CN104991056B - A kind of Serologic detection and the method for quantitative analysis - Google Patents

Abstract

The present invention relates to a kind of Serologic detection quantitative analysis method.On semilog coordinate image, the logarithm value being target substance concentration with X-axis, Y-axis is signal intensity and signal saturation exponent, records series reference standard experimental signal S1, S2 and SSDI according to T1, T2 time point and draws the two-way dose-effect curve of S1, S2 HB and SSI curve；Use the relation of function x Yu y and SSDI in the fitting process confirmation two-way curve of HB；Determine boundary point A1, A2 and Q that functional image is analyzed, constitute compound HB two-way dose-effect curve image analysis system；According to the experimental signal Y measured by analyzed specimen, and SSDI value and Q-value result of the comparison, look into reading what corresponding curve section carried out function x, the log result looking into reading is converted into actually detected concentration and reports experimental result.The experimental technique energy antagonist concentration dependency Hook effect interference set up, and can greatly widen target substance detection by quantitative scope.

Description

A kind of Serologic detection and the method for quantitative analysis

Technical field

The present invention relates to ion vitro immunization analysis technical field, dense more particularly to one energy antagonism when test experience sample Degree dependency Hook effect interference, and the detection method of target substance detection by quantitative scope can be widened.

Background technology

Serologic detection is combined into support with the immunity of antigen-antibody, under experimental state, participates in the antigen-antibody of reaction Characterization of molecules, the molecular size of immune complex (Immune comlement, IC), quantity number and generation experimental signal thereof Ability (strong and weak) is the main contents of Serologic detection dynamics research.Serologic detection is set up early stage, scholars find In same experimental system, the detected concentration of antigen and the intensity of experimental signal are proportionate, and (dependency relation is with Y=ax+b Describe), and this phenomenon is used for the evaluation to tested substance CONCENTRATION STATE.Researcher finds subsequently, experimental signal and target The linear correlation of material concentration can only be maintained at the narrowest and small concentration range, and the quantification range that thereby results in is narrow is current blood The clear major defect learning detection technique.Latent band (including front band and rear band) phenomenon is i.e. shown, i.e. by two beyond this concentration range There is directional curvature at high-concentration and low-concentration end in the function curve that person is constituted.The feature of this curve deviation is otherwise known as Hook effect (i.e. hook effect).After a while (early years in last century), this phenomenon is done systematic research by Heidelberger etc., finds along with target The sequential rising that material concentration is lasting, experimental signal by low rising, after reaching to a certain degree again by height reduce (and finally be expected return To threshold status), the two-way curve state that its functional image lifts in technique in calligraphy state sequence on constant coordinate, sit in semilog Put on the two-way curve state of the then sequential lifting that composition is similar with normal distribution.The Crack cause of this curve state is at low concentration End depends on that the concentration of substance to be measured in experimental specimen, middle and high concentration end then depend on antigen and antibody in experimental system Both molecular ratios change, and the sequential of the immune complex molecular signal generative capacity thereby resulted in reduces.Due to this One research is most important to the elaboration of immunoreaction kineties, and late comer, just with finder's naming, will paint according to this result The functional image of system is referred to as the two-way dose-effect curve of Heidelberger, and (the two-way dose-effect curve of HB is called for short HB two-way Curve).

It should be noted that the residing epoch experiment conditions such as Heidelberger extremely limit to, the research of scholar later is also Majority is confined to follow this result and verify, experimental result relative coarseness and fail to get rid of many factors interference, thus makes The research of this great theory value does not associates with SD engineering practice.

In sum, attaining in complete current in experimental technique development, Hook effect becomes restriction serologic test technology The principal element of development, a kind of form of expression of the narrow Hook effect for broad sense of quantification range.Along with labelled immune learns a skill Being substantially improved in sensitivity, the reactive mode of China's partial solid phase immunization experiment is by classics the most in a certain amount of time Two step method is changed into one-step method (such as one-step ELISA), and in Clinical Laboratory after a large amount of popularization and applications, Hook effect causes Assay distortion even mistake to become be a relatively conventional clinical picture.Its existence seriously hampers scientific data Accuracy, and the judgement to clinical diagnosis, treatment and prognosis thereof causes obvious negative effect.If it occur that it is special in some Clinical detection as used one-step ELISA or chemiluminescence to carry out serum HBsAg screening, then can cause 50% or face above The quantitative result deviation true value of bed specimen, part strong positive specimen measured value reduces or significantly reduces, even resulting in and have individually height Spend communicable strong positive specimen and false negative result occurs.This missing inspection once occurs to screen blood donor, then certainly will cause defeated The bad clinical event of blood dependency hepatitis.

One-step method, as a kind of mode to Hook effect antagonism, can be tied by two step method immunity (such as two step method ELISA etc.) In Guo, low signal high level and false negative result are converted into high signal overflow state, but it is inclined to change this part specimen quantitative result From the state of true value, its quantification range is also expanded.

The early 1990s in last century, there is scholar that the immunoreation time in Serologic detection is ground with experimental signal growth and decline Study carefully, find that, along with the prolongation in response time, experimental signal intensity increases, and detects, variable concentrations specimen within the same intervals period The amplitude that experimental signal increases is different, and researcher finds out the optimum signal rate of increase that can define when Hook effect occurs accordingly, Do boundary's point with it and judge that determined specimen whether occurs Hook effect, and to occurring that Hook effect person is diluted resurveying, obtain Preferably result.The method consequently as judge and correct Hook effect measure enter commercialization clinical practice, achieve relatively Good social benefit；Above-mentioned immunoreaction kineties phenomenon is made further research, further by Kornel Papik etc. While the above-mentioned phenomenon of system validation, propose an antagonism Hook effect and expand the new method of quantitative analysis scope simultaneously, will It, for the detection of serum ferritin, achieves very good experimental result；The domestic Liu Zhong people etc. (2000) use identical Laboratory facilities carry out serum IgG detection, while HB two-way dose-effect curve feature is expanded on further, it was found that with The experimental signal time-dependence that Kornel Papik research duplicates changes can simultaneously appear in antigen excess and antibody excess etc. two kinds Under experimental state, so that late comer is it is reasonable to conclude that this type of Hook effect antagonism method is expected to be simultaneously used for antigen and antibody two The detection of person.

The studies above result shows, the two-way dose-effect curve of HB is objective reality during an antigen antibody reaction Experimental phenomena, although under good trystate, the various piece of its curve possesses stable dosage phase the most to some extent Close feature, but previously researcher fails to use it for the quantitative ratiometric analysis of results of serological detection always.Its reason may bag The feature including 1. previously researcher dose-effect curve two-way to HB lacks deeply understanding；2. the Functional Analysis mode previously continued to use (rectilinear regression, Y=ax+b) can not carry out Hook phenomenon process, and simple curvilinear regression is difficult to reach in integrated curved calmly Experimental precision required for component analysis；3. previously most experiments method is suitable because the reasons such as experimentation, reagent, instrument are difficult to present The preferably curve state of reference；Two target substance concentration the most corresponding with same experimental signal (Y) on two-way reaction curve Mapping value confirms the target substance concentration true value of experimental specimen；And the most how to correct the two-way curve of HB and enclose summit section because of summit Effect and the partial Quantitative Analysis precision that causes are decreased obviously.The solution of these technological difficulties is to promote the two-way curve of HB by theory Move towards the premise of actual application.

Homogeneous immunity (including the class participated in by microsphere adsorption technology homogeneously immunity) experiment refers to whole reactions and result Observing the immunological detection method all carried out in same liquid phase environment, operating process is than the letter of other type of Serologic test Clean.Immunoprecipitation (with immune agglutination) is tested, and light excites chemiluminescence immune assay to test (Light Initiated Chemiluminescence assay, LiCA), and homogeneous enzyme immunoassay analysis is the main representative of this kind of method, is also current The main homogeneous immunological technique of (or) commercialization.Serum HBsAg detection the exciting of light that such as China releases in recent years Learn luminescence experiments, there is the highest sensitivity, convenience and stability, many experiment qualities such as detection by quantitative scope also with electrification Learn the outstanding detection technique such as luminous suitable, but because being very by Hook effects relatively solid-phase immunity experimental technique, and there is not portion Divide and correct the transitional arrangement (the two step method immunization experiment etc. as in solid phase immuno-assay) of Hook effect and cause and in a large number cannot Quantitatively and wrong quantity result, the deserved market share can be occupied the most far away, on the contrary with the conventional art one such as one-step ELISA Act the ranks of the application such as the scale market that is excluded from blood donor's screening.

Therefore, this area needs are a kind of is intended to correct Hooks effect and expand the easy to be easy of Serologic detection quantification range The Serological Detection method of row.

Summary of the invention

The purpose of the present invention is to propose to a kind of serology detection by quantitative and analyzing novel methods.The method is with homogeneous immunity or class Homogeneous immunological technique is support, and the technical characteristic of the energy original Serologic test of comprehensive holding can be used for the inspection of antigen or antibody Survey, antagonist concentration dependency Hook effect can disturb, and greatly widen target substance detection by quantitative scope.

Above-mentioned purpose is achieved through the following technical solutions:

A kind of Serology test being intended to correction Hook effect and expanding Serologic detection quantification range, including as follows Step:

1, experimentation: generate phase with unmarked corresponding immune response reagent (antigen and antibody) and signal there is labelling Close the experimental port (pipe, or other carrier containers) of reagent is separately added into reference standard, matching sample, yin and yang attribute comparison or Specimen to be measured, places after mixing, to bring it about immunoreation, and produces the experimental signal being available for measuring, in occurring immunity anti- T1, T2 time point answered carries out the detection of experimental signal S1, S2 respectively；It is inputted special Experiment Analysis System to process, and Output experimental result is instructed according to experimenter；

2, the matching of functional relationship:

1) ibid under experiment condition, utilizing more than 100 concentration point samples, concentration range sets and contains the two-way curve of HB and have Effect analyzes section image, and concentration ranges is set as between particle being spaced 0.025 0.10 transverse axis (X-axis) reading distance, to this reference Standard substance carry out 10 times or repeat experiment above, and the experimental signal meansigma methods taking each the detection of each concentration point is corresponding as this point Experimental signal carry out high density Function Fitting analysis；

2) the experimental signal input artificial intelligence analysis's system that will be generated, the path, backstage of artificial intelligence analysis's system is general Condition:

1. experimental signal value S1 and the S2 of the detection of T1 and T2 two time point are inputted；

2. calculate by inspection specimen T1 and the ratio of T2 time point S1 Yu S2, including experimental signal saturation etc.；Experiment is believed Number saturation is converted into saturation exponent (Signal Saturation degree index, SSDI), and conversion method is (S1/ S2) × 100 × e, e are conversion coefficient；

3. on plane semilog coordinate image, according to series reference standard measure acquired in experimental signal S1 and S2, draws out two two-way dose-effect curves of HeidelBerger of S1 and S2 respectively, and by saturation exponent according to the most right Answer target substance concentration to mark and draw successively in aforementioned semilog function image, draw out corresponding signal saturation exponent curve SSI, These three curves constitute compound HB two-way dose-effect curve image analysis system, and wherein Y-axis is experimental signal intensity or SSDI, X-axis is the logarithm numerical value of target substance concentration；

4. the relation of function x Yu y (and SSDI) in the fitting process confirmation two-way curve of HB is used.

By matching it is experimentally confirmed that in HB two-way dose-effect curve image the eyeball (y) on the two-way curve of each HB with Relation between its corresponding functional value (x) can use below equation to state:

$f\left(x\right)=a\×esp(-(\frac{x-b}{c})^2),$ Fitting effect R^2 >=0.99

Wherein x: for the natural logrithm of target substance concentration；α: corrected parameter；B: logarithmic average；C: standard deviation；F (x)=reality Test signal (y)；

In HB two-way dose-effect curve image, (SSDI limits two HB to the upper each eyeball of signal saturation exponent curve (SSI) Point value inside two-way curve image) and and corresponding functional value (x) between relation below equation can be used to state.

F (x)=a+b × x, fitting effect R^2 >=0.95

Wherein x: for the natural logrithm of target substance concentration；α: intercept (as x=0, the size of the value background signal of Y)； B: slope (the increase and decrease amount of Y when x changes a unit)；

Y=f (x)=SSDI=(S1/S2) × 100 × e (e is conversion coefficient)；

3, (matching) functional image analyzes the determination of boundary's point

1) calculating summit P1 and P2, formula: P (y)=f (the x)=μ of the two two-way curves of HB, wherein, μ=b is (see front curve Regression formula), i.e. the logarithmic average of the model parameter that experimental data simulates, for P1 or P2 at the log concentration of vertex correspondence Value；

2) two summits midpoint Pe (formula: Pe (x)=(P1 (x)+P2 (x))/2) in X-axis reading difference is confirmed, through Pe point X-axis is vertical line M, and with S1, S2 and SSI tri-curve intersect at A1, A2 and Q respectively, calculate respectively three experimental signal (y, Or SSDI) value；

3) programming is fixed to set up the homogeneous immune detection of serology with the two-way dose-effect curve of compound HB as reference subject Component analysis system；

4, concentration reference standard and setting

Set when time conventional reference standard substance, based on the two-way dose-effect curve of HB that step 2 is set up, its concentration With substantially reach two-way curve following linear position as target, concrete setting includes: 1. background verification reference, 0 target substance, threshold Value sets reference；2. sensitivity control reference, low concentration end, three order derivatives of curve normal distribution enclose starting point；The most linear State control, near the flex point of left and right, normal distribution curve enclose second dervative starting point；4. Y value height point control, normal distribution is bent Line enclose first derivative starting point (i.e. enclosing summit) place；5. class Hook effect control point set, its concentration set with generations test It is corresponding that signal controls experimental signal produced by reference product with sensitivity in intensity；

5, interpretation

1) by the detection of concentration reference standard, the input of data, recall suitably when time interpretation of result, i.e. recall and be corrected Abovementioned steps 2,3 set up data analysis system；Two-way for S1 curve asymmetric can be divided into two, left and right by A1 circle point Point, summit P1 is positioned at the left side of boundary point A1, and wherein the high x value section in right side is experimental result interpretation section；Can be by by A2 circle point Being divided into left and right two parts, summit P2 to be positioned at the right side of boundary point A2, wherein the low x value section in left side is real the two-way curve asymmetric of S2 Test result interpretation section；Lock experimental specimen to be analyzed actual measurement by the input of experimental specimen sequence number and calculate data；By surveying The comparison calibrating this SSDI and Q-value determines curve and the respective section of specimen ratiometric analysis to be measured, i.e. as the SSDI measuring specimen During more than Q-value, look into reading experimental result according to its T2 time point measured value y at the left side section of S2 curve；When measuring specimen SSDI less than Q During value, look into reading experimental result according to its T1 time point measured value y at the right side section of S1 curve；

2) in the data analysis system that the abovementioned steps 2,3 using input reference standard detection data to be recalled is set up Threshold value is to above-mentioned 5,1) item analysis result is further analyzed, and its experimental specimen S2 measured value is less than homologous thread threshold value (negative hole Average is multiplied by 2.1 times) person for feminine gender；

3) in the data analysis system that the abovementioned steps 2,3 using input reference standard detection data to be recalled is set up Class Hook effect control point is to above-mentioned steps 5,1) item analysis result is further analyzed, and its experimental specimen S1 measured value is less than right Answering class of a curve Hook effect control point reference signal person is the experimental specimen that there is class Hook effects, and this specimen target substance is dense Degree is higher than class Hook effect control point, and it is positive that its result is reported as ultrahigh concentration with qualitative fashion, and quantitative analysis results is by following Step 5,4) provide, only for reference；4) after above-mentioned selection analysis, according to the experimental signal Y measured by analyzed specimen, in phase What the curve section answered carried out function x looks into reading, the log result looking into reading is converted into actually detected concentration and reports experimental result. The composition flow process of compound HB two-way dose-effect curve image analysis system is shown in Fig. 1.

Preferably, this serology quantitative detecting method refers to homogeneous immunity, and class is the most immune, and other antigen, antibody And the three such as experimental signal generation system is stored in same experimental system, more than secondary experiment letter can be implemented within certain period Number detection, and immunoreation process in not obstruction system, do not affect the original detection of relied on experimental technique (i.e. conventional sense side Method) experimental technique of result quality.

Preferably, the specimen to be measured described in this serology quantitative detecting method means needs to carry out containing of immune detection Or without the solution of target substance (antigen or antibody), as serum, body fluid, culture supernatant and other comprise the reality of antigen and antibody Test material.

Preferably, the reagent employed in this serology quantitative detecting method include immunoreagent, signal generate reagent, Connect in reagent, mux--out signal exhibits and strengthening reagent one or more.

Preferably, in the Setup Experiments of this serology quantitative detecting method, the setting of its response time is depended on being relied on The process of experiment immunization reaction, dominant response time point is set as: the immunity that T0 time point, i.e. antigen and antibody specify at experimental technique Mix and start the time point of immunoreaction process under reaction condition；T1 time point, carries out experiment letter for the first time in immunoreaction process Number detection time point；T2 time point, in immunoreaction process, second time carries out the time point of experimental signal detection；The immunoreactive period It is planned to the T1 period: T0 to T1 time point institute interlude section；The T2 period: T0 to T2 time point institute interlude section；Immunity is anti- The total Period Length answered is otherwise varied because experimental technique is different and requirement to experimental result is different, and its scope is limited to Between 2-60min, the ratio of T1 Yu T2 Period Length is 0.1 to 0.95.

The technical program is in semilog coordinate image, and the signal growth and decline track of HB two-way dose-effect curve image is sequence The two-way change passed through, the dosage that experimental signal intensity is smooth to the function curve of target substance concentration change is relevant.It is right half Track on number coordinate shows as significant normal distribution image.Employing curvilinear regression processes, the two-way dose-effect curve of this HB On image, the functional value corresponding to each eyeball can use formulaMatching, and obtain fabulous Fitting effect (R^2 >=0.99).In actually detected, after obtaining specimen measured signal (y) to be measured, by above-mentioned formula, Interior (in addition to enclosing summit section) the arbitrary signal intensity experiment specimen of class Hook effect point on two-way curve can be obtained exactly Target substance concentration.This is found to be and two-way for HB dose-effect curve is used for Serologic detection quantitative analysis provides theoretical base Plinth.

In immunoreaction process experimental signal intensity time-dependence change main feature: in given period of time experimental signal with Response time extends and strengthens；Ratio between different time point signal intensitys is relevant to experimental specimen target substances concentration；When two The dependency relation of some measured value can be expressed with multiple manner of comparison, wherein experimental signal saturation index will be converted into saturation (Signal Saturation degree index, SSDI, conversion method in embodiment 1 is (S1/S2) × 100 to index ×10000).And be plotted in semilog coordinate image, it is found that along with the rising of target substance concentration, its curve (Curve OfSignal Saturation degree Index, SSI, signal saturation curves) image be the sequential of right low left high Downward trend.This trend is especially apparent at image core section (image-region between the most two-way curve two flex point).Through formula f (x)=a+b × x matching, fitting effect R^2 >=0.95.

Based on above technical characteristic, the technical program proposes compound HB two-way dose-effect curve image analytical method first. This method image is by two HB pairs drawn according to different time point measured values acquisition data (T1 and T2 time point, S1 and the S2 signal recorded) To dose-effect curve (S1 curve and S2 curve), a SSI curve, according to other boost line needing to add of quantitative analysis Section and boundary's point etc., its composition can be summarized in Fig. 1 mode with analysis path.The enforcement paving being established as this patent of this analysis system Put down road.

The present invention has a following innovation relative to prior art:

1, propose two-way for HB dose-effect curve as the reference subject of Serologic detection quantitative analysis；

2, by using semilog method to process, make the two-way dose-effect curve of HB by original straight-arc hybrid mode It is changed into simple curve mode.The ratiometric analysis of the two-way dose-effect curve of HB introduces curvilinear regression, and replaces with this Traditional rectilinear regression reference, in mathematical analysis, the foundation for this analysis mode provides theoretical basis；

3, the time dependence feature of experimental signal is introduced the compound two-way dose-effect curve of HB and analyze system, solve When making reference with single two-way curve of time point, " false mapping value " interference to experimental result (true value)；

4, the summit of two two-way dose-effect curves of HB in reference image is utilized to occur that the phenomenon that time-dependence swings is avoided Enclose the interference to experimental result of the summit effect, thus promote the experimental precision of quantitative analysis results on the whole；

5, set with reference to product target substance concentration according to the architectural feature of normal distribution three order derivative, for using the two-way curve of HB During reference method quantitative analysis, the preparation of reference standard provides theoretical support；

6, the setting of class Hook effect circle point, defines the analysis upper limit of experimental technique the most theoretically, again for find because of Occur that containing high concentration target substance the specimen that quantitative accuracy reduces provides possible.

7, two the two-way dose-effect curves of HB gathering different time points implement matching respectively, piecewise fitting and difference thereof Application, provides important guarantee for promoting the precision of quantitative analysis results.

Therefore, the present invention is not increasing substantive operations, on the premise of not affecting institute's basic quality of support method, it is possible to reach To correcting Hook effect, and significantly expand the target of the detection by quantitative scope of relied on experimental technique.

Accompanying drawing explanation

Fig. 1 constitutes block diagram for compound HB two-way dose-effect curve image analysis system core；

Fig. 2 is the first compound two-way dose-effect curve of HB of BA conjunction type many time points LiCA serum HBsAg detection；

The multiple different time points of Fig. 3 carry out the compound HB two-way dose-effect curve image of signals collecting acquisition；

Fig. 4 is that rear BA combines the compound two-way dose-effect curve of HB of LiCA serum Anti-HBc Serum detection；

Fig. 5 is routine immunization precipitation experiments standard curve schematic diagram；

Fig. 6 is the compound two-way curve of HB of serum human IgG time dependence many time points cotton-shaped immunoprecipitation detection；

Detailed description of the invention

By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement provided Example is only the explanation to the inventive method, and limits remaining content that the present invention discloses never in any form.

One, each term used herein is explained as follows:

1, antigen, antibody dosage-response curve (are also called the two-way dose-effect curve of Heidelberger, are called for short HB double To curve):

By Heidel berger in finding at the beginning of last century, under set antibody concentration, i.e. do immunoprecipitation experiment, precipitate Amount increase and proportional increase along with target antigen concentration, when after the increasing to a certain degree of antigen concentration, produced precipitate Quantity be not further added by, then reduce, eventually pass back to threshold status, the functional image of produced signal on constant coordinate in partially The two-way curve state of cutting edge of a knife or a sword state sequence lifting, then constitutes the double of the sequential lifting similar with normal distribution on semilog coordinate There is certain difference in the curve representation under curve state, different experiments state.The Crack cause of this curve state is at low concentration Section depends on the concentration of substance to be measured in experimental specimen, and middle and high concentration Duan Ze depends on antigen and antibody in experimental system Both molecular ratios, and the sequential reduction of the immune complex molecular signal generative capacity thereby resulted in.Use sensitivity Higher test method (such as LiCA) can make this change embody the most notable.Owing to immunoreation is moved by this research The elaboration of mechanics is most important, and the functional image drawn according to this result just with finder's naming is by late comer The two-way dose-effect curve of Heidelberger (the two-way dose-effect curve of HB).

2, Hooks effect:

Hooks effect (Hook effect, hook-shaped phenomenon) is relevant to " the band effect " in previously Serologic detection, is one Plant amount (target substance concentration) effect (detection signal) segregation phenomenon being present in multiple Serologic detection.It shows as working as reactant Being target substances, after being increased to a certain degree including the concentration of antigen or antibody, produced detection signal is at its function coordinates Can not extend further according to its past proportionate relationship on image, and be an off the linear relationship trend of original curve and show The experimental phenomena of directional curvature.Use previously quantitative assay, in this phenomenon showing as in two step method immune detection, High or ultra high concentration specimen mistake in detection by quantitative quantitatively (clear and definite quantitative result, i.e. overflow phenomenon can not be reported) state； In one-step method immune detection, its testing result occurs inspection at higher concentration status and appearance for significantly bending because of standard curve The dose-effect surveying result separates, and when its concentration raises further, then shows quantitative and the wrongest (the low letter of mistake of testing result Number high value state), false negative result even occurs.

3, homogeneous immunological technique:

Be a kind of synchronize in same experimental system or sequential carry out immunoreation, experimental signal forms reaction and experiment The immunological experiment technology of the multinomial experimentation such as signal detection.

4, solid-phase immunity technology:

Immune material (including other reagent) at least absorption participating in reacting is on solid phase carrier, and its immunity combines Reaction occurs between liquid phase and solid phase, and product is attached to the surface of solid phase carrier, tests the centre of reaction and completes rank Section needs to carry out product and separates with unreacting substance.Operating relatively cumbersome is the major defect of such method.

5, the homogeneous immunological technique of class:

Microsphere supported application gives part solid-phase immunity and tests with new technical characteristic.Immune agglutination experiment is this kind of The representative of special solid-phase immunity experiment.This experiment participates in the immune material (such as free antigen) of reaction and is respectively distributed to liquid phase and consolidates Phase carrier (latex particle, blood cell, or other particulate matter) surface, its immunoreation through liquid phase transhipment and at solid phase carrier Surface is carried out, and this process meets the technical characteristic of solid-phase immunity reaction.But experiment reaction centre or the stage of completing need not into Row product separates with nonreactant.Additionally, due to the detection of experimental signal can in above-mentioned entire reaction mass simultaneously Carry out under the state existed, not only make experimental implementation significantly simplify, also repeatedly weigh for the different time points within the same period Complex signal detection provides possibility.These features are identical with the Partial Feature of aforementioned homogeneous immunization experiment.

Owing to being provided simultaneously with the double characteristic of homogeneous immunity and solid-phase immunity experiment, such method in classification obviously between Between solid-phase immunity and homogeneous immunity.But because the homogeneous immune characteristic of its part can provide bigger sky for such technique extension tested Between, it is directly referred to " homogeneous immunization experiment " by many scholars.Applicant then admits some scholars " the homogeneous immunity test of class " Statement, and think that this statement can more objectively highlight the technical characteristic of such method.

6, summit effect is enclosed:

On the two-way curve of HB around summit and near curve section, the camber line constituted due to this section each point with Transverse axis is the most parallel, and position (point) this phenomenon closer to summit is the most obvious, uses this segment curve to carry out experimental result Reference quantitative analysis, other section of two-way for substantially less than HB curve, the impact thereby resulted in referred to as are enclosed by its quantitative accuracy Summit effect.

7, summit section is enclosed:

Use HB two-way curve ratiometric analysis time enclose summit effect as previously mentioned.Occur when ratiometric analysis significantly enclosing top The curve section of point effect is referred to as enclosing summit section.This segment curve is for the ratiometric analysis to experimental result, its quantitative accuracy It is substantially less than other section of the two-way curve of HB.

8, T0:

T0 refers to add whole immune response reagent and specimen in experimental system, blended and when starting to occur immunoreactive Point, the background signal that experimental signal is this experiment that this time point obtains.

9, T1 time point and the T1 period:

T1 refers to the time point gathered for the first time experimental signal of quantitative analysis.Its immunoreactive time is from T0 to T1 Time span between two time points, this length is referred to as the T1 period.

10, T2 time point and the T2 period:

T2 refers to the time point gathered for the second time experimental signal of quantitative analysis.Its immunoreactive time is from T0 to T2 Time span between two time points, this length is referred to as the T2 period；

11, S1 signal and the two-way curve of S1:

S1 is each experimental specimen experimental signal value obtained by T1 time point measures.At T1 time point according to the two-way curve of S1 The HB that acquired series concentration standard reference product experimental signal intensity level is drawn on the two-way curve image of compound HB is two-way Dose-effect curve.

12, S2 signal and the two-way curve of S2:

S2 is each experimental specimen experimental signal value obtained by T2 time point measures.At T2 time point according to the two-way curve of S2 The HB that acquired series concentration standard reference product experimental signal intensity level is drawn on the two-way curve image of compound HB is two-way Dose-effect curve.

13, signal saturation exponent (SSDI) and saturation exponent curve (SSI):

A certain experimental specimen or set concentration reference product series are detected at different time points, with high signal measured value S2 is Denominator compares and the S1/S2 ratio obtained is converted into percent (being signal saturation), then be multiplied by respectively suitable Transformation ratio (e, in embodiment 1, e is 10000), obtained numerical value is signal saturation exponent (Signal Saturation degree index, SSDI).It is referred to as signal according to the curve that this index and transverse axis respective function x are drawn Saturation exponent curve (SSI).

14, compound HB two-way tracing analysis system:

A kind of HB two-way tracing analysis image mode set up for adapting to this patent mode.It includes with experimental signal real Actual value is vertical coordinate, and the logarithm of target substance concentration is one group of function coordinates system of abscissa；Article two, detect time point according to difference Experimental signal value acquired in T1, T2 detection and the two-way dose-effect curve of HB drawn, the i.e. two-way curve of S1, S2；Glycine tomentella Signal saturation exponent curve (SSI) that the number of it is believed that saturation exponent SSDI draws with each self-corresponding target substance concentration, at song The low target substance concentration section of line, SSDI index is higher, along with gradually rising of target substance concentration, its signal saturation exponent curve Gradually reducing, this variation tendency is as a complete unit in smooth and slightly S shape image, the stage casing inside two-way curve image The linear trend of (between two flex points of the most two-way curve) curve meets Y=a+b × x rule.It is specifically shown in Fig. 2.

15, summit:

The peak of experimental signal intensity, referred to as summit on the two-way curve of HB.In the compound two-way curve image of HB, S1 and S2 The summit of two two-way curves is respectively P1, P2.

16, summit dancing:

In the two-way curve image of compound HB, gather, according to different time points, two two-way songs of HB of S1 Yu S2 that signal is drawn The summit of line not at same on the vertical line of transverse axis, corresponding function x (the target substance concentration on transverse axis) is also different, by This causes hump to present left and right along with prolongation or the shortening in response time, and (i.e. the low concentration end of transverse axis target substance is with highly concentrated Degree end) phenomenon that swings is referred to as summit dancing.Within the reaction period that same experimental system is set, the width that its summit swings Degree depends on the gap length of two curve signal acquisition times.It is specifically shown in Fig. 2 and 3.

17, boost line M:

Determine summit (P1 and P2) and both projection values on transverse axis of the two-way curve of HB, confirm the X of two subpoints The midpoint Pe of axle reading difference, is boost line M through Pe point vertical line done to X-axis, and boost line M is because of itself and transverse axis and S1, S2, SSI Deng intersect and produce the important analysis circle points such as Pe, A1, A2 and Q, be also called boundary's point lifting-line.It is specifically shown in Fig. 2.

18, A1:

In compound two-way curve image, boost line M is referred to as A1 with the intersection point of the two-way curve of S1, and this point is by two-way for S1 song Line is divided into left and right two parts, and curve right part without summit and enclose summit section, effectively judges section as S1 curve Analyze experimental result and can avoid enclosing the interference of summit effect, be specifically shown in Fig. 2.

19, A2:

In compound two-way curve image, boost line M is referred to as A2 with the intersection point of the two-way curve of S2, and this point is by two-way for S2 song Line is divided into left and right two parts, and curve left part is without flex point and encloses summit section, as effective interpretation section of S2 curve Analyze experimental result and can avoid enclosing the interference of summit effect, be specifically shown in Fig. 2.

20, Q:

In compound two-way curve image, boost line M is referred to as Q (being also called boundary point Q) point with the intersection point of SSI curve, should Point is to discriminate between determined specimen experimental result interpretation curve and boundary's point of curvilinear portion.According to determined specimen SSDI value and Q-value Be relatively classified as two groups, SSDI value looks into reading experimental result, SSDI value according to S2 measured value more than Q person on the left of the two-way curve of S2 On the right side of the two-way curve of S1, look into reading experimental result according to S1 measured value less than Q person, be specifically shown in Fig. 2.(transverse axis functional value between relevant boundary point Relation: Pe (x)=(P1-P2)/2=Q=A1=A2)

21, first BALiCA and rear BALiCA:

Current commercial LiCA introduces Biotin-Avidin system (BAS) in experiment reagent system, its object is to Strengthen speed and the stability of experimental signal generation that immune material combines.The intervention of BAS makes the experimentation of LiCA contain Two main biologicallies such as antigen-antibody combination and biotin antibiotin combination.Theoretically, both reactions can Same system synchronizes occur, it is possible to the sequential generation because of the difference of addition sequence.In actual applications, we will contain respectively Biotin is pre-mixed before immunoreation with the reagent of antibiotin, allow its occur BA receptor association reaction, be subsequently adding by Detection material carries out the most first BALiCA of immunoreactive experiment method, and will be initially charged immunoreagent and (contain for biotin mark The immunoreactant of note) do immunoreation with tested substance, then add antibiotin labelling microsphere and carry out BA combination BA immunoreation after the LiCA experiment referred to as of reaction.Many time points detection LiCA is simultaneously suitable for above two experiment reaction side Formula, can obtain roughly the same implementation result, but be many because multi-period reaction and multiple signal detect time-consuming more conventional LiCA, and Being implemented in of first BALiCA reacts in the arrangement of period BALiCA after relatively can provide bigger space for the application method.Above-mentioned point Other receptor response suitable and antigen antibody reaction simultaneous experimental technique time similar.

22, true value and false mapping value

When using two-way curve to carry out reference, each specimen is bent in correspondence at the single experimental signal acquired by certain time point All there are two target substance concentration values in mirrored state and the most totally different on line image.Applicant can represent target substance The numerical value of concentration is referred to as target substance concentration true value, and another numerical value is referred to as " false mapping value ".

[embodiment 1] time dependence point analysis of many times method excites chemiluminescence (LiCA) (time the most at light Point LICA) application (first BA conjunction type LICA experiment) of detection HBsAg in serum

(1) experiment material

1. instrument and consumptive material

1) experimental apparatus: LiCAHT light-induced chemiluminescent detector, rich sun biological (Shanghai) Science and Technology Ltd. of China system Make.

2) artificial intelligence's data handling system: Homebrew processing system.

3) experiment consumptive material: ibid provided by biological (Shanghai) Science and Technology Ltd. of rich sun of China.

2. experiment reagent and specimen

1) LiCAHBsAg detectable is produced by biological (Shanghai) Science and Technology Ltd. of rich sun of China, commercially available acquisition.Bag Include:

Reagent 1:R1.Anti-HBs is coated luminous microsphere；

The biotinylated anti-HBs of reagent 2:R2.；

Reagent 3:R3. is coated the photosensitive microsphere of streptavidin (SA)；

Reagent 4: experiment dilution system, commercial reagent system is carried；

2) reference standard

1. self-control reference product stores liquid: in self-infection person's serum, (density－gradient centrifuga－tion method, BSA reference spectrum absorbs purification Standard measure), concentration 7.62mg/ml, put-30 DEG C frozen.

2. reference product is made by oneself: this extract uses experiment dilution system to adjust concentration (2400.0 μ g/ml), through concentration calibration Rear dilution, appropriate subpackage ,-30 DEG C frozen, and for indoor standard reference, and recovery experiment specimen uses.Reference product concentration and quantity Set rule curvilinear structures two-way with HB and fit approach associate the comfort level (table 1) of also reference Clinical detection, specifically Concentration is shown in Table 2.The setting of recovery experiment specimen is with reference to table 3.

3. commercial reference product: commercially available LiCA test kit with band, totally 6 parts.HBsAg concentration is respectively as follows: 0.0ng/ml, 0.6g/ Ml, 4.6ng/ml, 18.3ng/ml, 200.0ng/ml, 500.0ng/ml (upper limit of quantification 500ng/ml).

3) control serum

Negative control and positive control serum: commercial reagent box carries, confirm for thresholding, and reagent reacting is evaluated.

4) detection specimen: serum, blood plasma, body fluid or In vitro culture thing.

3. artificial intelligence analysis's system

" compound HB two-way dose-effect curve image method analyzes system ", its macroscopic view logical framework is shown in summary of the invention.For The special intelligent analysis system of serum HBsAg detection builds and also completes (to see below analysis system in the framework of above-mentioned analysis system Build).

(2) experimental procedure

1. Preparatory work of experiment:

Take out all reagent and specimen, recover to room temperature, on-demand adjust to working concentration.

2. experimental implementation

1) taking reagent R2, R3 equal-volume, mixing, room temperature places 15min；

2) add in the LiCA experiment each hole of Sptting plate, every hole (pipe) 200 μ l；

3) R1 (100 μ l/ hole (pipe)) is added, mixing；

4) taking specimen to be measured, positive and negative compare, and each 25 μ l of HBsAg quantitative criterion serum serial, add to LiCA respectively anti- Answering in each hole of plate, every part of specimen adds a hole；

5) combined experiments system, puts on LiCA luminometer, educates temperature 15min (this time point is T1).(note eliminating Immunoreation time difference between each hole, lower same)；

6) excite at 680nm, under the conditions of 610nm wavelength detecting, detect each hole experimental signal (S1), and by defeated for each signal value Deliver to artificial intelligence's data handling system；

7) continue to place 15min (reaching T2 time point, 30min after i.e. specimen mixes with experimental system)；

8) putting on LiCA luminometer, 680nm excites, 610nm wavelength detecting each hole experimental signal (S2), and by signal It is delivered to artificial intelligence's data handling system；

9) according to experimenter's instruction, above-mentioned experimental signal is comprehensively analyzed by artificial intelligence system, and report detection knot Really.

(3) Experiment Analysis System builds and application

1, the building process of special target substance (i.e. HBsAg) testing and analysis system

1) utilizing more than the serial HBsAg dilution standard reference product of 100 concentration point, concentration range setting is contained whole HB two-way curve effective image, the concentration ranges of sample is set as between particle being spaced 0.025 0.05 transverse axis readings (X-axis), Carrying out 10 times or above repetition LICA detection, the experimental signal meansigma methods taking each the LICA detection of each concentration point is right as this point The experimental signal answered；

2) in whole reaction system mixed different time points (time point of immunoreation experience, T1 and T2) test experience letter Number (S1 and S2)；

3) experimental data is included artificial intelligence analysis's system in, curve simulation program in startup system, carry out common and high Density particle Function experiment reference curve image simulation (one or many, tie mutually with dividual simulation by overall simulation, separation simulation Close).And form the interpretation system for serum HBsAg detection accordingly；

4) use height Quality Control thing of tracing to the source, with same upper type, above-mentioned curve is simulated verification (available low-density matching)；

5) selectively this program is used for different regions detection unit, is verified by concrete field experiment and revise Analysis system.

2, compound HB two-way dose-effect curve image method artificial intelligence analysis's systems soft ware data handling path:

1) obtain also experiment with computing result, obtain tri-groups of data of S1, S2, SSDI；

2) application experiment reference product detection data input, recalls and stores experimental system in straightener, constructing and be suitable for working as The compound HB two-way dosage analysis curve image that secondary experiment uses analyzes system；

3) by dose-effect curve two-way to HB and the application of curvilinear regression analysis method thereof, for preventing of Hook effect And notable expansion of quantification range provides effective quantitative analysis space；

4) the two-way curve of integrated application S1, the two-way curve of S2, and SSI curve, the analysis result of relation between three, confirm inspection The curve section that reading is used looked into by mark this target substance concentration (true value), solves the mapping value in two-way curve ratiometric analysis with this The phenomenon interference to interpretation.

5) experimental phenomena of application apex offset (or claim summit drift) solves to enclose the partial section that summit effect causes and (encloses Summit section) accuracy of quantitative analysis decline problem.(concrete grammar is as described in schematic diagram 2)；

6) utilize class Hook effect to sentence a little, identify the specimen that on the right side of curve, low signal section quantification analysis precision declines (this phenomenon only occurs in the experimental specimen of the rarest superelevation target substance concentration)；

3, the image method of artificial intelligence system software path shows

The elementary path of this System Operation can be shown by compound HB two-way dose-effect curve image.It is specifically painted Method processed can brief and comprehensive summary be: 1. inputs and processes total data, draws out three curves (S1, S2, SSI)；2. confirmed by calculating The summit of S1 and S2 curve；3. under artificial intelligence software guides, draw N and M boost line；4. according to boost line M and three curves Intersection point location A1, A2 and Q 3 point, and confirm its function point value (y and x)；5. with these 3 point values for reference to determining that result is looked into Reading area section, and look into the target substance concentration reading detected specimen.Above-mentioned boundary point is all to be calculated by analytical function method to try to achieve.

4, interpretation Technology Ways

1) input experimental specimen sequence number, artificial intelligence system is automatically looked into and is read detected specimen when the signal detection in time experiment Strength S 1, S2, calculate its SSDI, and it compared, to determine experimental result with when the selected Q circle point value of time analysis system Image analysis system looks into reading area section；

2): result is looked into reading (calculating) section and accepted and believed standard:

SSDI value is more than Q-value: look into reading testing result according to T2 time point signal intensity in the left side of S2 two-way curve A2 circle point；

SSDI value is less than Q-value: look into reading testing result according to T1 time point signal intensity on the right side of S1 two-way curve A1 circle point；

3) judgement of yin and yang attribute result

It is that boundary's point judges with threshold value (i.e. blank well experimental signal × 2.1), is negative less than this value person；Higher than this value person it is The positive, positive must do the quantitative analysis of target substance concentration.

4) quantitative analysis of positive findings

According to respective determination data, (experimental signal y), uses equation to calculate (being performed by the system of analysis) tested calibration This specify on two two-way curves of S1 and S2 look into target substance log concentration in reading area (i.e. respective function x of experimental signal Y, Computing formula:Computational methods are set up with reference to the system of analysis, and are reversed.

5) x function that is acquired and that accept and believe (the logarithm numerical value of concentration) is converted to target substance concentration value (ng/ml), and root Output experimental result is instructed according to experimenter.

(4) experimental result

1, serum HBsAg detection time dependence many time points light excites chemiluminescence to test (hereinafter referred to as many time points LiCA) Experimental result quantified system analysis see Fig. 2.

2, several main analytical data

Relevant intermediate data required for implementing HBsAg many time points LiCA detection is obtained by after analyzing system operations, this Boundary's point data of experimental section key is shown in Table 1.

The first BA conjunction type many time points LiCA interpretation intermediate data of table 1 HBsAg detection

*: in table, the computational methods system of threshold value continues to use according to the past document custom, also can confirm according to alternate manner；#: auxiliary Index contour M is boundary's point lifting-line, identical (Pe (x)=(P1 (x)-P2 (x))/2=Q=A1=of the x value of each boundary's point value on this line A2), y value takes from the intersection point of M and each curve.

3, many time points LiCA examination criteria reference product concentration and quantitative analytical experiment thereof is used to the results are shown in Table 2

Table 2 serum HBsAg Patent Law LiCA examination criteria reference product concentration sets and experimental result

4, recovery experiment result

Use many time points LICA that the self-control control serum of one group of serial dilution is detected, and with aforementioned foundation (test Stage) artificial intelligence system testing result is analyzed.Experimental result is shown in Table 3.

Table 3 serum HBsAg elder generation BA conjunction type many time points LiCA detects recovery experiment result

*: saturation exponent: computational methods are SSDI=(S1/S2) × 100 × 10000, and different experiments method is used Coefficient is different；

5, Clinical detection result

1) use many time points LiCA that one group of clinical samples is detected, and compare with conventional LiCA, the results are shown in Table 4.The 72 unscreened clinical patients of example, two method HBsAg positive rates are 16.7%.Conventional LiCA detection is imitated because of Hooks The overall bias ratio of detection by quantitative result that should cause reaches 50.0%.Wherein quantitatively failure prediction ratio (i.e. can not be given because of overflow state Definite quantitative data specimen) it is 16.67%；Quantitatively misrepresent deliberately rate (result deviation true value more than 5 times) 33.3%；

The first BA conjunction type many time points LiCA Clinical detection of table 4 and the comparison with conventional LICA thereof

*: serum collection detected the experiment numbers of unit the same day.#: conventional LiCA detection fails to report the mark of exact concentration This.

2) use many time points LiCA to one group of Abbott Laboratories' chemiluminescence detection by quantitative overflow (two step immunizations, > 250IU/ml, 67 parts) serum specimen detect, and compare with conventional LiCA.All the detection of serum two method HBsAg is the most positive, and it is dense Degree average is respectively 6729.32 ± 1396.26 and 217.85 ± 69.73, and the former average is the latter 31.03 times.Do not find concentration Dependency false negative phenomenon.The concentration ranges distribution of its testing result is shown in Table 5.

Table 5 uses two kinds of LiCA to one group of Abbott Laboratories' reagent HBsAg overflow Virus monitory result

Experimental technique	First BA combines many time points LiCA*	Conventional LiCA*
			Low concentration true value (200-500ng/ml) district	9/13.43	9/13.43
High concentration true value district (Patent Law)	58/86.57	Nothing
			High signal overflow (conventional LiCA) district	Nothing	6/8.96
Low signal high concentration (conventional LiCA) district	Nothing	52/77.61
			Add up to	67/100%	67/100%

*: number of cases/percentage rate (with all experimental subjecies for 100%)

(5) the experimental signal development feature of many time points LiCA detection

Use ibid experimental technique to detect, test in immunoreactive different time points (T1, T2, T3, T4, T5) Signal (S1 S5) gathers, and is plotted in compound HB two-way dose-effect curve image (Fig. 3) with same upper type, result Display, within the set period, the experimental signal Strength Changes caused because reaction time point changes is not limited to aforementioned patent The specific time point of certain in method, and summit dancing also occurs in immunoreactive overall process of observed period.

(6) conclusion:

1, method sensitivity: two methods (many time points LiCA and conventional LiCA) are identical, ＜ 1.0ng/ml；

2, quantification range:

Conventional LiCA:1.0---500 (1000) ng/ml；

Many time points LiCA:1.0---100000～1000000ng/ml.

Both compare: the more conventional LiCA of Patent Law LiCA expands 100～1000 times.

3, implementation result:

1) by dose-effect curve two-way to HB and the application of curvilinear regression analysis method thereof, the conventional LiCA of notable expansion Quantitative analysis space；

2) the two-way curve of integrated application S1, the two-way curve of S2 and the analysis result of SSI curve triadic relation thereof, confirms detection Specimen target substance concentration (true value) calculates and looks into the curve section that reading is used, and solves the vacation in two-way curve ratiometric analysis with this The property mapping value interference to quantitative analysis；

3) experimental phenomena of application apex offset (or claiming summit drift), solves to enclose the partial section ginseng that summit effect causes The problem declined than accuracy of quantitative analysis；

4) utilize class Hook effect to sentence a little, identify the specimen that on the right side of curve, in low signal section, experimental precision declines and (be somebody's turn to do Phenomenon only occurs in the experimental specimen of the rarest superelevation target substance concentration).

5) two the two-way dose-effect curves of HB gathering different time points implement matching respectively, piecewise fitting and difference thereof Application, provides important guarantee for promoting the precision of quantitative analysis results.

[embodiment 2] time dependence point analysis of many times method is anti-in light excites chemiluminescence (LiCA) detection serum The application (experiment of rear BA conjunction type LiCA) of HBc

The present embodiment difference from Example 1 is that detecting object is Anti-HBc Serum antibody, and experiment method is rear BA reaction LiCA。

(1) experiment material

1, instrument and consumptive material

1) experimental apparatus: LiCAHT light-induced chemiluminescent detector；

2) artificial intelligence's data handling system: Homebrew processing system, back-end data process path see patent description and Embodiment 1；

3) experiment consumptive material: ibid provided by biological (Shanghai) Science and Technology Ltd. of rich sun of China.

2, experiment reagent and specimen

1) commercial reagent (Bo Yang bio tech ltd of China manufactures) is used.Including:

The biotinylated HBcAg of reagent 1:R1.；

Reagent 2:R2. is coated the photosensitive microsphere of streptavidin (SA)

Reagent 3:R3.HBcAg is coated luminous microsphere；

Reagent 4: experiment dilution system；Ibid commercial reagent system is carried；

2) reference standard: can simultaneously use commercially available and self-control standard reference product.

1. commercial standard: commercial reagent box with band, totally 6 parts.Anti-HBc Serum concentration is respectively as follows: 0.0IU/ml, 0.3IU/ml, 2.4IU/ml, 13.2IU/ml, 80.0IU/ml, 200.0IU/ml

2. self-control reference standard category: use Anti-HBc Serum strong positive previously HBV infection person's serum, demarcate through Anti-HBc Serum activity Rear preparation.Prepared product includes matching experimental article, recovery experiment articles for use, reference experiments articles for use, and conventional reference standard substance etc.. Preparation principle and method are with embodiment 1；

3) control serum

Negative control carries for ibid commercial reagent box with positive control serum, confirms for thresholding and reagent reacting is commented Valency.

4) detection specimen: serum, blood plasma, body fluid or In vitro culture thing

3. artificial intelligence analysis's system

Anti-HBc Serum detection special " compound HB two-way dose-effect curve image method analyzes system ", its macroscopic view logical framework ginseng See summary of the invention.It builds also introduces in the framework of above-mentioned analysis system.

(2) experimental procedure

1. Preparatory work of experiment: take out all reagent and specimen, recovers to room temperature, on-demand adjusts to working concentration and state；2. Experimental implementation

1) taking reagent R1 and R3 reagent is appropriate, equal-volume mixes, and adds in each hole of LiCA Sptting plate, every hole (pipe) 100 μ l；

2) taking specimen to be measured, positive and negative compare, and each 25 μ l of Anti-HBc Serum quantitative criterion serum serial, add to respectively above-mentioned respectively Reacting in each hole, every part of specimen adds a hole (or any hole as required), mixing, educates temperature 10min；

3) adding reagent R3, every hole 175 μ l, mixing, at machine reaction 15min (reaching T1)；

4) excite at 680nm, under the conditions of 610nm wavelength detecting, detect each hole experimental signal (S1), and by defeated for each signal value Deliver to artificial intelligence's data handling system；

5) continue to place 15min, reach T2 time point；

6) putting on LiCA luminometer, 680nm excites, 610nm wavelength detecting each hole experimental signal (S2), and by signal It is delivered to artificial intelligence's data handling system；

7) according to experimenter's instruction, above-mentioned experimental signal is comprehensively analyzed by artificial intelligence system, store and report inspection Survey result.The concentration of Anti-HBc Serum represents (the present embodiment is as follows) with IU/ml.

(3) interpretation path

1, transfer and revise the special Experiment Analysis System of deposit according to control serum experimental data, formed for when secondary Serum Anti-HBc Serum many time points LiCA compound HB bidirectional measuring of detection analyze system；

2, in special artificial intelligence's data handling system, input T1 and T2 two time point detection data S1 and S2, calculate and be subject to The SSDI value of inspection specimen；

3, the relevant boundary point data (threshold value, A1, A2 and Q circle point etc.) in experimental system is confirmed according to the method for embodiment 1；

4, according to the method for embodiment 1, experimental result is carried out the analysis of target substance concentration, and by instruction output experiment knot Really；

5, serum Anti-HBc Serum detection LiCA experiment compound HB two-way dose-effect curve image analytical method analysis system simulation is shown in Shown in Fig. 4.

(4) experimental result

1, the light that time dependence point analysis of many times method is set up excites chemiluminescence (hereinafter referred to as many time points LICA) to examine The image method of the compound HB two-way dose-effect curve analysis system surveying Anti-HBc Serum in serum shows sees Fig. 4.

2, several main analytical data

The first BA conjunction type many time points LiCA interpretation intermediate data of table 6 serum Anti-HBc Serum detection

*: in table, the computational methods system of threshold value continues to use (blank well signal average × 2.1) according to the past document custom, also may be used Use alternate manner.

3, when secondary experiment reference experiments data (many time points LICA sets EXPERIMENTAL EXEMPLIFICATIONThe with reference to product)

After table 7 is simple, BA combines LiCA serum Anti-HBc Serum examination criteria curve and relies on data

4, recovery experiment result

Table 8 serum Anti-HBc Serum Patent Law (after simple, BA combines) LiCA recovery experiment result

*: Anti-HBc Serum concentration, IU/ml

(5) conclusion:

1, method sensitivity: conventional LiCA and many time points LiCA two method is identical, ＜ 0.10IU/ml；

2, specificity, stability and simple operation: two methods are identical；

3, quantification range:

Conventional LiCA:0.1～200IU/ml；

Patented method: 0.1～100000IU/ml or more than；

4, Hooks effect antagonistic effect (Patent Law compares with conventional LiCA):

Quantitative analysis scope can be expanded the most significantly, Hook effects of substantially preventing, and possess discovery extremely Rare class Hook effect phenomenon ability.Concrete introduction is same with embodiment 1.

The detection time dependence many time points cotton-shaped immune agglutination experiment of [embodiment 3] serum IgG

The serum IgG detection many time points of time dependence detect cotton-shaped immunoprecipitation experiment and (call many time points in the following text and detect cotton-shaped exempting from Epidemic disease precipitation experiments).Compared with embodiment 1,2, there is the following main distinction, the experiment skill that i.e. this embodiment is relied in embodiment 3 Art (immunoprecipitation experiment) sensitivity is the most on the low side, and the relative Repeat of detection by quantitative is also the narrowest；Immunocomplex formation shape State is the biggest on the impact of experimental result, and the time-dependence variation characteristic of its experiment signal intensity is also notable compared with the above two.But this Have no effect on the quantitative analysis to experimental result a bit；It is high that human IgG expresses radix in serum, concentration change scope relative narrower, Using patented method to detect, the analysis to experimental result need not use the two-way dose-effect curve of whole HB；Therefore, should The target that embodiment is reached is only that using the experimental index of this kind of economical and convenient to prove this patent method sinks in cotton-shaped immunity Form sediment experiment, the feasibility of application in immune agglutination experiment and the method such as synergetic immunity coagulation experiment thereof, simultaneously heavy for seroimmunity The development of the experimental techniques such as shallow lake (as used same dilution factor specimen synchronous detecting serum IgG, IgA and IgM, and uses same Serum and the multiple humoral specimen of concentration difference great disparity are done synchronous detecting etc. by test method) new technique direction is provided.

(1) experiment material

1, instrument and consumptive material

1) experimental apparatus: HITACHI 7170A automatic biochemistry analyzer, Hitachi company of Japan manufactures；

2) artificial intelligence's data handling system: Homebrew processing system, background process path sees enforcement 1；

2, reagent

1) serum IgG detection immunoturbidimetry measures test kit: Shanghai Magnolia denudata biochemical reagents institute produces；

Reagent 1:R1. anti-human igg solution；

Reagent 2:R2. diluent；

2) reference standard

Standard reference product: conventional criteria serum.Ibid test kit is with band, totally 6 parts；Ig concentration be respectively as follows: 0.00g/L, 0.20g/L、2.60g/L、4.00g/L、16.00g/L、64.0g/L；

3) self-control reference product

Reference product raw material (human IgG): purification in Healthy Human Serum, BSA reference spectrum absorption process is quantitative, through demarcating and adjusting Its concentration whole is 128.00mg/ml (128g/L).Take this purified with ibid commercial reagent with the diluted of band, divide in right amount Dress ,-20 DEG C frozen.The concentration range of constructed reference series is:

Reference experiments product: 0.00g/L, 2.00g/L, 4.00g/L, 16.00g/L, 64.0g/L, 256g/L

Recovery experiment sample: concrete concentration is shown in Table 9；

4) detection specimen: serum (and blood plasma etc.).

(2) experimental procedure

1, Preparatory work of experiment: take out all reagent and specimen, recovers to room temperature, on-demand adjusts to corresponding concentration；

2, experimental implementation

1) experiment reagent (R1 and R2) and experimental specimen are placed on by operating instruction the corresponding site of instrument, start instrument Device, sets according to specimen classification and Guan Xu (single pipe method) and gradually performs experimental implementation；

2) the 20th (T1,6min) and the 33rd (T2,10min) measuring point in instrument setting carry out experimental signal detection (450nm Wavelength colorimetric), and the two-way dose-effect curve of compound HB that the experimental signal input gathered is the establishment of this method is analyzed system；

3) experimental specimen is processed successively according to detection sequence number, and by instruction report experimental result；

4) detection of conventional cotton-shaped immunoprecipitation experiment serum IgG strictly is illustrated to carry out (Fig. 5) by reagent.

(3) interpretation path

1, transfer according to control serum experimental data and correction analysis system stores information, formed as the secondary compound HB used Bidirectional measuring analyzes picture system；

2, in artificial intelligence's data handling system, input T1 (the 20th measuring point) and T2 (the 33rd measuring point) two time point detection number According to S1 and S2, calculate the SSDI value by inspection specimen；

3, according to the method correction experimental analysis system of embodiment 1, and the relevant boundary point data (threshold in experimental system is confirmed Value, A1, A2 and Q point etc.)；

4, with reference to the analysis mode of embodiment 1, experimental result is carried out the quantitative analysis of target substance concentration, and defeated by instruction Go out experimental result；

5, serum IgG detects cotton-shaped immunoprecipitation experiment and is combined HB two-way dose-effect curve image analytical method analysis system Fig. 6 is shown in display.

(4) experimental result

1, the target substance concentration of standard curve and standard reference product

Use patent with conventional methods, the standard reference product of same set of serial dilution to be detected, be inputted specially Item analyzes system, and is formed when time compound HB two-way dose-effect curve quantified system analysis of use.The results are shown in Table 9, table 11, Fig. 5 and Fig. 6.

The table 9 many time points of serum human IgG time dependence cotton-shaped immunoprecipitation experiment reference standard testing result

*: the SSDI of conventional method quantitative limit definite value, it is can quantitative measured value specimen more than this value person.

2, several important intermediate data

Several for quantitative analysis important intermediate data acquired in the present embodiment detection, are shown in Table 10.

Several middle experiment data * of table 10 serum human IgG time dependence many time points cotton-shaped immunoprecipitation detection

*: for working as the judgement of time experimental result and looking into reading experimental result

3, Patent Law detection recovery experiment result

Use many time points to detect the experiment of cotton-shaped immune agglutination the serial dilution reference product of one group of concentration known is detected, And with artificial intelligence system, testing result is analyzed.Experimental result (three hole averages) is shown in Table 11.Examine at experimental small sample Under the conditions of survey, its response rate is between 90.9%---105%.

The table 11 serum human IgG detection many time points of time dependence cotton-shaped immunoprecipitation experiment testing result

Note: SSDI computing formula is (S1/S2) * 100*100；The response rate=(reclaiming concentration/setting concentration) × 100；

Result computing formula:The same 2 embodiments of computational methods.

4, use Patent Law and conventional cotton-shaped immunization experiment (with 64g/L concentration for setting the concentration analysis upper limit) thereof to one group Clinical samples carries out comparison and detection.Experimental result is shown in Table 12.Do not assume a marked difference between two method testing results P > 0.05. Due to this comparison and detection use Clinical detection sample on the low side, only 1 example specimen measured value slightly higher go out normal quantitative analysis limits.

Inspection and the comparison with conventional sense thereof are surveyed in the many time points of table 12 human serum IgG cotton-shaped coagulation experiment

*: Hook effect 1 example, statistics is not counted.

(5) conclusion:

1) method sensitivity: two methods are identical, ＜ 100.0mg/L；

2) quantification range:

Conventional cotton-shaped coagulation: 1.0---64g/L；

Patent Law LiCA:1.0---300g/L or more than.

Both compare: the more conventional method of Patent Law expands 10～100 times (being affected by specimen target substances concentration limit).

3) Hooks effect antagonistic effect (Patent Law compares with conventional LiCA):

1. detection by quantitative scope can significantly be expanded；

2. the Hook effects specimen in normal experiment detection is found；

3. can prevent Hook effect.

Claims (5)

1. the method for a Serologic detection quantitative analysis, it is characterised in that: comprise the steps:

1) experimentation: there is labelling and unmarked corresponding immune response reagent and the experiment container of signal generation related reagent In be separately added into when time reference standard, matching sample, yin and yang attribute comparison or specimen to be measured, described corresponding immune response reagent Refer to antigen and antibody, place after mixing, to bring it about immunoreation, and produce the experimental signal being available for measuring, in generation Immunoreactive T1, T2 time point carries out the detection of experimental signal S1, S2 respectively；It is inputted at special Experiment Analysis System Reason, and instruct output experimental result according to experimenter；

2) matching of functional relationship:

(1) ibid under experiment condition, utilizing more than 100 concentration point samples, concentration range sets and contains the two-way curve of whole HB In effectively analyze section image, the concentration ranges of sample is set as between particle being spaced 0.025 0.10 transverse axis reading distances, to this Reference standard carries out 10 times or repeats experiment above, takes the experimental signal meansigma methods of each detection of each concentration point as this point Corresponding experimental signal carries out high density Function Fitting analysis；

(2) the experimental signal input artificial intelligence analysis's system that will be generated, path, the backstage overview of artificial intelligence analysis's system:

1. experimental signal value S1 and the S2 of the detection of T1 and T2 two time point are inputted；

2. calculating by the ratio examining specimen T1 and T2 time point S1 and S2 measured value, i.e. experimental signal saturation, satisfies experimental signal Be converted into saturation exponent (Signal Saturation degree index, SSDI) with degree, conversion method be (S1/S2) × 100 × e, e are conversion coefficient, and SSDI function on the longitudinal axis is worth same with experimental signal y；

3. on plane semilog coordinate image, according to experimental signal S1 and S2 acquired in series reference standard mensuration, point Do not draw out two two-way dose-effect curves of HeidelBerger of S1 and S2, and by saturation exponent according to the most corresponding target Matter concentration is marked and drawed in aforementioned semilog function image successively, draws out corresponding signal saturation exponent curve SSI, these three Curve constitutes compound HB two-way dose-effect curve image analysis system, and wherein Y-axis is experimental signal intensity or SSDI, and X-axis is The logarithm numerical value of target substance concentration；

4. use in the fitting process confirmation two-way curve of HB in function x and y, and signal saturation exponent curve SSI function x with The relation of SSDI:

By matching it is experimentally confirmed that the eyeball (y) on the two-way curve of each HB and its institute in HB two-way dose-effect curve image The corresponding relation between functional value (x) can use below equation to state:

Fitting effect

Wherein x: for the natural logrithm of target substance concentration；α: corrected parameter；B: logarithmic average；F (x)=experimental signal (y)；C: mark Accurate poor；

Each eyeball SSDI on signal saturation exponent curve SSI, limits point value inside the two two-way curve images of HB, and with right Relation between the functional value (x) answered can use below equation to state:

F (x)=a+b × x, fitting effect

Wherein x: for the natural logrithm of target substance concentration；α: intercept, refers to as x=0, the size of the value background signal of Y；B: Slope, refers to the increase and decrease amount of Y when x changes a unit；

Y=f (x)=SSDI=(S1/S2) × 100 × e (e is conversion coefficient)；

3) determination of fitting function graphical analysis circle point

(1) summit P1 and P2, formula: P (y)=f (the x)=μ of the two two-way curves of HB, wherein, μ=b, i.e. experimental data mould are calculated The logarithmic average of the model parameter drawn up, for P1 or P2 in the log concentration value of vertex correspondence；

(2) two summits midpoint Pe (formula: Pe (x)=(P1 (x)+P2 (x))/2) in X-axis reading difference is confirmed, through Pe point to X Axle is vertical line M, and with S1, S2 and SSI tri-curve intersect at A1, A2 and Q respectively, calculate the experimental signal of three respectively, i.e. y or SSDI value；

(3) programming quantitatively divides to set up the homogeneous immunization experiment of serology with the two-way dose-effect curve of compound HB as reference subject Analysis system；

4) concentration reference standard and setting

Setting when time conventional reference standard substance, based on the two-way dose-effect curve of HB that step 2 is set up, its concentration is with substantially The following linear position reaching two-way curve is target, and concrete setting includes: 1. background verification reference, 0 target substance, threshold value sets Reference；2. sensitivity control reference, low concentration end, three order derivatives of curve normal distribution enclose starting point；3. linear condition control System, near the flex point of left and right, normal distribution curve enclose second dervative starting point；4. Y value height point control, enclosing of normal distribution curve First derivative starting point (i.e. enclosing summit) place；5. class Hook effect control point set, its concentration set with generation experimental signal exist Experimental signal produced by controlling reference product with sensitivity in intensity is corresponding；

5) interpretation:

(1) by the detection of concentration reference standard, the input of data, recall suitably when secondary interpretation of result, i.e. abovementioned steps 2), 3) The data analysis system set up；Left and right two parts, summit P1 can be divided into be positioned at two-way for S1 curve asymmetric by A1 circle point The left side of boundary point A1, wherein the high x value section in right side is experimental result interpretation section；Can be by the most right for two-way for S2 curve by A2 circle point Claiming ground to be divided into left and right two parts, summit P2 to be positioned at the right side of boundary point A2, wherein the low x value section in left side is experimental result interpretation district Section；Lock experimental specimen to be analyzed actual measurement by the input of experimental specimen sequence number and calculate data；By measuring specimen SSDI and Q The comparison of value determines curve and the respective section of specimen ratiometric analysis to be measured, i.e. when the SSDI measuring specimen is more than Q-value, according to Its T2 time point measured value y looks into reading experimental result at the left side section of S2 curve；When measuring specimen SSDI less than Q-value, according to its T1 Time point measured value y looks into reading experimental result at the right side section of S1 curve；

(2) use the input reference standard detection abovementioned steps 2 that recalled of data), 3) in the data analysis system set up Threshold value is to above-mentioned 5) (1) item analysis result is further analyzed, and its S2 measured value be feminine gender less than homologous thread threshold value person, described right Answering curve threshold value is that negative hole average is multiplied by 2.1 times；

(3) use the input reference standard detection abovementioned steps 2 that recalled of data), 3) in the data analysis system set up Class Hook effect control point is to above-mentioned steps 5) (1) item analysis result is further analyzed, and its S1 measured value is less than homologous thread class Hook effect control point reference signal person is the experimental specimen that there is class Hook effects, and target substance concentration is imitated higher than class Hook Answering control point, quantitative analysis results is only for reference, and it is positive that this result is reported as ultrahigh concentration with qualitative fashion；

(4) after above-mentioned analysis, according to the experimental signal Y measured by analyzed specimen, function x is carried out at corresponding curve section Calculating, the log result of calculating is converted into actually detected concentration and reports experimental result.

The method of Serologic detection quantitative analysis the most according to claim 1, it is characterised in that: described Serologic detection Referring to homogeneous immunity, class is the most immune, and other antigen, antibody and experimental signal thereof generate system three and be stored in same experiment body In system, the detection of secondary above experimental signal, and immunoreation process in not obstruction system can be implemented within certain period, do not affect The experimental technique of the original testing result of institute's conventional experimental technique.

The method of Serologic detection quantitative analysis the most according to claim 1, it is characterised in that: step 1) described in be measured Specimen means the solution with or without target substance needing to carry out immune detection.

The method of Serologic detection quantitative analysis the most according to claim 1, it is characterised in that: step 1) examination that used Agent includes that immunoreagent, signal generate one or more in reagent, connection reagent, mux--out signal exhibits and strengthening reagent.

The method of Serologic detection quantitative analysis the most according to claim 1, it is characterised in that: step 1) described in reaction Time setting depends on that the process that relied on experiment immunization reacts, dominant response time point are set as: T0 time point, i.e. antigen and antibody Under the conditions of the immunoreation that experimental technique specifies, mix and start the time point of immunoreaction process；T1 time point, immunoreation Journey carries out the time point of experimental signal detection for the first time；T2 time point, in immunoreaction process, second time carries out experimental signal detection Time point；The immunoreactive period is planned to the T1 period: T0 to T1 time point institute interlude section；The T2 period: T0 to T2 time point Institute's interlude section；Immunoreactive total Period Length is because experimental technique is different and has the requirement difference of experimental result Being distinguished, its scope is limited between 2-60min, and the ratio of T1 Yu T2 Period Length is 0.1 to 0.95.