CN101762688A - Experimental method for synchronously detecting relative content and absolute content of various antigen-specific associated antibodies - Google Patents
Experimental method for synchronously detecting relative content and absolute content of various antigen-specific associated antibodies Download PDFInfo
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Abstract
The invention relates to an experimental method for synchronously detecting the relative content and the absolute content of various antigen-specific associated antibodies. In the method, different types of antibodies competitively react with the corresponding antigens in the same system, the absolute content of at least two kinds of antibodies with the same antigenic specificity in the same specimen is quantitatively detected in one experiment by combining the detection method that the number corresponds to the respective content in the specimen, then the relative content between the antibodies is calculated according to the absolute content, wherein the absolute content refers to the content level of the various determined antibodies in the serum of the examinee, and the relative content refers to the relative percentage comparison of the content of the various determined antibodies in the experimental specimen. The method is suitable for classification and determination and quantitative detection of various antibodies and determination of the relative content between the antibodies, simultaneously, is also suitable for detecting other related substances with the common antigen determinant such as the material with isoform and sequential degradation characteristics, the material series thereof and the like.
Description
Technical field
The present invention relates to the sero-immunity detection range, relate in particular to the relative absolute content synchronous detection of a kind of antigen-specific associated antibodies experimental technique.This method is applicable to that also other has the mensuration of the related substances of common antigenic determinant (having material series with worker's isomery and sequential degraded feature thereof as some) when the classification qualitative and quantitative detection that is applicable to each antibody-like and relative content detect.
Technical background
The general technical background:
Antibody be called again immunoglobulin (Ig) (Immunoglobulin, Ig) be one group under the stimulation of antigenic substance, by producing in the biosome body, can with the immune molecule of corresponding antigen generation association reaction.Human antibodies comprises class five classes (and subclass) such as IgG, A, M, D and E, and inhomogeneity antibody structure, function, content, generation position and generation thereof are all distinct opportunity.This class material can be removed antigen or pathogenicity material in vivo, prevent of the again infection of pathogenic antigen to body, the visible immune response phenomenon that maybe can detect can take place with specific antigen external, thereby the specific diagnosis index as body pathogen (or some antigen) the past and existing disease are infected plays an important role in clinical diagnosis, epidemiology and preventive medicine research.Some in particular cases this antibody-like can also cause some special pathologic processes.
The clinical detection of antibody is divided into non-specific and specific antibody and detects two big classes, and the former is used for (comprise the detection of serum gamma globulin, and the detection of variety classes Ig etc.) general evaluation of immune status.Specific antibody detects the middle and later periods that the research of medical diagnosis on disease meaning was started from 18th century, and the purpose of early detection is intended to prove conclusively the infection whether person under inspection exists corresponding pathogen.Detection method comprises the ring-type precipitation, multiple agar diffusion experiment, electrophoresis experiment, aggegation experiment, complement fixation test, and adhesion and adhesion inhibition experiment etc. are multiple.These methods are subjected to multiple restrictions such as its susceptibility, specificity and stability, only can carry out preliminary qualitative or half-quantitative detection.And only gradually ripe in the development of modern detecting such as labelled immune, the detection by quantitative of antibody and classification and Detection just obtain the technical foundation of clinical practice, and are subjected to clinical and the concern epidemiological study scholar.In surplus nearest 30 year, along with the inspection technology level progressively improves enhancing day by day with national economy strength, the scope of serological specificity antibody test and intension constantly develops and expansion.Up to now, be seen in the report project at the classification and Detection of the specific antibody of same antigen and comprised IgM, IgG and IgG
1, IgA, IgA
1With IgA
2, and various dissimilar and subclass such as IgE, its testing result estimate disease process and somatotype, morbid substance invasion approach and infringement position, and corresponding antigen exists the effect of aspects such as situation all to be not quite similar at diagnosis patient pathogenesis.
In the These parameters, because time of occurrence is the earliest after accepting antigenic stimulus for specific IgM antibodies, IgG type antibody content in vivo is the highest and lasting life period is the longest, thereby they learn diagnosis implementing disease pathogen, set forth disease somatotype and process, epidemiology state and in preventive medicine research role be that other any index all can't replace.Be technical difficulty that reduces early detection and the susceptibility that improves method, the total antibody activity of serum detects (promptly not sub-category, the polytype antibody activity detects simultaneously) index and arises at the historic moment, and be used widely in clinical detection.For deeply excavating the value of antibody test in epidemic disease and preventive medicine research, the method of multiple specific IgM and IgG classification and Detection is able to successively set up, both obtain systematic research at the dynamic change of content in peripheral blood and body fluid, and the meaning of discontinuity surface ratio (relative content) change in epidemiology and preventive medicine also was subjected to a certain degree concern and evaluation when both were same.But limited by experimental technique, the detection of both relative contents can not carried out always to this.
Labelled immune is learned and is comprised radio-immunity, enzyme immunity and fluorescence immunoassay three classes thereof, all kinds of experiments comprise multiple different reactive mode simultaneously, they are in the convenience of specificity, susceptibility, stability, operation, and have nothing in common with each other on the multiple technologies features such as resistance to environmental impact.(over nearly 40 years) labelled immune learns a skill to develop and also can be divided into qualitative detection with regard to its macro development process, several Main Stage such as detection by quantitative and the detection of many index set reduction thereof, different phase technology mutual intersection the in clinical practice permeates but and not exclusively mutual the replacement.Wherein maximum with the application of two class methods in antibody test such as ELISA and fluorescence (or chemiluminescence) detection techniques.The analysis of streaming immune microsphere, protein chip technology and light-induced chemiluminescent technology thereof etc. then are typical case's representatives of modern technologies progress.Each aspect technology all has its classical separately response procedures with regard to its reaction pattern, original advantage and shortcoming, and thereby for separately development has stayed independent or cross one another space, and provide wide and diversified technology stage for the foundation of new experimental technique.
Single detection technique background is given an example:
Now be described below with regard to the anti-HBc Mark Detection of serum technical background
1. hepatitis B virus infection situation
Hepatitis B is to spread the extensivelyst in the world, and number of the infected is the infectious diseases of (more than 4.5 hundred million) at most.To the propagation of this disease, treatment has constituted maximum in the world at present public health problem with prevention.
China is one of hepatitis B virus infection the widest popular country in the world, its number of the infected is (existing disease infection surpasses 300,000,000) at most, preventative task is the heaviest, to having the greatest impact of Chinese national economy, has become the primary public health problem that country and hygiene department thereof pay close attention to the most.
2. serum hepatitis B virus Mark Detection situation
First line detects the anti-HBc of the sign HBsAg anti-HBe of anti-HBs HBeAg
Total antibody
Second line detects the anti-HBc-IgM of sign, pre S1; HBV DNA
Three-way detection indicates other antigen sign: (DNAPase; HBcAg; HBxAg; )
Other antibody sign:
Molecular biology sign: dna sequencing; Hypotype detects; Variation type detects
3. experimental index and method that anti-HBc detects
The total antibody of anti-HBc: a clinical line index
Anti-HBc-IgM: clinical two line index
Anti-HBc-IgG, G1, A, A1, A2, E: scientific research and special clinical picture are explained
The detection of anti-HBc-IgG/M ratio: in the elaboration of antagonist generation rule, once had related in early days.Do not see special detection method (the off line deduction is its main evaluation method on the basis that each monospecific antibody detects).Do not enter clinical practice, also not as formal scientific research index.
Summary of the invention
The objective of the invention is the deficiency that exists in the above-mentioned background technology, propose the relative absolute content synchronous detection of a kind of antigen-specific associated antibodies experimental technique in serological specificity antibody test field, and promote the value of serological specificity antibody test in clinical, epidemiology and preventive medicine research field thereof energetically with this.
For achieving the above object, the present invention adopts following technical scheme: the relative absolute content synchronous detection of antigen-specific associated antibodies experimental technique, this method utilize dissimilar antibody in same system with corresponding antigen generation competitive reaction, it is in conjunction with the corresponding detection method of content separately in quantity and the sample, carry out detection by quantitative at the absolute content that has the specific antibody of same antigen with at least two classes in once testing to same sample, and calculate relative content between them in view of the above, described absolute content is meant the above-mentioned determined contents level of each antibody-like in person under inspection's serum, and the relative percentage that relative content refers to above-mentioned determined each antibody-like content in experimental specimen relatively.
In such scheme, described have the specific antibody of same antigen for being present in individual serum of human or animal the infected or the body fluid, has IgM, IgG, IgA, IgD or the IgE type antibody and their subclass thereof etc. of same antigen binding characteristic.Concrete detect combination often to be confined to its content approaching relatively, and have some (two to three kinds) antibody type or subclass of special clinical and scientific value.
Described detection method is:
The relative absolute content synchronous detection of antigen-specific associated antibodies can adopt all labelled immunes to learn a skill in principle, and the experiment of streaming fluorescence immunoassay microballoon is as the technical foundation of setting forth this invention.Its testing process comprises and will be mixed with sample to be checked by the solid phase carrier of target antigen bag quilt, after treating that dissimilar antibody competitions in the same sample are attached on the immobilization antigen on the same solid phase carrier, again with the tracer antibody reaction of adopting several different material marks, on the relevant detection instrument, detect, and compare with negative control sample, positive control sample and blank sample, calculate absolute content and relative content.
Detecting instrument, reagent and equipment are fixed according to the selection of test method, are example with streaming fluorescence immunoassay microballoon technology, and described detecting instrument can be flow cytometer.
Described solid phase carrier can be a kind of size and quality homogeneous rule, and high-molecular polypeptide is had special adsorptive power, the microballoon of diameter between the 0.1-50 micron, and its application characteristic is identical with the streaming immune microsphere.
Immobilization antigen on the described solid phase carrier can be the target antigen product with full genetic recombination eukaryotic or procaryotic cell expression of purified processing, or the similar or identical albumen or the polypeptide of expression, non-full gene expression and chemosynthesis naturally of its immunogenicity.
Described tracer antibody can be anti-human IgG-FICT and anti-people IgM-PE, or is anti-human IgG-PE and anti-people IgM-FICT; The fluorescence molecule of mark is PE, FICT, PE-cy15 on its tracer antibody, or other multiple similar fluorescence molecule.
Narrate negative control, positive control, blank and the detection by quantitative positive criteria serum etc. that are used for all kinds of specific target antibody tests and conventional labelled immune to detect test (as ELISA and chemiluminescence etc.) roughly the same, the demarcation strictness of its quality of the pharmaceutical preparations and target content of material is with reference to WHO and Chinese medicine and the biological products evaluation fixed standard of issuing.
In such scheme, the concrete experimental procedure of described detection method is: solid phase carrier is assigned in each developmental tube; In the different experiments pipe, add sample to be checked respectively, the negative control sample, the quantitative measurement standard items series of positive control sample, blank sample and variable concentrations is educated the temperature reaction; In each experiment tube, add fluorescently-labeled mixing tracer antibody respectively, educate the temperature reaction, wash and it is suspended in the mensuration liquid; Adopt flow cytometer that each experiment tube is detected, draw the typical curve of all kinds of Ig according to quantitative criterion pipe testing result; On typical curve separately, find the absolute content of inhomogeneity Ig respectively according to sample detection signal to be checked; Calculate the ratio (relative content) of the content of different I g in the sample to be checked according to absolute content, and report experimental result.
The present invention adopts single antigen coated microballoon, can draw the ratio (relative content) of absolute content between them simultaneously at same sample with once detecting the absolute content that at least two classes have the specific antibody of same antigen simultaneously in the experiment; Adding two covers in same system (or more than) vary in size, the immune microsphere that envelope antigen is different, can be in same experimental specimen, through finish with experiment once at the polytype antibody of the corresponding antibody of two kinds of (or multiple) antigenic substances relatively with the analysis of absolute content, its detection intension can be doubled and redoubled as required.
Experimental facilities used in the present invention (as flow cytometer) should have powerful memory, analyze and output function by all kinds of means, can show the absolute content of several antibody simultaneously, and calculates both relative contents (%) at machine (but also off line).
Absolute content of the present invention is meant the above-mentioned contents level of antibody in person under inspection's serum of being examined, and uses antibody activity unit (international unit or milli-international unit usually.The anti-HBc of serum detects and adopts NCU/ml at present) expression, also can adopt the signal intensity that they are showed in detection, or represent it with its other metering method (as mass unit or microequivalent unit) that serve as foundation calculates; The relative percentage that relative content refers to above-mentioned determined each antibody-like content in experimental specimen relatively, or single detected antibody accounts for the ratio of its detected antibody gross activity (or content).
Effect of the present invention and advantage:
1, the present invention can solve the synchronous detection by quantitative problem of two or more type antibody well, and it detects effect and is parity with or superiority over (by in the machine corrective action) other monospecific antibody detection technique;
2, enforcement of the present invention can be support with a few class specific antibody absolute contents detections, at machine or the off-line computation relative content between them, compare with the method that previously detects off-line computation respectively, on method, greatly simplify, on result's comparability, increase substantially the practicality that it is clinical thereby significantly strengthened.
3, each antibody-like ratio of the present invention determines that be foundation to the ability of the competitive combination of solid phase antigen with them in reaction system, antibody quantity is how much irrelevant in its ratio and the reaction system, even if lack quantitative criterion accurately, under the prerequisite of relative control experiment condition, only can estimate relative content between them objectively with the intensity of detection signal each other.By introducing relative content examination criteria system (its exploitation is relevant with this patent), the also relative content of interpretation testing result intuitively.
4, adopt traditional prize law to carry out the classification and Detection of serum antibody, its susceptibility is not good enough, and is subject to the interference of rheumatoid factor in the serum; The susceptibility of the inventive method significantly improves than prize law, and can avoid the interference of rheumatoid factor effectively.
5, be support with streaming fluorescence immunoassay microballoon experimental technique and flow cytometer thereof, can make experimental technique obtain following advantage simultaneously.
1) susceptibility height: can compare favourably (be used for the anti-HBc of serum and detect, susceptibility exceeds 5.0NCU/ml) with other existing reagent (comprising chemical illuminating reagent etc.);
2) controllability that the quantitative scope of absolute value is widened and tool is suitable (utilizing this instrument signal intensity adjustments function);
3) the detection information capacity is big.Utilize the powerful information acquisition and the analytic function of flow cytometer, not only can the multiclass antibody of single antigentic specificity be detected, introduce different antigen coated and microballoons that vary in size and experimentize, also can in same serum, detect the multiclass specific antibody of several different antigentic specificities simultaneously;
6, do the classification antibody test with indirect antibody response technology (as indirect ELISA), described absolute content is several antibody-likes reacted results that vie each other, and its measured value departs from true value, and its departure degree is different.The present invention when several antibody-likes are made the absolute content synchronous detection since can synchronous detection relative content between them, for the correction of its absolute content provides foundation.Therefore, relative conventional art, this law testing result can be proofreaied and correct as required, and the more existing testing result of the measured value after the correction is more near true value.
7, detection theory proposed by the invention all has wide expanding space at aspects such as experiment material, experimental technique, detected object and ranges of application thereof.
Range of application of the present invention:
Sensing range comprises that the classification qualitative and quantitative detection of each antibody-like and relative content detect;
Absolute and relative content with related substances of common antigenic determinant detects.
The research field clinical diagnosis: disease by stages with somatotype;
Epidemiology survey; Subclinical infection, existing disease are infected, the evaluation of previous infection;
Preventive medicine research: the previously evaluation of protective capacities and existing preventive effect;
Zoology and veterinary science research: its application and above-mentioned three are together.
Patented technology is relied on: based on streaming fluorescence immunoassay microballoon technology, its application can be extended to existing
The whole labelled immunes of row learn a skill.
Using value is estimated the application that absolute content detects: same with conventional quantitative detecting method;
The application that relative content detects: with the hepatitis B virus infection is example, different infected individuals can have very big difference to the immune response intensity of viral antigen, do not propose different idea but the bulkage formula that disappears in the order that its corresponding antibodies (as all kinds of anti-HBc such as G, A, M etc.) is produced and the body both regular at present still has the author.That is to say, no matter whether there is corresponding antigenic substance in the body, in person under inspection's body the height of dissimilar antibody relative contents (%) for explain that the infected is clinical, in epidemic disease and the preventive medicine value of some situation significantly greater than mensuration respectively to this antibody-like absolute content (, being considered one of important clinical sign that infects into the existing disease of HBV) as the anti-HBc-IgM content detection of serum.Therefore, although still there is not the report that dissimilar antibody relative contents detect so far, the foundation of this class index and application, to relevant disease clinical diagnosises such as hepatitis B, the development of epidemiology and preventive medicine research will be played very strong impetus.
Description of drawings
Fig. 1 is the overlapping analysis chart of serial quality controlled serum testing result.
Fig. 2 is the analysis chart of serial quality controlled serum target content of material and detection signal dose-effect relationship.
Embodiment
Set forth the present invention clearly for more succinct, being technology carrier with " indirectly streaming fluorescence immunoassay microballoon art " in the elaboration of following embodiment, is object of experiment with the detection with the specific IgG of same antigen and relative and absolute content of IgM two immunoglobulin like protein.Reach the purpose of annotating content of the present invention by elaboration to the anti-HBc IgM/IgG of serum phase, the synchronous streaming immune microsphere of absolute content detection technique.
The particular content of embodiment 1 comprises: 1. the preparation of related antigen and antibody with and purifying; 2. the preparation of experiment reagent (microballoon preparation; The preparation of fluorescence labeling tracer antibody; The preparation of kinds of experiments standard and reference substance); 3. streaming immune microsphere analytical technology is set up and is optimized (preparation of reaction system and optimization indirectly; The setting of response procedures and optimization; The application of reagent and equipment, adjustment and development); 4. clinical verification; Test section clinical samples on the basis of the above is by existing hardware and software systems show in real time and the report testing result.
The indirect immuno fluorescent microballoon method that sets under above-mentioned prerequisite, its concrete experimental procedure are with the broad processing of microballoon (or not handling), to be assigned to then in each developmental tube; In the different experiments pipe, add sample to be checked respectively, the negative control sample, the quantitative measurement standard items series of positive control sample, blank sample and variable concentrations is educated temperature reaction and washing; In each experiment tube, add the not potpourri of isolabeling tracer antibody respectively, the same reaction and washing; Each experiment tube is detected on flow cytometer, draw each typical curve in view of the above; Calculate the absolute content that sample two classes to be checked have the same antigen specific antibody on this basis, and the ratio of both content (relative content).
In the same reaction system, introduce different fluorescein-labeled anti-human IgGs, IgM and IgA antibody, and suitably revise the routine analyzer that streaming fluorescence immunoassay microballoon detects, can carry out the detection of the absolute and relative content of the different anti-HBc antibody of three classes.Embodiment 2 is seen in the brief introduction of this method.
Embodiment 1: the anti-HBc-IgM of serum and anti-HBc-IgG antibody mutually, the absolute content synchronous detection
Experimental technique
Detect target:
Anti-HBc specific IgM of serum and IgG content, and both content ratios (being relative content).
Reagent, instrument and experiment material:
1, microballoon (or claiming solid phase carrier): special preparation, homogeneous grain diameter, the about 0.5-50 μ m of size, the solid phase adsorption materials similar of its function and streaming fluorescence immunoassay microballoon.Method for preparing microsphere is not in this patent scope.Main application is as follows:
1) blank correction microballoon (background correction, the double as microsphere volume is proofreaied and correct) bag is participated in the experiment reaction synchronously by no dried albumen.
2) experiment detects microballoon: HBcAg is antigen coated, is used for the competitiveness combination and the spike of dissimilar antibody; (above-mentioned microballoon prepares respectively, mixes in proportion, is suspended in 5～10% ratios and preserves in the liquid 0-4 ℃ of cryopreservation. and face with after before shaking up and take a sample, be diluted to 0.05-2.00% concentration, be used for experiment and detect.)
3) solid phase antigen: the complete pure product of genetic recombination HBcAg eukaryotic cell expression product;
2, standard items preparation standard product as required.The preparation method is associated with this patent.
Main kind is as follows.
1) negative control sera: from social crowd's random acquisition, its feature and conventional anti-HBc detect together;
2) anti-HBc IgG positive control serum; From the hepatitis B infected relevant people collection of mining massively, principal character and conventional anti-HBc detect the Quality Control thing with.Its preparation divides high concentration (quantitatively or non-quantitative), or two kinds of set concentration instants, and anti-HBc IgG, IgM and both relative contents thereof are controlled in set scope.
3) anti-HBc IgM positive control serum; From the hepatitis B infected relevant people collection of mining massively, it is identical that principal character and conventional anti-HBc detect the Quality Control thing, and its preparation divides high concentration (quantitatively or non-quantitative), or two kinds of set concentration instants, and anti-HBc IgG, IgM and both relative contents thereof are controlled in set scope.
3, fluorescence labeling (spike) antibody:
Mix tracer antibody, include anti-human IgG-FICT and anti-people IgM-PE, demarcate both respectively and tire, proper proportion is mixed, and cryopreservation faces the time spent dilution, (or be diluted to the state of promptly using, cryopreservation uses in the phase in effect).
4, experiment buffer system:
Sample diluent
The reagent dilution
The experiment cleansing solution
5, experimental apparatus and equipment;
1) flow cytometer (or wait research and develop special checkout equipment)
2) related experiment equipment (same) with the used experiment equipment of flow cytometry
3) proprietary experimental analysis software architecture (waiting to research and develop)
Experimental technique:
1, the anti-HBc IgM/IgG of serum relatively and the synchronous detection of absolute content
1) experiment reagent is prepared
Microballoon is prepared: it is an amount of to get the mixed immunity microballoon, adds in the experimental analysis system, and swelling 15min adopts same system to be made into 0.1% concentration, and (Quality Control is done same processing with reference to microballoon) is dispensed in each developmental tube, every pipe 50-100 μ l; In 12 hours, use;
The microballoon that solution state is preserved down, first before use mixing is quantitatively drawn, and centrifuge washing once adopts same system to be made into 0.1% concentration, is dispensed in each developmental tube, and every pipe 50-100 μ l used standby in 12 hours;
The Quality Control thing is prepared: take out needed standard items in refrigerator, Quality Control thing or control serum are put behind the equilibrium at room temperature 15-30min standby.
Sample is prepared: fresh serum, 4 degree are preserved serum, or the frozen serum of low temperature.Both need of back are put room temperature, treat to use after the temperature balance.
2) experimental implementation
(1) numbering contains the experiment tube of all kinds of microballoons;
(2) in the different experiments pipe, add test serum respectively, various criterion product, or Quality Control and each 10 μ l of contrast (positive and negative control) serum, mixing; Room temperature jolts placement (or leave standstill every 3-5 minute shake up once) 30min;
(3) add cleansing solution 0.5-2.0ml, mixing, 500g * 5min centrifuge washing 3 times returns to original volume (50-200 μ l);
(4) add tracer antibody (the anti-human IgG of enzyme labeling/IgM potpourri) 10 μ l/ pipe, the samely shake up, react and wash (3 times);
(5) add dilution to 0.2-1.0ml, shake up, can go up machine testing;
(6) go up machine testing and interpretation of result:
The negative control sample: the calibration equipment baseline is in machine memory work data;
The positive control sample: according to the bag by with non-bag by particle calibration equipment amplification coefficient,
And with store linear data and be associated.At machine memory work number
According to;
Typical curve is formulated: start typical curve and formulate program, successively to each experimental specimen
Reaction tube is measured, and measures the end back and shows at once at machine
Curve, analytic curve statistics feature, and camera carries out
Curve statistics revision (this is for being avoided great dominance error).Warp
Confirm that the back is in machine memory curve data.Detect sample for this
Interpretation of result, and keep and to do (the simulation of next laboratory reference
Curve is seen Fig. 1).
The detection of clinical samples: input detects the numbering and the clinical data of sample; Implement sample
(limit detection is guaranteed weighing of check and analysis in limited time in detection
Renaturation); Detect data in machine memory, analysis and demonstration in real time;
Signal reanalyses function: the reanalysing of data (locking is separated in the locking of data,
And reanalyse mode once again slightly);
The object of experiment result: the detection target index that this experimental project is given an example comprises that serum is anti-
HBc-IgG content; HBc-IgM content; The anti-HBc of serum
Three of IgM/IgG ratios etc.
The absolute content detection sensitivity is not less than 5.0NCU/ml; Phase
To content detection scope 0.1%～99.9%;
Report manner as a result: with the above-mentioned experimental result of the direct printout of the mode of laboratory test report.
The online output and the electronic document page way of output
Screen display mode prompting interpretation of result data.
Experimental result and evaluation
1, the detection level of anti-HBc absolute content, and clinical and EPDML meaning is with previously the related detection report is similar;
2, anti-HBc detects the detection level of relative content, is not subjected to the restriction of its absolute content testing result usually.The theoretical numerical value that its anti-HBc IgM/IgG detects is usually between 0-99%;
3, the actual detected result of anti-HBc IgM/IgG relative content detection is not seen in bibliographical information so far, and preliminary test shows following three quasi-representative situations can occur.Its value in clinical and epidemiology remains further to be studies confirm that.
1) anti-HBc IgM/IgG relative content is greater than 50%: acute infection, and existing disease is infected window phase;
2) anti-HBc IgM/IgG relative content 10-30%: existing disease is infected, and previous infection is early stage;
3) anti-HBc IgM/IgG relative content is less than 5.0%: previous infection.
4) evaluation method of experimental result diagram
(1) detection and the overlapping demonstration (see figure 1) of signals collecting series quality controlled serum detection signal.
(2) signal analysis series quality controlled serum detection signal dose-effect relationship is analyzed (see figure 2).
Embodiment 2: the anti-HBc-IgG of serum, anti-HBc-IgM and anti-HBc-IgA antibody three mutually, absolute content synchronous detection experimental technique
Detect target: the anti-HBc specific IgG of serum, A and M three's content, and between the three
The detection of content ratio (being relative content).
The anti-HBc detection technique of serum background: the same embodiment 1.
Reagent, instrument and experiment material: main contents are the same.Difference is:
Fluorescence labeling (spike) antibody: mix tracer antibody, include anti-human IgG-FICT, anti-people IgM-PE, three kinds of fluorescent-labeled antibody of anti-people IgA-PE-CY15.The three demarcates respectively and tires, and proper proportion is mixed, and cryopreservation faces time spent dilution (or be diluted to the state of promptly using, cryopreservation uses in the phase in effect).
The standard items difference: the preparation method is the same, but increases the absolute of anti-HBcIgA and relative content detection reference index in the standard items system.
Proprietary analysis software: have certain difference with embodiment 1, specifically wait to develop.
Experimental technique: the same, reach the absolute and relative content that draws the different anti-HBc of three classes after the analysis after testing.
Negative control sample of the present invention is: from serum or its preparation product of the target antibody negative patient of social crowd screening, do not contain and have and IgG, the IgM of target material specific binding capacity and IgA etc.;
Described positive reference sample is: from the positive person's of target antibody of social crowd screening serum or its preparation product, contain and IgG, the IgM of target material specific binding capacity and IgA etc.;
The quantitative reference quality controlled serum of anti-HBc IgG is: above-mentioned positive reference sample, and the content (or ability) that its IgG antibody combines with target material specificity is accurately demarcated, and with the standard items system of containing of this serum preparation of known anti-HBc IgG concentration;
The quantitative reference quality controlled serum of anti-HBc IgG is: above-mentioned positive reference sample, and the content (or ability) that its IgM antibody combines with target material specificity is accurately demarcated, and with the standard items system of containing of this serum preparation of known anti-HBc IgM concentration;
The quantitative reference quality controlled serum of anti-HBc IgG is: above-mentioned positive reference sample, and the content (or ability) that its IgA antibody combines with target material specificity is accurately demarcated, and with the standard items system of containing of this serum preparation of known anti-HBc IgA concentration;
The quantitative reference quality controlled serum of anti-HBc IgM/IgG is: above-mentioned positive reference sample, its IgM is accurately demarcated with the content (or ability) that IgG antibody combines with target material specificity, and prepare on this basis, its two antibody-like is the standard items system of different activities ratio;
The demarcation strictness of above-mentioned IgG and IgM type target antibody content is with reference to WHO and Chinese medicine and the biological products evaluation fixed standard of issuing;
Described blank sample is: do not contain the dilution buffer experimental system of target material antibody or the animal blood serum that has nothing to do with HBV host.
The raw material serum that is used for the preparation of above-mentioned contrast and standard serum, before collection the blood donor except that section H BV infects the serum marker relative positive, HCV antibody.A detection such as HIV-1/HIV-2 antibody, microspironema pallidum is all negative, and the Serum ALT measured value is roughly normal.But, must be noted that personal protection in use because of section H BV infects the sign positive.
Claims (8)
1. the relative absolute content synchronous detection of antigen-specific associated antibodies experimental technique, this method utilize dissimilar antibody in same system with corresponding antigen generation competitive reaction, it is in conjunction with the corresponding detection method of content separately in quantity and the sample, carry out detection by quantitative at the absolute content that has the specific antibody of same antigen with at least two classes in once testing to same sample, and calculate relative content between them in view of the above, described absolute content is meant the above-mentioned determined contents level of each antibody-like in person under inspection's serum, and the relative percentage that relative content refers to above-mentioned determined each antibody-like content in experimental specimen relatively.
2. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 1 experimental technique, it is characterized in that describedly having the specific antibody of same antigen, have IgM, IgG, IgA, IgD or the IgE type antibody and their subclass thereof etc. of same antigen binding characteristic for being present in individual serum of human or animal the infected or the body fluid.
3. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 1 experimental technique, it is characterized in that described detection method is: will be mixed with sample to be checked by the solid phase carrier of target antigen bag quilt, after treating that dissimilar antibody competitions in the same sample are attached on the immobilization antigen on the same solid phase carrier, again with the tracer antibody reaction of adopting several different material marks, on the relevant detection instrument, detect, and with the negative control sample, positive control sample and blank sample compare, and calculate absolute content and relative content.
4. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 3 experimental technique is characterized in that described detecting instrument is a flow cytometer.
5. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 3 experimental technique, it is characterized in that described solid phase carrier is a kind of size and quality homogeneous rule, high-molecular polypeptide had special adsorptive power, the microballoon of diameter between the 0.1-50 micron, its application characteristic is identical with the streaming immune microsphere.
6. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 3 experimental technique, it is characterized in that the immobilization antigen on the described solid phase carrier is the target antigen product with full genetic recombination eukaryotic or procaryotic cell expression of purified processing, or the similar or identical albumen or the polypeptide of expression, non-full gene expression and chemosynthesis naturally of its immunogenicity.
7. the relative absolute content synchronous detection of antigen-specific associated antibodies according to claim 3 experimental technique is characterized in that described tracer antibody is anti-human IgG-FICT and anti-people IgM-PE, or is anti-human IgG-PE and anti-people IgM-FICT; Fluorescent tag molecule adopts FICT, PE or PE-CY 15 on the tracer antibody.
8. according to the relative absolute content synchronous detection of the described antigen-specific associated antibodies of arbitrary claim experimental technique in the claim 1 to 7, it is characterized in that the concrete experimental procedure of described test experience method is: will be assigned in each developmental tube by the solid phase carrier of target antigen bag quilt; In the different experiments pipe, add sample to be checked respectively, the negative control sample, positive control sample and blank sample are educated temperature reaction and washing; In each experiment tube, add tracer antibody respectively, educate the temperature reaction; Each experiment tube is detected on flow cytometer, draw the typical curve of all kinds of Ig, and try to achieve the relative and absolute content of each antibody-like in each developmental tube according to typical curve according to testing result.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102375052A (en) * | 2010-08-17 | 2012-03-14 | 复旦大学附属华山医院 | Method for detecting IgM antibody and IgG antibody in a same system |
| CN108291909A (en) * | 2015-04-28 | 2018-07-17 | 奥菲迪亚有限公司 | Analyze analyte detection and its method |
| CN109115739A (en) * | 2018-08-06 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of hole internal calibration test method |
| CN109142743A (en) * | 2018-07-25 | 2019-01-04 | 滴准生物科技（常州）有限公司 | A kind of multinomial detection kit for exempting from liver antibody certainly |
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- 2009-06-12 CN CN200910062674A patent/CN101762688A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375052A (en) * | 2010-08-17 | 2012-03-14 | 复旦大学附属华山医院 | Method for detecting IgM antibody and IgG antibody in a same system |
| CN108291909A (en) * | 2015-04-28 | 2018-07-17 | 奥菲迪亚有限公司 | Analyze analyte detection and its method |
| CN109142743A (en) * | 2018-07-25 | 2019-01-04 | 滴准生物科技（常州）有限公司 | A kind of multinomial detection kit for exempting from liver antibody certainly |
| CN109142743B (en) * | 2018-07-25 | 2022-02-08 | 宙斯生命科技(常州)有限公司 | Multiple detection kit for self-immune liver antibody |
| CN109115739A (en) * | 2018-08-06 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of hole internal calibration test method |
| CN109115739B (en) * | 2018-08-06 | 2023-11-10 | 宙斯生命科技(常州)有限公司 | Method for testing in-hole calibration |
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