in 1 hour after, filtration sterilization divides under aseptic condition in the 1.5ml eppendorf pipe of packing into, every pipe 0.5ml.Be stored in 4 ℃.
2. make LP monoclonal antibody bag by plate:
1) LP Monoclonal Antibody:
A) structure (immunogene) of large protein pre-S surface antigen for hepatitis B virus district recombinant antigen: in the hepatitis b virus s antigen code area, the preS1 section is the exclusive section of large protein, is the inevitable choice that detects LP.For obtaining the conformation type epi-position of this section, choose preS1 and preS2 coded sequence complete section, in baculoviral,,, obtain the conformation type hepatitis B surface antibody large protein preS antigen of purifying through nickel post affinity chromatography with affinity purification label his-tag amalgamation and expression.
B) preparation of hepatitis b virus s antigen large protein (immunogene): with a) the preS antigen immune rabbit of affinity purification.The polyclonal antibody that produces utilizes the sepharose 4B of PreS coupling to do purifying.The polyclonal antibody of affinity purification is coupled on the sepharose 4B more subsequently, obtains affinity column, is used for the natural HBV preS antigen of purifying from acute HBV infected patient, obtains the pure product of HBV surface antigen large protein.
C) immunity and Fusion of Cells: as b) preparation immunogene (hepatitis b virus s antigen large protein) mix with Freund's complete adjuvant, obtain oil emulsion.With this emulsion with 0.2 milliliter dosage subcutaneous be applied to BALB/C mice (product of CLEAJapan, 6 the week ages male) the site, back.The enhance immunity after 7 days or 14 days of immunity for the first time in Fusion of Cells preceding 3 days then, is used 0.2 milliliter of above-mentioned immunogene in the peritonaeum.The last enhancing after 3 days downcut spleen cell from each mouse, and (the SP2/0-Ag14 strain RikenGeneBank) mixed with 10: 1, merged with 50% Macrogol 4000 with the myeloma cell.Go up selectivity at HAT nutrient culture media (product of Gibco) and cultivate hybridoma.
D) hybridoma is selected: after fusion the 12nd day, measure antibody activity in each culture supernatant with the ELISA method.The culture supernatant of respectively getting the fused cell of 200 microlitres is added to the product of 96 hole elisa plate Coaster) the hole in, wherein every hole is fixed with the antigen of 10 mcg/ml, be respectively the preceding S district of the different section HBV surface antigens recombinant antigen of baculovirus expression, comprise preS1, preS2 and preS1+preS2 antigen.Be reflected at 37 ℃ and carried out 1 hour,, in each hole, add the anti-mouse IgG (Cappel product, 1: 20,000) of 200 microlitre peroxidase labellings then with PBS (washing lotion) washing that contains 0.05%Tween 20.Be reflected at 37 ℃ carry out 1 hour after, with above-mentioned washing lotion wash plate.In each hole, add 200 microlitre substrate solutions (0.1M benzidine and 0.012% aqueous hydrogen peroxide solution) then, reaction was at room temperature carried out 15 minutes.Then, in each hole, add 50 microlitre 3.5mol sulfuric acid and stop enzyme reaction, measure the absorbance of 492 nanometers, selection can produce reaction with preS1 and preS1+preS2 antigen and not with the aitiogenic antibody of preS2, and the results absorbance is not less than 0.15 hybridoma clone.With the restriction dilution method each clone is carried out two time clonings.Hybridoma behind the clone is transplanted in the BALB/C mice, found that 32 hybridoma clones produce the various monoclonal antibodies that ascites reclaims that can be used as.
2) bag quilt:
12 * 8 removable battens that ELISA Plate adopts Costar company to produce.It is to add each hole of ELISA Plate behind the 20 μ g/ml that step 1) is obtained monoclonal anti body and function 0.05mol/L carbonate buffer solution dilution, every hole 100 μ l, and absorption is spent the night, wash plate with cleaning buffer solution, spend the night with this confining liquid damping fluid sealing again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing.
3. the anti-HBsAg polyclonal antibody for preparing horseradish peroxidase (HRP) mark
1) oxidation of enzyme (overall process lucifuge)
A) take by weighing HRP 5mg, add ddH
2O 250 μ l dissolving.
B) take by weighing NaIO
45mg adds ddH
2O 250 μ l dissolve, and are mixed with the concentration of 20mg/mL.
C) in HRP solution, dropwise add NaIO
4Solution, the limit edged stirs.
D) solution that mixes is placed 4 ℃, left standstill 30 minutes.
E) get 5ml ethylene glycol and be dissolved in 25 μ l ddH
2Among the O, dropwise add in the above-mentioned mixed solution, the limit edged stirs.
F) room temperature left standstill 30 minutes.
G) the oxydasis process is finished, and the HRP final concentration is 10mg/ml.
2) preparation of polyclonal antibody and mark (lucifuge)
A) adjust antibody concentration (protein concentration cross low then concentrate) to about the 5mg/ml, with 50mmol/L CB (the 1mol/L NaHCO about pH9.5 with PEG20000
3With 1mol/L Na
2CO
3Mix in 10: 1 ratios, use preceding with 20 times of distilled water dilutings) to dialyse and remove glycerine or impurity (as Tris), 4 ℃ of following dialysed overnight are wherein changed liquid 3 times.
B) polyclonal antibody is mixed by 1: 4 with HRP, dialysis two hours was changed liquid once more than 6 hours in 50mmol/L pH9.5 CB.
C) with the 1mg NaBH of fresh configuration
4The solution cessation reaction.Shake up, 4 ℃ left standstill 2 hours, and shake once per half an hour, NaBH
4The amount that solution adds is suitable.
D) the 10mM PBS (Na of pre-configured 0.01mol/L of usefulness pH7.2
2HPO
4And NaH
2PO
4Storing solution, the two becomes the PBS damping fluid pH value mixing as required) dialysed overnight.Changing liquid once gets final product.
3) purifying HRP enzyme mark anti-HBsAg polyclonal antibody
A) finish in the polyclonal antibody solution of mark and dropwise add saturated ammonium sulfate solution, stir while adding, be reduced to 1/3 until saturated ammonium sulfate concentration.
B) 4 ℃ left standstill 1 hour.
C) 8000rpm is centrifugal 10 minutes, and supernatant is moved in the new pipe, and precipitation suspends again with equal-volume PBS.
D) repeat above operation, saturated ammonium sulfate concentration is brought up to 40%, collect respectively and go up cleer and peaceful precipitation.
E) repeat above operation, saturated ammonium sulfate concentration is brought up to 50%, collect respectively and go up cleer and peaceful precipitation.
F) repeat above operation, saturated ammonium sulfate concentration is brought up to 60%, collect respectively and go up cleer and peaceful precipitation.
G) collect each component of separating, SDS-PAGE identifies purity.
H) the HRP-polyclonal antibody of Ti Chuning is to the PBS dialysed overnight.
I) the centrifugal concentrated and purified HRP-polyclonal antibody of ultrafiltration pipe obtains mole ratio and marks the anti-HBsAg polyclonal antibody near 1: 8 enzyme.
4) assembling: mark the anti-HBsAg polyclonal antibody to suitable working concentration with the enzyme that the damping fluid dilution that contains 10% hyclone is obtained by step 3), press the packing of 6ml/ bottle, be stored in 4 ℃.
4. prepare substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (PH5.0) preparation, press the packing of 5ml/ bottle.
5. preparation colour developing liquid B:TMB (0.1mg/ml) methanol solution is pressed the packing of 5ml/ bottle.
6. preparation reaction terminating liquid: 2mol/L H
2SO
4, press the packing of 3ml/ bottle.
7. 1% polysorbas20 solution of preparation cleaning buffer solution (20 times of concentrates): PBS (pH7.4) preparation is pressed the packing of 15ml/ bottle.
Use the quality testing that the technology of the present invention prepares the qualitative enzyme-linked immunologic detecting kit of LP
1) accuracy: the testing result of 15 parts of negative quality controlled serum (comprising the specificity control serum) reference materials of LP, non-false positive occurs.The reference material testing result of 10 parts of LP positive quality control serum does not have false negative and occurs.The positive terminal point that the positive reference material of serial dilution is detected all was not less than 1: 8.
2) precision: randomly draw 20 box different batches kits, use with a LP positive quality control serum by specification operation steps and carry out replication.Calculate each measurement result, obtain average, SD and coefficient of variation CV.CV＜＜15% between the Precision test result demonstration is criticized.
3) detection sensitivity: according to the recombinant expressed antigen gradient dilution of LP measurement result, the detection sensitivity of this kit is 2.4ng/ml.
4) specificity: be divided into four parts of pooled serum samples, every part of 1ml after getting four parts of testing samples mixing.Make interference test serum specimen #1, #2, #3 after adding tissue-type plasminogen activator (tPA), plasmin (Plasmin) or the fibronectin (FN) of 50ng dosage respectively, the #4 pooled serum sample that does not add any chaff interference is as basic sample.The by specification operation steps is measured and result of calculation.Calculate jamming rate by the interference test computing formula then.The mushing error of sample #1, #2, #3 is all less than 1.5%.
The use of the qualitative enzyme-linked immunologic detecting kit of embodiment 2:LP
1. cleaning buffer solution preparation: 20 times of dilutions of concentrated cleaning buffer solution adding distil water that kit is provided.
2. antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add 50 μ l positive or negative quality controlled serums respectively, or the test serum sample, HRP-polyclonal antibody solution is added each hole, every hole 50ul, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 5 times, each 3 minutes.
3. chromogenic reaction: every hole adds each 50 μ l of substrate solution A, TMB colour developing liquid B successively, 37 ℃ of water bath heat preservations 10 minutes, and every hole adds the 50ul reaction terminating liquid again and finishes reaction.
4. integrated enzyme reaction: HRP-polyclonal antibody solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 60 minutes.Repeat to wash plate operation 4 times, each 3 minutes.
5. colorimetric:, measure OD mean value at 450nm with microplate reader with the light absorption value zeroing in blank hole; Write down each hole light absorption value; Calculate the mean value of the diplopore positive, negative quality controlled serum OD value.
6. the result judges
Positive quality control serum OD value is more than 2.0, and negative quality controlled serum OD value is 0.05 when following, and this experiment effectively.Testing sample OD value is greater than being judged to the hepatitis b virus s antigen large protein positive at 0.105 o'clock.
The making of the quantitative enzyme-linked immunologic detecting kit of embodiment 3:LP
A kind of hepatitis b virus s antigen large protein enzyme-linked immune quantitative detection reagent box (96 person-portion), its composition comprises:
1 bottle of LP standard items;
LP monoclonal antibody bag is by 1 of plate (96 hole);
1 bottle of the HBsAg polyclonal antibody of horseradish peroxidase (HRP) mark, the 6ml/ bottle;
Each 1 bottle of substrate solution A, colour developing liquid B, each 5ml/ bottle;
Reaction terminating liquid bottle 1, the 5ml/ bottle;
1 bottle of cleaning buffer solution (20X concentrates), the 30ml/ bottle.
Concrete operations are as follows:
1. prepare the LP standard items:
1) LP is recombinant expressed: PCR obtains the HBV large protein full length sequence of preS1, preS2 and S coding, inserts in the rhabdovirus expression vector, utilizes baculovirus expression system to express structure facies pattern LP albumen, utilizes the His label of amalgamation and expression to do affinity purification again;
2) the pure product of HBV surface antigen large protein by first required concentration packing of typical curve in the kit instructions (75ng/ml), freeze drying, be stored in 4 ℃.
2. make LP monoclonal antibody bag by plate:
1) LP Monoclonal Antibody:
A) structure (immunogene) of large protein pre-S surface antigen for hepatitis B virus district recombinant antigen: in the hepatitis b virus s antigen code area, the preS1 section is the exclusive section of large protein, is the inevitable choice that detects LP.For obtaining the conformation type epi-position of this section, choose preS1 and preS2 coded sequence complete section, in baculoviral,,, obtain the conformation type hepatitis B surface antibody large protein preS antigen of purifying through nickel post affinity chromatography with affinity purification label his-tag amalgamation and expression.
B) preparation of hepatitis b virus s antigen large protein (immunogene): with a) the preS antigen immune rabbit of affinity purification.The polyclonal antibody that produces utilizes the sepharose 4B of PreS coupling to do purifying.The polyclonal antibody of affinity purification is coupled on the sepharose 4B more subsequently, obtains affinity column, is used for the natural HBV preS antigen of purifying from acute HBV infected patient, obtains the pure product of HBV surface antigen large protein.
C) immunity and Fusion of Cells: as b) preparation immunogene (hepatitis b virus s antigen large protein) mix with Freund's complete adjuvant, obtain oil emulsion.With this emulsion with 0.2 milliliter dosage subcutaneous be applied to BALB/C mice (product of CLEAJapan, 6 the week ages male) the site, back.The enhance immunity after 7 days or 14 days of immunity for the first time in Fusion of Cells preceding 3 days then, is used 0.2 milliliter of above-mentioned immunogene in the peritonaeum.The last enhancing after 3 days downcut spleen cell from each mouse, and (the SP2/0-Ag14 strain RikenGeneBank) mixed with 10: 1, merged with 50% Macrogol 4000 with the myeloma cell.Go up selectivity at HAT nutrient culture media (product of Gibco) and cultivate hybridoma.
D) hybridoma is selected: after fusion the 12nd day, measure antibody activity in each culture supernatant with the ELISA method.The culture supernatant of respectively getting the fused cell of 200 microlitres is added to the product of 96 hole elisa plate Coaster) the hole in, wherein every hole is fixed with the antigen of 10 mcg/ml, be respectively the preceding S district of the different section HBV surface antigens recombinant antigen of baculovirus expression, comprise preS 1, preS2 and preS1+preS2 antigen.Be reflected at 37 ℃ and carried out 1 hour,, in each hole, add the anti-mouse IgG (Cappel product, 1: 20,000) of 200 microlitre peroxidase labellings then with PBS (washing lotion) washing that contains 0.05%Tween 20.Be reflected at 37 ℃ carry out 1 hour after, with above-mentioned washing lotion wash plate.In each hole, add 200 microlitre substrate solutions (0.1M benzidine and 0.012% aqueous hydrogen peroxide solution) then, reaction was at room temperature carried out 15 minutes.Then, in each hole, add 50 microlitre 3.5mol sulfuric acid and stop enzyme reaction, measure the absorbance of 492 nanometers, selection can produce reaction with preS1 and preS1+preS2 antigen and not with the aitiogenic antibody of preS2, and the results absorbance is not less than 0.15 hybridoma clone.With the restriction dilution method each clone is carried out two time clonings.Hybridoma behind the clone is transplanted in the BALB/C mice, found that 32 hybridoma clones produce the various monoclonal antibodies that ascites reclaims that can be used as.
2) bag quilt:
12 * 8 removable battens that ELISA Plate adopts Costar company to produce.It is to add each hole of ELISA Plate behind the 20 μ g/ml that step 1) is obtained monoclonal anti body and function 0.05mol/L carbonate buffer solution dilution, every hole 100 μ l, and absorption is spent the night, wash plate with cleaning buffer solution, spend the night with this confining liquid damping fluid sealing again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing.
3. the anti-HBsAg polyclonal antibody for preparing horseradish peroxidase (HRP) mark
1) oxidation of enzyme (overall process lucifuge)
A) take by weighing HRP 5mg, add ddH
2O 250 μ l dissolving.
B) take by weighing NaIO
45mg adds ddH
2O 250 μ l dissolve, and are mixed with the concentration of 20mg/mL.
C) in HRP solution, dropwise add NaIO
4Solution, the limit edged stirs.
D) solution that mixes is placed 4 ℃, left standstill 30 minutes.
E) get 5ml ethylene glycol and be dissolved in 25 μ l ddH
2Among the O, dropwise add in the above-mentioned mixed solution, the limit edged stirs.
F) room temperature left standstill 30 minutes.
G) the oxydasis process is finished, and the HRP final concentration is 10mg/ml.
2) preparation of polyclonal antibody and mark (lucifuge)
A) adjust antibody concentration (protein concentration cross low then concentrate) to about the 5mg/ml, with 50mmol/L CB (the 1mol/L NaHCO about pH9.5 with PEG20000
3With 1mol/L Na
2CO
3Mix in 10: 1 ratios, use preceding with 20 times of distilled water dilutings) to dialyse and remove glycerine or impurity (as Tris), 4 ℃ of following dialysed overnight are wherein changed liquid 3 times.
B) polyclonal antibody is mixed by 1: 4 with HRP, dialysis two hours was changed liquid once more than 6 hours in 50mmol/L pH9.5 CB.
C) with the 1mg NaBH of fresh configuration
4The solution cessation reaction.Shake up, 4 ℃ left standstill 2 hours, and shake once per half an hour, NaBH
4The amount that solution adds is suitable.
D) the 10mM PBS (Na of pre-configured 0.01mol/L of usefulness pH7.2
2HPO
4And NaH
2PO
4Storing solution, the two becomes the PBS damping fluid pH value mixing as required) dialysed overnight.Changing liquid once gets final product.
3) purifying HRP enzyme mark anti-HBsAg polyclonal antibody
A) finish in the polyclonal antibody solution of mark and dropwise add saturated ammonium sulfate solution, stir while adding, be reduced to 1/3 until saturated ammonium sulfate concentration.
B) 4 ℃ left standstill 1 hour.
C) 8000rpm is centrifugal 10 minutes, and supernatant is moved in the new pipe, and precipitation suspends again with equal-volume PBS.
D) repeat above operation, saturated ammonium sulfate concentration is brought up to 40%, collect respectively and go up cleer and peaceful precipitation.
E) repeat above operation, saturated ammonium sulfate concentration is brought up to 50%, collect respectively and go up cleer and peaceful precipitation.
F) repeat above operation, saturated ammonium sulfate concentration is brought up to 60%, collect respectively and go up cleer and peaceful precipitation.
G) collect each component of separating, SDS-PAGE identifies purity.The HRP-polyclonal antibody of purifying is to the PBS dialysed overnight.
H) the centrifugal concentrated and purified HRP-polyclonal antibody of ultrafiltration pipe obtains mole ratio and marks the anti-HBsAg polyclonal antibody near 1: 8 enzyme.
4) assembling: mark the anti-HBsAg polyclonal antibody to suitable working concentration with the enzyme that the damping fluid dilution that contains 10% hyclone is obtained by step 3), press the packing of 6ml/ bottle, be stored in 4 ℃.
4. prepare substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (PH5.0) preparation, press the packing of 5ml/ bottle.
5. preparation colour developing liquid B:TMB (0.1mg/ml) methanol solution is pressed the packing of 5ml/ bottle.
6. preparation reaction terminating liquid: 2mol/L H
2SO
4, press the packing of 3ml/ bottle.
7. 1% polysorbas20 solution of preparation cleaning buffer solution (20 times of concentrates): PBS (pH7.4) preparation is pressed the packing of 15ml/ bottle.
Use the quality testing that the technology of the present invention prepares the quantitative enzyme-linked immunologic detecting kit of LP
1) accuracy: be divided into three parts of pooled serum samples, every part of 1ml after getting three parts of testing samples mixing.Add 0,20 respectively, the pure product of LP of 100ng, make recovery test serum specimen #1, #2, #3.The by specification operation steps is measured and result of calculation.Then by recovery computing formula calculate recovery rate.The recovery of sample #2, #3 is respectively 96.4% and 98.7%, and average recovery rate is 97.5%, and promptly the proportional jitter of kit is 2.5%, and accuracy is 97.5%.
2) precision: randomly draw 20 box different batches kits, use with a sample to be tested body by specification operation steps and carry out replication.Calculate each measurement result, obtain average, SD and coefficient of variation CV.CV " 15% between the Precision test result demonstration is criticized
3) range of linearity: the pure product solution that is diluted to a series of variable concentrations with the pure product of LP: 300ng, 15ong, 75ng, 37.5ng, 18.8ng, 9.5ng, 4.8ng, 2.4ng, 1.2ng.Operation steps is measured to specifications.With concentration is that horizontal ordinate, absorbance are the ordinate curve plotting.The highest detection higher limit is 150ng/ml in the range of linearity, and the lowest detection lower limit is 2.4ng/ml.The range of linearity of kit is 2.4～75ng/ml.
4) detection sensitivity: according to above-mentioned range of linearity measurement result, the detection sensitivity of this kit is 2.4ng/ml.
5) specificity: be divided into four parts of pooled serum samples, every part of 1ml after getting four parts of testing samples mixing.Make interference test serum specimen #1, #2, #3 after adding tissue-type plasminogen activator (tPA), plasmin (P1asmin) or the fibronectin (FN) of 50ng dosage respectively, the #4 pooled serum sample that does not add any chaff interference is as basic sample.The by specification operation steps is measured and result of calculation.Calculate jamming rate by the interference test computing formula then.The mushing error of sample #1, #2, #3 is all less than 1.5%.
The use of the quantitative enzyme-linked immunologic detecting kit of embodiment 4:LP
1. cleaning buffer solution preparation: 20 times of dilutions of concentrated cleaning buffer solution adding distil water that kit is provided.
2. first freeze-dried powder of the standard items that kit provided (concentration 75ng/ml) carry out doubling dilution 5 times then with the dissolving of 500ul cleaning buffer solution.Typical curve in the kit is made up of the LP standard items of 6 variable concentrations, and typical curve each point concentration is respectively 75ng/ml, 37.5ng/ml, 18.8ng/ml, 9.5ng/ml, 4.8ng/ml, 2.4ng/ml.
3. antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the standard items that 100 μ l have diluted good variable concentrations respectively, or the test serum sample, 37 ℃ of water bath heat preservations 60 minutes.Repeat to wash plate 4 times with cleaning buffer solution then, each 3 minutes.
4. integrated enzyme reaction: HRP-polyclonal antibody solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 60 minutes.Repeat to wash plate operation 4 times, each 3 minutes.
5. chromogenic reaction: every hole adds each 50 μ l of substrate solution A, TMB colour developing liquid B successively, 37 ℃ of water bath heat preservations 10 minutes, and every hole adds 50 μ l reaction terminating liquids again and finishes reaction.
6. colorimetric:, measure OD mean value at 450nm with microplate reader with the light absorption value zeroing in blank hole; Write down each hole light absorption value; Calculate the mean value of diplopore standard items OD value.
7. the result calculates
1) drafting of typical curve: with standard items concentration is that the average OD value that horizontal ordinate, standard items are measured is an ordinate, draws the typical curve of this mensuration; Basis of calculation curvilinear regression coefficients R 2, this experiment effectively when R2＞0.98.
2) calculate testing sample concentration: when typical curve and quality-control product all are determined when effective, calculate LP concentration the test serum sample from typical curve according to the OD value of sample to be tested.
Embodiment 5: the qualitative enzyme-linked immunologic detecting kit of LP of the present invention is to the detection of HBV-DNA positive
For judging the coincidence rate of qualitative enzyme-linked immunologic detecting kit of LP of the present invention and HBV-DNA PCR testing result, get the preceding S1 ELISA reagent of qualitative enzyme-linked immunologic detecting kit of LP of the present invention and Alpha of the same type and compare.Ditan hospital's clinical research data: 200 parts of known HBV-DNA positive samples, 100 parts of healthy serum of health check-up, 100 parts of hepatitis C serum.Detect and judgement with S1 kit before qualitative enzyme-linked immunologic detecting kit of LP and the Alpha respectively, the results are shown in Table 1.
The HBV recall rate of the different detection methods of table 1. relatively
| The serum source | The example number | Number positive (positive ratio) | ||
| ?HBV-DN ????A | The LP enzyme-linked immunologic detecting kit | S1 kit before the Alpha | ||
| Hepatitis B is suffered from | ??200 | ???200 | ??????192??(96％) | ???123??(61.5％) |
| Person HCV health examination | ????100 ????100 | ????0 ????0 | ????0 ????0 | ????0 ????0 |
Research in theory, hepatitis B large protein and virus replication are closely related, and therefore the existence with viral DNA should highly meet, and this experimental result shows, the present invention up to 96%, obviously improves a lot than the detection coincidence rate of S1 before the original dependence for the coincidence rate of HBV-DNA.
Embodiment 6: the qualitative enzyme-linked immunologic detecting kit of LP of the present invention is to the detection of hepatitis B great three positive sample and "small three positive" sample
The contrast of the testing result of LP testing result and five index of hepatitis b, to judge HBV virus in vivo development and the prognosis of treatment situation important directive significance is arranged.According to the virology principle, its serum HBV surface antigen large protein of hepatitis B great three positive patient must be positive; And in the "small three positive" patient, LP is positive to show that virus is in replication status, and the probability that develops into hepatitis is very big.Detect hepatitis B great three positive and "small three positive" sample respectively with the qualitative enzyme-linked immunologic detecting kit of LP of the present invention, get before the Alpha of the same type the S1 kit and do reference, the result sees Table 2 and table 3 respectively.
The different detection methods of table 2. are to hepatitis B great three positive test result of samples.
Reference reagent
S1 the present invention before the great three positive Alpha
1?????????0.419?????????0.395
2?????????2.18??????????2.664
3??????
??0.0096????????2.687
4?????????0.428?????????2.725
5?????????2.288?????????2.539
6?????????0.904?????????2.799
7?????????2.229?????????2.64
7?????????2.103?????????2.724
8?????????1.874?????????2.597
9?????????0.645?????????2.862
10????????0.824?????????2.874
11????????2.165?????????2.744
12-A??????2.05??????????2.471
12-B??????2.199?????????2.52
12-C??????2.161?????????2.306
13????????0.644?????????2.708
14????????1.505?????????1.281
15????????2.249?????????2.661
16-A??????0.192?????????2.654
16-B???
??0.08??????????2.658
17?????
??0.012?????????0.034
The result shows that two kinds of reagent testing results of most of hepatitis B great three positive sample are all positive.The diverse measurement result in two kinds of detectable at 16-A and 16-B determines with the PCR method that again find that two samples are the PCR positive, LP enzyme-linked immunologic detecting kit testing result of the present invention conforms to it, but S1 reagent omission before the Alpha.
The different detection methods of table 3. are to hepatitis B "small three positive" test result of samples.
"small three positive" Alpha the present invention
1???????0.128?????0.95
2???
??0.403?????0.092
3???????0.958?????0.35
4???????0.025?????0.015
5???????2.077?????1.714
6???????2.234?????1.957
7???????0.025?????0.028
8????
??0.103?????1.584
9???????2.241?????2.033
10???????2.113?????1.151
11????
??0.024?????0.149
12???????0.028?????0.072
13???????1.974?????0.806
14???????1.015?????0.513
15???????0.965?????0.438
16????
??0.021?????0.169
17????
??0.078?????0.062
18???????0.988?????2.415
19???????0.016?????0.047
20???????0.009?????0.022
21????
??0.013?????1.027
22???????0.022?????0.017
23???????0.296?????1.552
24???????0.341?????0.128
25???????0.216?????1.347
The result shows, LP enzyme linked immunosorbent detection reagent of the present invention detects 216 positives in 25 "small three positive" samples, positive rate 64%, S1 reagent only has 60% positive rate before the Alpha, with reference to the high coincidence rate of LP enzyme linked immunosorbent detection reagent of the present invention and HBV-DNA PCR detection, still there is the height possibility of viral massive duplication in the sample of S1 reagent omission before the Alpha.
Claims (7)
1. large protein pre-S surface antigen for hepatitis B virus district ELISA measuring reagent kit, it is characterized in that it contains the monoclonal antibody of using large protein pre-S surface antigen for hepatitis B virus district antigen immune mouse with conformation type epi-position, screening hybridoma clone and obtaining.
2. large protein pre-S surface antigen for hepatitis B virus district ELISA measuring reagent kit, it is characterized in that, it contains the monoclonal antibody of preparing by following method: with the pure product of the hepatitis b virus s antigen with conformation type epi-position that have the large protein pre-S surface antigen for hepatitis B virus district recombinant antigen of conformation type epi-position or extract from serum the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, the hybridoma clone that screening LP is special, the injection mouse peritoneal, collect ascites, monoclonal antibody purification.
3. kit according to claim 1 and 2, it also contains the LP positive, negative control, ELISA Plate, horseradish peroxidase-labeled polyclonal antibody, auxiliary reagent, it is characterized in that, and described monoclonal antibody is to be coated on the ELISA Plate.
4. kit according to claim 1 and 2, it also contains LP standard items, ELISA Plate, horseradish peroxidase-labeled polyclonal antibody, auxiliary reagent, it is characterized in that, and described monoclonal antibody is to be coated on the ELISA Plate.
5. method of making the described kit of claim 3, it preparation, monoclonal antibody bag that comprises the LP positive, negative control is by the preparation of the making of plate, enzyme mark Polyclonal Antibody Preparation and auxiliary reagent.
6. method of making the described kit of claim 4, it preparation, monoclonal antibody bag that comprises the LP standard items is by the preparation of the making of plate, enzyme mark Polyclonal Antibody Preparation and auxiliary reagent.
7. method that detects the hepatitis B replication situation is comprising each the described kit and serum or the contacted step of plasma sample that make among the claim 1-6.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108344869A (en) * | 2018-02-05 | 2018-07-31 | 苏州长光华医生物医学工程有限公司 | A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application |
| CN109988241A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of antibody and its preparation method and application of specific recognition people immune complex |
| CN110003326A (en) * | 2019-04-15 | 2019-07-12 | 郑州伊美诺生物技术有限公司 | The preparation method and applications of PreS1 Ag specific antibody |
| CN116199774A (en) * | 2023-01-05 | 2023-06-02 | 北京科跃中楷生物技术有限公司 | Monoclonal antibody for hepatitis B virus surface antigen mutant strain |
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2005
- 2005-06-23 CN CN 200510077451 patent/CN1700010A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988241A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of antibody and its preparation method and application of specific recognition people immune complex |
| CN108344869A (en) * | 2018-02-05 | 2018-07-31 | 苏州长光华医生物医学工程有限公司 | A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application |
| CN110003326A (en) * | 2019-04-15 | 2019-07-12 | 郑州伊美诺生物技术有限公司 | The preparation method and applications of PreS1 Ag specific antibody |
| CN116199774A (en) * | 2023-01-05 | 2023-06-02 | 北京科跃中楷生物技术有限公司 | Monoclonal antibody for hepatitis B virus surface antigen mutant strain |
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