CN1888904A - Method for indicating xenotropic antibody interference in immune reaction - Google Patents

Abstract

The invention is concerned with the out-body examination field that indicates the interferential method and the corresponding reagent box of the Heterophilic antibody. The invention can improve the accuracy of the double-position filling immunoassay, and reduce the false positive ratio of the double-position filling immunoassay.

Description

A kind of method of indicating heterophile antibody interference in the immune response

Technical field

The present invention relates to the vitro detection technical field, relate to a kind of method that the indication heterophile antibody disturbs when the immune detection serum sample particularly.

Background technology

Heterophile antibody disturb refer to finger in immune double antibodies sandwich reaction capture antibody with detect antibody, not sandwich, and directly link together the protein antibodies material of formation false positive results by forming.Common heterophile antibody has HAMA (Human Anti-mouse Antibody) antibody, rheumatoid factor etc.

A kind of common heterophile antibody disturbs by the HAMA reaction and causes.HAMA (Human Anti-mouse Antibody) is reflected in the immune detection and often takes place, and its incidence is about 10%.For example, once contacted or inoculated in the individuality of murine antibody, can there be the antibody of the anti-mouse of people in its body.Because these individual serum samples contain the antibody of the anti-mouse of people, so be called the HAMA serum sample.That is adopted when immune detection is one anti-and two anti-when all being murine antibody, the antibody of the anti-mouse of contained somebody in the HAMA serum sample, can be directly one anti-and two anti-crosslinked, the normal double antibodies sandwich that influence serum sample reacts, demonstration makes the mistake, form false positive testing result (.Hazra DK, Britton KE, Lahiri VL, Gupta AK, KhannaP, Saran S.Nucl Med Commun.1995 Feb; 16 (2): 66-75.).

At present, common immunologic detection method can't be judged HAMA sample serum and the normally difference of sample serum, therefore can't judge that testing result is true positives or false positive, finally very easily causes mistaken diagnosis.

Another kind of common heterophile antibody disturbs and is caused by rheumatoid factor.Rheumatoid factor (RF) is the specific antibody of antigen decision family on the anti-human IgG molecule FC fragment, is the autoantibody of anti-IgG, can combine with the sex change IgG molecule of people or animal, usually causes the false positive of detected sample.

In contrast, also exist some easily to cause false-negative phenomenon in immune response, the HD-HOOK effect is one of them.High dose hook (HD-HOOK) effect is meant in the experiment of dibit point sandwich immunoassay, the high dose of its dose-effect curve (HIGH DOSE, HD) section, linear trend is not to be platform-like to prolong after unlimited, but be bent downwardly shape, like hook or reaping hook (HOOK), according to this phenomenon realism be referred to as " HD-HOOK " effect (Miles LEM, Lipschitz DA, Bieber CP and Cook JD:Measurementof serum ferritin by a 2-site immunoradiometric assay.Analyt Biochem 61:209-224,1974.)

The molecule mechanism that produces the HD-HOOK effect has " the molecule allosteric is said " and inferences such as " concentration effects " (" progress of the clinical immunology check " entire PLA of Nanjing General Hospital, Nanjing Military Area Command, PLA medical test center, the military foundation; " quality management of ELISA " clinical examination center, Jiangsu Province, Xu Bin; " influence factor and application of principle that tumor markers detects " Wuhan Union Hospital, Wu Jianmin).

The HD-HOOK effect often takes place in immune detection, and its incidence is accounting for positive sample about 30%.Its concentration exceeds the range of linearity or the concentration own of detection kit because the existence of HD-HOOK effect causes detected sample not to be divided into by right area is exactly this value, to such an extent as to the experiment mistaken diagnosis especially causes false negative rate to rise.

Therefore, this area press for exploitation new easy, effectively indicate the method for heterophile antibody, improve the accuracy of dibit point sandwich immunoassay, reduce the false positive rate of dibit point sandwich immunoassay simultaneously.Simultaneously, this area also presses for the method for effective indication HD-HOOK, so that reduce the false negative rate of dibit point sandwich immunoassay.

Summary of the invention

Purpose of the present invention just provide a kind of easy, effectively indicate the method for heterophile antibody, improve the accuracy of dibit point sandwich immunoassay, reduce the false positive rate of dibit point sandwich immunoassay simultaneously.

Another object of the present invention be provide simultaneously a kind of easy, effectively indicate the method for HD-HOOK effect, thereby reduce the false negative rate of dibit point sandwich immunoassay.

In a first aspect of the present invention, the immune sandwich detection method of target antigen in a kind of test sample is provided, it may further comprise the steps:

(a) with sample, detect microballoon, heterophile antibody disturb indication microballoon and mark the second antibody of detectable signal mix, form a reaction system,

Wherein said detection microballoon is the binary complex of the first antibody-microballoon shown in the I formula,

anti ₁X-bead (I)

In the formula, " X " represents target antigen, " anti ₁X " represent first antibody at described target antigen " X ", " bead " represents microballoon, the combination between "-" expression first antibody and the microballoon,

And described second antibody and first antibody can be incorporated into the different epi-positions of target antigen simultaneously,

Thereby form " second antibody-antigen-first antibody-microballoon " tetraplex;

Wherein, it is the binary complex of the different preferendum interference indicant-microballoon shown in the III formula that wherein said heterophile antibody disturbs the indication microballoon,

Z-bead″ (III)

In the formula, the different preferendum of " Z " expression is disturbed indicant (preferably, described different preferendum disturbs indicant to be selected from mouse antibodies, rat antibody, chicken source antibody, rabbit source antibody, sheep source antibody, horse source antibody, ox source antibody or rheumatoid factor), " bead " " expression can with " bead " the mutual different microballoons of difference; the different preferendum of "-" expression is disturbed the combination between indicant and the microballoon

Thereby in sample, exist under the situation of heterophile antibody, form " second antibody-heterophile antibody-different preferendum is disturbed indicant-microballoon " tetraplex;

(b) detectable signal on the microballoon in " second antibody-antigen-first antibody-microballoon " described in detection reaction system tetraplex, and with standard value or typical curve relatively, thereby whether and/or quantity the existence of determining target antigen in the reaction system;

And the detectable signal in " second antibody-heterophile antibody-different preferendum is disturbed indicant-microballoon " described in detection reaction system tetraplex on the microballoon, obtain the signal value that heterophile antibody disturbs the indication microballoon, and the heterophile antibody when being as good as preferendum antibody interferes with effect disturbs indication microballoon signal value (normal value) relatively, when heterophile antibody disturbs indication microballoon measured value to disturb 1.5 times of indication microballoon normal values greater than heterophile antibody, judge that then the measurement result of target antigen is unreliable; If heterophile antibody disturbs indication microballoon measured value to be less than or equal to heterophile antibody when disturbing 1.5 times of indication microballoon normal values, then judge to be as good as the preferendum antibody interferes with in the detected sample.

In another preference,, also comprise step in step (a) with (b):

(b1) HD-HOOK is indicated microballoon join in the system of step (a), wherein HD-HOOK indication microballoon is the binary complex of the target antigen-microballoon shown in the formula II,

X-bead’ (II)

In the formula, " bead ' " expression can with " bead " and " bead " " the different microballoons of difference mutually, "-" represents the combination between target antigen X and the microballoon,

Thereby having in the presence of the second antibody of detectable signal, forming " second antibody-antigen-microballoon " ternary complex; With

(b2) detectable signal on the microballoon in " second antibody-antigen-microballoon " ternary complex in the detection reaction system, obtain the signal value of HD-HOOK indication microballoon, and the HD-HOOK indication microballoon signal value (normal value) during with no HD-HOOK effect relatively, when HD-HOOK indication microballoon measured value is indicated the microballoon normal value less than HD-HOOK, judge that then the measurement result of target antigen is unreliable; If when HD-HOOK indication microballoon measured value is indicated the microballoon normal value more than or equal to HD-HOOK, judge that then the concentration of target antigen is in measurable range in the sample.

In another preference, first antibody, different preferendum disturb the combination between indicant or target antigen and the microballoon that covalent bond or aglucon reaction or non-specific adsorption are arranged.

In another preference, when described heterophile antibody disturbs indication microballoon measured value to disturb 2 times of indication microballoon normal values greater than heterophile antibody, judge that then the measurement result of target antigen is unreliable, wherein said normal value is following definite:

(a ') concentration is known and target antigen standard items series that be in measurement range is mixed with described detection microballoon, the described second antibody that has detectable signal respectively, form a reaction system, thereby form " second antibody-antigen-first antibody-microballoon " tetraplex of different target antigen standard items concentration;

(b ') disturbs the indication microballoon to join in the system of step (a ') described heterophile antibody;

(c ') detects described heterophile antibody and disturbs detectable signal on the indication microballoon, with P+2SD is indication microballoon normal value, when P represented different target antigen standard items concentration in the formula, heterophile antibody disturbed the mean value of indication microballoon signal value, and 2SD is 2 times a standard deviation.

In another preference, in step (b2), when HD-HOOK indicates microballoon measured value≤0.9 * HD-HOOK to indicate the microballoon normal value, judge that then there is the HD-HOOK effect in sample, and described HD-HOOK indication microballoon normal value is in order to method is definite down:

(a ') concentration is known and target antigen standard items series that be in measurement range is mixed with described detection microballoon, the described second antibody that has detectable signal respectively, form a reaction system, thereby form " second antibody-antigen-first antibody-microballoon " tetraplex of different target antigen standard items concentration;

(b ') joins described HD-HOOK indication microballoon in the system of step (a '), thereby having in the presence of the second antibody of detectable signal " second antibody-antigen-microballoon " ternary complex of formation different target antigen standard items concentration;

(c ') detects the detectable signal on the microballoon in the described ternary complex, with P+2SD is HD-HOOK indication microballoon normal value, when P represented different target antigen standard items concentration in the formula, the mean value of microballoon signal value in the ternary complex, 2SD were the standard deviation of 2 times microballoon signal value.

In another preference, target antigen quantity is the 1-10000 kind, preferably is the 1-1000 kind, more preferably is the 2-1000 kind, is the 4-1000 kind best.

In another preference, described antigen is protein.

In another preference, first antibody is 1 with the mol ratio of corresponding second antibody: 0.1-1: 2.

In another preference, described various microballoon bead, bead ' and bead " are the microballoons with different fluorescence.

In a second aspect of the present invention, a kind of kit that is used to detect target antigen is provided, it comprises: container, and be loaded on following material in the container respectively:

(a) detect microballoon, wherein said detection microballoon is the binary complex of the first antibody-microballoon shown in the I formula,

anti ₁X-bead (I)

In the formula, " X " represents target antigen, " anti ₁X " represent first antibody at described target antigen " X ", " bead " represents microballoon, the combination between "-" expression first antibody and the microballoon,

(b) have the second antibody of detectable signal, wherein, described second antibody and first antibody can be incorporated into the different epi-positions of target antigen simultaneously,

(c) heterophile antibody disturbs the indication microballoon, and it is the binary complex of the different preferendum interference indicant-microballoon shown in the III formula that wherein said heterophile antibody disturbs the indication microballoon,

Z-bead″ (III)

In the formula, the different preferendum of " Z " expression is disturbed indicant, described different preferendum disturbs indicant to be selected from: mouse antibodies, rat antibody, chicken source antibody, rabbit source antibody, sheep source antibody, horse source antibody, ox source antibody or rheumatoid factor, " bead " " expression can with " bead " the mutual different microballoons of difference, the different preferendum of "-" expression is disturbed the combination between indicant and the microballoon.

In another preference, described kit also comprises:

(d) HD-HOOK indication microballoon, described HD-HOOK indication microballoon is the binary complex of the target antigen-microballoon shown in the formula II,

X-bead’ (II)

In the formula, " bead ' " expression can with " bead ", " bead " " the different microballoons of difference mutually, the combination between "-" expression target antigen X and the microballoon.

In another preference, described kit contains at 1-1000 kind (preferably 2-500 kind, more preferably 3-100 kind) the relevant detection microballoon of target antigen, the second antibody that has detectable signal and HD-HOOK indication microballoon; And described various microballoon bead, bead ' and bead " are the microballoons with different fluorescence.

Other aspects of the present invention after having read the application, are conspicuous to those skilled in the art.

Embodiment

The inventor is through extensive and deep discovering, by setting up heterophile antibody to disturb the indication microballoon, can get rid of the false positive rate that heterophile antibody caused in the dibit point sandwich immunoassay simple and effectively, thereby improve the accuracy of dibit point sandwich immunoassay.

In addition, by setting up HD-HOOK indication microballoon, can get rid of the false negative rate that the HD-HOOK effect is caused in the dibit point sandwich immunoassay simple and effectively, thereby further improve the accuracy of dibit point sandwich immunoassay further.

As used herein, term " first antibody ", " one is anti-" are used interchangeably, but refer to that specificity is incorporated into a kind of antibody of a certain antigen (as tumor markers).

As used herein, term " second antibody ", " two is anti-" are used interchangeably, but refer to that specificity is incorporated into the another kind of antibody of a certain antigen (as tumor markers).For example, for for a kind of antigen (as tumor markers), corresponding first antibody is different with second antibody, and can be incorporated into the different epi-positions of described antigen simultaneously.

As used herein, term " antigen " refers to have immunogenic material, for example protein, polypeptide.Representational antigen example comprises (but being not limited to): cell factor, tumor markers, metalloprotein class, cardiovascular diabetes associated protein etc.

As used herein, term " tumor markers " is meant in the generation and breeding of tumour, and by tumour cell itself produced or by body the tumour cell reaction is produced, the reflection tumour exists and a class material of growth.Representational tumor markers comprises (but being not limited to): and alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), T-PSA (PSA), free prostate gland specificity antigen (f-PSA), neuron specificity olefinic alcohol enzyme (NSE), carbohydrate antigen (CA242), cancer antigen (CA15-3), human chorionic gonadotrophin (β-HCG).

As used herein, term " different preferendum interference indicant " is meant in the reaction of immune double antibodies sandwich capture antibody and detects antibody, and is not sandwich by forming, and directly links together the protein antibodies material of formation false positive results.Representational example comprises (but being not limited to): mouse antibodies, rat antibody, chicken source antibody, rabbit source antibody, sheep source antibody, horse source antibody, ox source antibody or rheumatoid factor.

Ultimate principle

(a) double antibodies sandwich ratio juris

The ultimate principle of double antibodies sandwich immunodetection is well-known to those skilled in the art.Conventional way is with an anti-solid phase carrier that is fixed in, an anti-and antigen-reactive then, after the washing again with the two anti-reactions that indicate enzyme, chemiluminescence or enzyme connection chromogenic reaction detection signal is carried out in washing at last.

(b) Luminex xMAP ratio juris

Luminex xMAP is a multi-functional very flexibly technology platform.Its principle is that small latex particle (Beads abbreviates " microballoon " as) is dyed different iridescent respectively, and then the albumen (as antigen-antibody) that detects thing at difference is attached on the microballoon of particular color with covalent manner.During application, mix detect microballoon thing, that encode with different colours at difference earlier, add detected material (measured object can be antigen, antibody or the enzyme etc. in the serum) again.Microballoon in suspension combines specifically with detected material, and adds fluorescence labeling.Then, microballoon becomes single-row and passes through two bundle laser, thus the specificity (qualitative) of the color of a branch of judgement microballoon decision measured object; Thereby another bundle is measured the amount (quantitatively) of the fluorescence labeling intensity decision measured object on microballoon, and resulting data can directly be used for judged result after the computer processing.

In a preference, anti-characteristics that are fixed on the different microballoons (Beads) that flow have been made full use of, optimize two concentration that resist of phycoerythrin (PE) mark simultaneously, with two anti-solution of crosslinked one anti-microspheres solution and serum sample or antigen standard quality-control product liquid, PE mark successively or add in the reaction vessel in the lump simultaneously, thus following the reaction taken place:

1. one on the microballoon is anti-combines with corresponding antigen (being tumor markers antigen) in serum or the standard quality-control product liquid, forms " tumor markers-first antibody-microballoon " ternary complex,

2. two anti-combine with corresponding antigen (being tumor markers antigen) in serum or the standard quality-control product liquid, final " second antibody-tumor markers-first antibody-microballoon " tetraplex (compound that comprises the antigen-PE mark of the compound of micro-sphere crosslinked anti--serum corresponding antigens-PE mark or micro-sphere crosslinked anti--standard quality-control product liquid) that forms, must centrifuge washing in the course of reaction, in liquid phase, can detect the fluorescence of compound by Luminex xMAP, reach from being reacted to qualitative and quantitative analysis one and go on foot the effect of finishing, that is: single stage method.

On the Luminex detector, these microballoons are lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the coding of a branch of judgement microballoon determines the kind of tested tumor markers; Another bundle is measured the fluorescence intensity of PE on the microballoon, draws the content of tested tumor markers through data processing.

Technology platform particulars about Luminex xMAP see also product description or document, (1) Cancer Chemotherapy and Pharmacology, 51:321-327, (2) Journal oflmmunological Methods, 227:41-52; (3) www.luminexcorp.com.

(c) principle of the different preferendum interference of indication

On the one hand, in the methods of the invention, at first upward resist at one of certain antigen so that covalent is crosslinked at a kind of fluorescence-encoded microballoon (being called ball No. 1); The two anti-solution corresponding to this kind antigen (normally detecting this antigen concentration, identical) that add serum sample or standard quality-control product liquid, phycoerythrin (PE) mark with method common, that do not have HD-HOOK effect deixis.At this moment following reaction takes place simultaneously:

1. one on No. 1 microballoon is anti-combines with corresponding antigen in serum or the standard quality-control product liquid,

2. two anti-combine with corresponding antigen in serum or the standard quality-control product liquid,

The tetraplex that final formation " anti--serum corresponding antigens-PE mark that No. 1 fluorescence-encoded micro-beads is crosslinked two anti-" constitutes, or " antigen-PE mark of No. 1 fluorescence-encoded micro-sphere crosslinked anti--standard quality-control product liquid two anti-" tetraplex of constituting.

On the Luminex detector, fluorescence-encoded microballoon is lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the coding of a branch of judgement microballoon determines the kind of tested antigen; Another bundle is measured the fluorescence intensity of PE on the microballoon, draws the content of tested antigen through data processing.

On the other hand, be example with indication HAMA reaction, on No. 2 fluorescence-encoded Beads with the crosslinked mouse source monoclonal antibody of covalent at HAMA; With crosslinkedly have the beads of antibody to add simultaneously in the reactive system No. 1, following reaction takes place simultaneously:

1. one on No. 2 Beads anti-with serum sample in the HAMA antibodies;

2. the HAMA antibodies in any two of the mouse source anti-and the serum samples,

The two anti-compounds of anti--HAMA antibody-PE mark that No. 2 fluorescence-encoded Beads of final formation are crosslinked.

Simultaneously on the Luminex detector, handle simultaneously with No. 1 for fluorescence-encoded No. 2, Beads is lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the coding of a branch of judgement Beads determines tested HAMA antibody; Another bundle is measured the fluorescence intensity that Beads goes up PE, judges according to fluorescence signal is strong and weak whether this serum sample is the HAMA serum sample.

On the other hand, on No. 2 fluorescence-encoded microballoons with the pure product of the crosslinked detected antigen of covalent.Crosslinkedly have after an anti-microballoon and sample, two anti--PE reactions finish at No. 1, in described crosslinked No. 2 fluorescence-encoded microballoons (being called " HD-HOOK indicate microballoon ") the adding reactive system that antigen arranged.

At this moment, if to be in non-HD-HOOK district (be to contain the quantity of antigen to be detected and not obvious greater than two anti-quantity in the detection architecture in the sample to the concentration of antigen to be detected in the sample, therefore some or the two more anti-unbound states that are), so following reaction can take place: the antigen on No. 2 microballoons combines with remaining two free anti--PE in the reactive system, the ternary complex of final formation " antigen-PE mark that No. 2 fluorescence-encoded micro-beads are crosslinked two anti-".This causes in the subsequent detection, can detect the described ternary complex of some.

In contrast, if the range of linearity that the concentration of antigen to be detected surpass to detect in the sample and to be in HD-HOOK district (be to contain the quantity of target antigen in the sample obviously greater than two quantity that resist in the detection architecture, the two then nearly all anti-target antigens that all are incorporated in the sample, so do not have or do not have basically two of unbound state to resist in the system), antigen on No. 2 microballoons will be difficult to run into remaining two free anti--PE in the reactive system and combine so, therefore be difficult to form the ternary complex that " antigen-PE mark that No. 2 fluorescence-encoded micro-beads are crosslinked two anti-" constitutes.This causes in the subsequent detection, and it is very low to detect the ternary complex or the reading that constitute less than " antigen-PE mark that No. 2 fluorescence-encoded micro-beads are crosslinked two anti-".

On the Luminex detector, to handle simultaneously with No. 1 microballoon for fluorescence-encoded No. 2, microballoon is lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the decision of the coding of a branch of judgement microballoon is indicated microballoon for HD-HOOK; Another bundle is measured the fluorescence intensity of PE on the microballoon, judges according to fluorescence signal is strong and weak whether this serum sample is the HD-HOOK serum sample.If the fluorescence intensity from No. 2 microballoons (HD-HOOK indicates microballoon) lower (as the indication microballoon fluorescence signal value (normal value) when being lower than no HD-HOOK effect 90%, preferably be lower than 50%, more preferably be lower than 30%, be lower than 10% best), then the concentration of determined antigen is in the HD-HOOK district in the decidable sample, and promptly sample is the HD-HOOK sample.

Below, be that the situation of tumor markers is an example with antigen, further specify associative operation details of the present invention.

The binary complex of first antibody-microballoon (detection microballoon)

In the present invention, the binary complex of first antibody-microballoon has formula (I) structure:

anti ₁X-bead (I)

In the formula, X represents tumor markers, anti ₁X represents the first antibody of antitumor mark X, and bead represents microballoon, the covalent bond between-expression first antibody and the microballoon;

One anti-and microballoon crosslinked

Anti-(anti at different tumor markers antigens ₁X, X represent tumor markers antigen) can use conventional method with the DOP detailed operating procedure of microballoon covalent cross-linking, for example according to the product description or the website of Luminex company: Www.luminexcorp.comDescribed in method carry out coupled, thereby obtain different microballoons and the corresponding one anti-coupled thing anti of formation ₁X-Beads.

Get anti respectively at different tumor markerses ₁X-Beads mixes just to obtain first antibody solution (abbreviating A liquid as) by a certain percentage.

The binary complex of mouse source antibody-microballoon (HAMA indicates microballoon)

By above-mentioned similar approach,, form the binary complex (being HAMA indication microballoon) of mouse source antibody-microballoon with the microballoon covalent cross-linking of mouse source antibody and another kind of number.HAMA is indicated microballoon and detects the microballoon mixing, is A liquid together.

Two anti-marks

Though two anti-available various detectable signals known in the art carry out mark.Yet, preferably carry out mark, especially by biotin-avidin connected mode mark PE with fluorescence signal.

In a preference, two biotin (Biotin) labeling methods that resist are as follows: get two anti-(anti at different tumor markers antigens respectively ₂X, X represent tumor markers antigen) add biotin dimethyl sulfoxide (DMSO) (DMSO) solution behind the dialysis purifying, the lucifuge reaction, unreacted biotin is removed in dialysis, preserves standby.

Get biotin labeled anti respectively at different tumor markers antigens ₂X mixes in proportion, adds the PE of Avidin (Streptavidin) mark, and biotin is combined with Streptavidin, and generating and being with fluorescein-labeled second antibody (is PE-anti ₂X, wherein PE represents phycoerythrin), obtain second antibody solution (abbreviating C liquid as).

Quality Control or standard

In order to eliminate false positive and false negative, Quality Control should be set in testing process.Standard solution with in the standard items preparation finite concentration scope of certain antigen is B liquid.

In addition, in order to obtain quantitative result, the standard items of a plurality of tumor markerses that contain concentration known can be set in testing process.

For example, antigen standard (STD _n, n=0～5) and quality-control product (Quality Control 1, Quality Control 2) but the preparation of the preparation according to the form below of solution

Table 1: hybrid antigen standard quality-control product solution preparation table

Tumor markers TM	STD0	STD1	STD2	STD3	STD4	STD5	Quality Control 1	Quality Control 2
									1	0	C 1-1	C 1-2	C 1-3	C 1-4	C 1-5	C 1-6	C 1-7
2	0	C 2-1	C 2-2	C 2-3	C 2-4	C 2-5	C 2-6	C 2-7
									3	0	C 3-1	C 3-2	C 3-3	C 3-4	C 3-5	C 3-6	C 3-7
4	0	C 4-1	C 4-2	C 4-3	C 4-4	C 4-5	C 4-6	C 4-7
									5	0	C 5-1	C 5-2	C 5-3	C 5-4	C 5-5	C 5-6	C 5-7
6	0	C 6-1	C 6-2	C 6-3	C 6-4	C 6-5	C 6-6	C 6-7

· · · · · ·	0	· · · · · ·	· · · · · ·	· · · · · ·	· · · · · ·	· · · · · ·	· · · · · ·	· · · · · ·
									40	0	C 40-1	C 40-2	C 40-3	C 40-4	C 40-5	C 40-6	C 40-7

The 1st classifies different tumor markerses as in the table, and STD0 represents that the concentration of all tumor markerses in this standard solution is 0, is the starting point of typical curve; STD1 represents that the concentration of the different tumor markerses in this standard solution is respectively C _1-1, C _2-1, C _3-1... C _40-1, be the 2nd point of typical curve; The implication of the rest may be inferred STD2, STD3, STD4, STD5; The concentration of the different tumor markerses in Quality Control 1 this standard solution of expression is respectively C _1-6, C _2-6, C _3-6... C _40-6, between STD0 and STD5, be inner Quality Control point; The concentration of the different tumor markerses in Quality Control 2 these standard solution of expression is respectively C _1-7, C _2-7, C _3-7... C _40-7, between STD0 and STD5, be other 1 inner Quality Control point.STD0～STD5 and Quality Control 1 and Quality Control 2 constitute control liquid (abbreviating B liquid as).

With above-mentioned first antibody solution, control liquid and second antibody solution (being A, B and C liquid) mixing successively or simultaneously, fully reaction then (as at 37 ± 5 ℃ of reaction 10-100min), reading on luminex100 can obtain many typical curves (concrete number is by tumor markers number decisions different in the tumor markers combination) subsequently.

2.5HAMA the detection by quantitative of certain antigen in the judgement of serum sample and the normal serum sample

With A, B and C liquid mixing, 37 ℃ of reaction 30min, reading on luminex100 obtains different antigen concentrations on No. 1 microballoon (detection microballoon) and makes typical curve with corresponding fluorescence signal; What No. 2 microballoons (HAMA indicates microballoon) were gone up correspondence is the fluorescence signal value of HAMA reaction negative;

With A, human serum sample and C liquid mixing, 37 ℃ of reaction 30min, the fluorescence signal on the reading on luminex100, No. 1 microballoon converses the concentration of certain antigen according to typical curve.The fluorescence signal on No. 2 microballoons and the fluorescence signal value of HAMA reaction negative be its size relatively.If it is bigger more than 5 times then judge that serum sample is positive than the fluorescence signal value of HAMA reaction negative.

(d) principle of indication HD-HOOK method

In another preference, on the one hand, also can indicate the HD-HOOK effect simultaneously in the inventive method.

At first, on No. 3 fluorescence-encoded microballoons with the pure product of the crosslinked detected antigen of covalent.Crosslinkedly have after an anti-microballoon and sample, two anti--PE reactions finish at No. 1, in described crosslinked No. 3 fluorescence-encoded microballoons (being called " HD-HOOK indicate microballoon ") the adding reactive system that antigen arranged.

At this moment, if to be in non-HD-HOOK district (be to contain the quantity of antigen to be detected and not obvious greater than two anti-quantity in the detection architecture in the sample to the concentration of antigen to be detected in the sample, therefore some or the two more anti-unbound states that are), so following reaction can take place: the antigen on No. 3 microballoons combines with remaining two free anti--PE in the reactive system, the ternary complex of final formation " antigen-PE mark that No. 3 fluorescence-encoded micro-beads are crosslinked two anti-".This causes in the subsequent detection, can detect the described ternary complex of some.

In contrast, if the range of linearity that the concentration of antigen to be detected surpass to detect in the sample and to be in HD-HOOK district (be to contain the quantity of target antigen in the sample obviously greater than two quantity that resist in the detection architecture, the two then nearly all anti-target antigens that all are incorporated in the sample, so do not have or do not have basically two of unbound state to resist in the system), antigen on No. 3 microballoons will be difficult to run into remaining two free anti--PE in the reactive system and combine so, therefore be difficult to form the ternary complex that " antigen-PE mark that No. 3 fluorescence-encoded micro-beads are crosslinked two anti-" constitutes.This causes in the subsequent detection, and it is very low to detect the ternary complex or the reading that constitute less than " antigen-PE mark that No. 3 fluorescence-encoded micro-beads are crosslinked two anti-".

On the Luminex detector, to handle simultaneously with No. 1 microballoon for fluorescence-encoded No. 3, microballoon is lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the decision of the coding of a branch of judgement microballoon is indicated microballoon for HD-HOOK; Another bundle is measured the fluorescence intensity of PE on the microballoon, judges according to fluorescence signal is strong and weak whether this serum sample is the HD-HOOK serum sample.If the fluorescence intensity from No. 3 microballoons (HD-HOOK indicates microballoon) lower (as the indication microballoon fluorescence signal value (normal value) when being lower than no HD-HOOK effect 90%, preferably be lower than 50%, more preferably be lower than 30%, be lower than 10% best), then the concentration of determined antigen is in the HD-HOOK district in the decidable sample, and promptly sample is the HD-HOOK sample.

With antigen is that the situation of tumor markers is an example, and the associative operation details of indication HD-HOOK effect is basically with indication HAMA reaction, and difference is that mainly the preparation of HD-HOOK indication microballoon and HD-HOOK indication microballoon are just to add after reaction finishes.

The binary complex of antigen-microballoon (HD-HOOK indicates microballoon)

By above-mentioned similar approach, with the microballoon covalent cross-linking of pure product of detected antigen and another kind of number, form the binary complex (being HD-HOOK indication microballoon) of antigen-microballoon, corresponding solution abbreviates H liquid (HD-HOOK effect indication microballoon suspension) as.

The detection of sample

The sample that available the inventive method detects is not particularly limited, and can be any sample that contains tumor markers, and representational example comprises serum sample, urine specimen, saliva sample etc.Preferred sample is a blood serum sample.

(A) (detection by quantitative that is certain antigen in the judgement of serum sample and the normal serum sample) disturbed in indication HAMA reaction

With A, B and C liquid mixing, 370C reacts 30min, and reading on luminex100 obtains different antigen concentrations and makes typical curve with corresponding fluorescence signal on No. 1 beads; It is the fluorescence signal value of HAMA reaction negative that No. 2 beads go up corresponding;

With A, human serum sample and C liquid mixing, 370C reacts 30min, and the fluorescence signal on the reading on luminex100, No. 1 beads converses the concentration of certain antigen according to typical curve.The fluorescence signal on No. 2 beads and the fluorescence signal value of HAMA reaction negative be its size relatively.If it is bigger more than 5 times then judge that serum sample is positive than the fluorescence signal value of HAMA reaction negative.

B. indicate the HD-HOOK effect

The fluorescence signal value of No. 2 microballoons when (b1) measuring no HD-HOOK effect

Only to detect a kind of antigen is example, and No. 1 microballoon is the crosslinked anti-microballoon (be called and detect microballoon) that has; No. 3 microballoons are the crosslinked microballoon (HD-HOOK indicates microballoon) that antigen is arranged, and wherein the coding fluorescence of HD-HOOK indication microballoon is different from the detection fluorescence that detects microballoon.With A, B and C liquid mixing, 37 ℃ of reaction 30min add H liquid then, 37 ℃ of reaction 10min, and reading on luminex100 obtains different antigen concentrations and makes typical curve with corresponding fluorescence signal on No. 1 microballoon; Corresponding indication microballoon signal value (normal value) during for no HD-HOOK effect on No. 3 microballoons;

(b2) detection by quantitative of antigen in the indication of HD-HOOK effect and the sample

With A, human serum sample and C liquid mixing, 37 ℃ of reaction 30min add H liquid then, 37 ℃ of reaction 10min, and the fluorescence signal on the reading on luminex100, No. 1 microballoon converses the concentration of certain antigen according to typical curve.The fluorescence signal value that fluorescence signal on No. 3 microballoons and above-mentioned steps are determined HD-HOOK effect feminine gender is its size relatively.

If 90% of the indication microballoon signal value (normal value) when the fluorescence signal value of indication microballoon is equal to or less than no HD-HOOK effect (preferably is equal to or less than 50%, more preferably be equal to or less than 30%, be equal to or less than 10% best)), judge that then this sample is the HD-HOOK effect positive.

If the multiple antigen in the while test sample, can correspondingly use multiple detection microballoon and HD-HOOK indication microballoon, promptly can use a kind of detection microballoon and HD-HOOK indication microballoon to each, and the different coding fluorescence that has of various detection microballoons and indication microballoon, thereby can distinguish mutually.

Major advantage of the present invention is:

(a) by using the microballoon of indication heterophile antibody interference, can significantly improve the accuracy of dibit point sandwich immunoassay, reduce the false positive rate of dibit point sandwich immunoassay simultaneously.

(b) simple to operate.

(c) be used the microballoon of indicating the HD-HOOK effect, can further significantly improve the accuracy of dibit point sandwich immunoassay, reduce the false negative rate of dibit point sandwich immunoassay simultaneously.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

Material:

Antigen and antibody sources:

Raw material	Lot number/article No.	Manufacturer
			CA125-antibody	M86306M(groupA)	Biodesign
CA125-antibody	M86294M(groupB)	Biodesign
			CA125 Ag	30AC20	Fitzgerald
CA153-antibody	M37901M(clone695)	Biodesign
			CA153-antibody	M37552M(clone552)	Biodesign

Raw material	Lot number/article No.	Manufacturer
			CA153 Ag	30AC16	Fitzgerald
F-PSA-antibody	M86209M(total)	Biodesign
			F-PSA-antibody	M86806M(PEree)	Biodesign
PSA antigen	A86878H Pure	Biodesign
			T-PSA-antibody	M86506M(total)	Biodesign
T-PSA-antibody	M86209M(total)	Biodesign
			PSA antigen	A86878H Pure	Biodesign
CA199-antibody	M8073022	Fitzgerald
			CA19-9 Ag	30AC14	Fitzgerald
NSE-antibody	9601	Medix
			NSE-antibody	9602	Medix
NSE Ag	30-AN10	Fitzgerald
			β-HCG-antibody	5012	Medix
β-HCG-antibody	5006	Medix
			β-HCG Ag	A81455M	Biodesign
CA242-antibody	101-01	Canag
			CA242 Ag		Canag
AFP-antibody	G4	The Shanghai The 2nd Army Medical College
			AFP-antibody	C2	The Shanghai The 2nd Army Medical College
AFP Ag		Biodesign
			CEA-antibody	A1	The Shanghai The 2nd Army Medical College
CEA-antibody	C9	The Shanghai The 2nd Army Medical College
			CEA Ag	A86808H	Biodesign

Microballoon (Beads) is available from U.S. Luminex company, and specification is 5.0 μ m, and microsphere surface is-the COOH modification that other conventional reagent are commercially available product.

Embodiment 1:

10 kinds of tumor markers parallel detections (no HID-HOOK indication is as good as the indication of preferendum antibody interferes with)

1. prepare

1.1 anti-a removal in advance contains amino micromolecule and foreign protein (dialysis or mistake chromatographic column), measures its concentration.

1.2 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-hydroxysulfosuccinimide (NHS), standby (protection against the tide).

1.3 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-ethyl-N ' (3-dimethylainopropyl)-carbodiimide (EDC), standby (protection against the tide).

2. microballoon (Beads) activation

2.1 microballoon stoste vortex DL instrument suspendible 20 seconds pipettes 200 μ L microballoons and (is equivalent to 2.5 * 10 ⁶Microballoon) in the 1.5ml polypropylene centrifuge tube.

2.2 centrifugal 2 minutes of 15000rpm (being provided with 3 minutes) removes supernatant.

2.3 add 100 μ L distilled water, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm removes supernatant.

2.4 repeat 2.3.

2.5 add 80 μ L 0.1mol/L phosphate buffers (PBS), pH6.2, vortex DL instrument suspendible 20 seconds.

2.6 (NHSS) is diluted to 50mg/ml with distilled water.(now with the current)

Be added in the Beads solution vortex DL instrument suspendible 20 seconds 2.7 get 10 μ L 50mg/ml (NHSS).

2.8 EDC is diluted to 50mg/ml with distilled water.(now with the current)

Be added in the Beads solution vortex DL instrument suspendible 20 seconds 2.9 get 10 μ L 50mg/ml EDC.

2.10 lucifuge, 37 ℃ were hatched 20 minutes.

2.11 centrifugal 2 minutes of 15000rpm removes supernatant.

2.12 add (MES) pH5.0 of 250 μ L 50mmol/L 2-(N-morpholino) ethanesulfonic acid (MES).

2.13 centrifugal 2 minutes of 15000rpm removes supernatant.

2.14 repeat 2.12,2.13, descend the step experiment at once.

3. crosslinked, sealing and store

3.1 what add that 20 μ g have handled is one anti-in the Beads that has activated, vortex DL instrument suspendible 20 seconds.

3.2 lucifuge, 37 ℃ the reaction 2 hours, per 15 minutes mixings are once.

3.3 add 1ml PBS-TBN, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant (bSA of the phosphate buffer that consists of 10mmol/L pH7.4 of PBS-TBN, 0.02% polysorbas20,1mg/ml and 0.05% Sodium azide).

3.4 add 500 μ L PBS-TBN, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant.

3.5 add 500 μ L PBS-TBN, vortex DL instrument suspendible 20 seconds.

3.6 2-8 ℃ keeps in Dark Place.

4. two resist (anti ₂X) biotin labeling

4.1 two anti-pre-service

Two contain Sodium azide, glycocoll etc. in anti-contains amino micromolecule and other micromolecule	1 * PBS with pH7.4 fully dialyses.
		Two contain big molecule such as bovine serum albumin(BSA) in anti-	Protein A post or other pillar purifying.
Two anti-concentration calibrations	Its OD280 of spectrophotometric instrumentation (1OD280 is equivalent to the 0.7mg/ml monoclonal antibody).Finally with 1 * PBS, pH7.4 schedules 2mg/ml (concentrate and use the centrifugal post of Pall company's desalination) with concentration.

4.2 biotin labeling reaction

Get above-mentioned pretreated two anti-25 μ L, add the 1mg/ml NHSS-biotin DMSO solution of 25 μ L, mixing, 4 ℃ of refrigerator lucifuges were reacted 2 hours.

5. the preparation of hybrid antigen standard, quality-control product (B liquid)

TM	STD0	STD1	STD2	STD3	STD4	Quality Control 1	Quality Control 2
								β-HCG	0mIU/ml	0.5mIU/ml	2mIU/ml	20mIU/ml	100mIU/ml	2mIU/ml	20mIU/ml
CA19-9	0U/ml	5U/ml	40U/ml	200U/ml	800U/ml	40U/ml	200U/ml
								free-PSA	0ng/ml	0.5ng/ml	2ng/ml	20ng/ml	100ng/ml	2ng/ml	20ng/ml
total-PSA	0ng/ml	0.5ng/ml	2ng/ml	20ng/ml	100ng/ml	2ng/ml	20ng/ml
								NSE	0ng/ml	5ng/ml	20ng/ml	60ng/ml	120ng/ml	20ng/ml	60ng/ml

CA125	0U/ml	40U/ml	200U/ml	400U/ml	800U/ml	200U/ml	400U/ml
								CA15-3	0U/ml	1U/ml	5U/ml	30U/ml	240U/ml	5U/ml	30U/ml
CA242	0U/ml	10U/ml	50U/ml	200U/ml	400U/ml	50U/ml	200U/ml
								AFP	0ng/ml	5ng/ml	20ng/ml	200ng/ml	500ng/ml	20ng/ml	200ng/ml
CEA	0ng/ml	5ng/ml	50ng/ml	200ng/ml	800ng/ml	50ng/ml	200ng/ml

Phosphate buffer with pH7.4 is prepared B liquid by last table.

6.A the preparation of liquid

Get the following anti of 10 kinds of different B eads ₁β-HCG-Beads, anti ₁PEree-PSA-Beads, anti ₁Total-PSA-Beads, anti ₁NSE-Beads, anti ₁CA15-3-Beads, anti ₁CA19-9-Beads, anti ₁CA125-Beads, anti ₁CA242-Beads, anti ₁APEP-Beads, anti ₁CEA-Beads solution is by 4 * 10 ⁵Individual/kind of mixing, be added among the pH7.4 PBS, volume is 5ml, 4 ℃ keep in Dark Place standby.

7. mix two anti--PE potpourri (C liquid) preparations

Get the PEree-PSA of the good biotin of mark respectively, total-PSA, NSE, CA242, CA19-9, CA125, β-HCG, CA15-3, APEP is among the CEA two anti-adding pH7.4 PBS, every kind two anti-final concentration is 5 μ g/ml, adding the PE total concentration simultaneously is 60 μ g/ml, mix two anti--PE potpourri cumulative volume is 5ml, 4 ℃ keep in Dark Place standby.

8. patients serum's tumor in digestive tract mark content detection

8.1 collect 10 parts of 2ml/ parts of patient's blood sample, 5000rpm * 5min gets supernatant, and is standby.

8.2 add A liquid respectively in 96 hole ELISA Plate, 50 μ l/ holes; Add standard items (STD0, STD1, STD2, STD3, STD4, STD5), quality-control product (Contol1, Contol2), blood serum sample 1-9 number, 5 μ l/ holes then; Add C liquid again, 50 μ l/ holes.Abundant mixing is put into 37 ℃ of shaking tables and is hatched 40min. on vortex DL instrument

8.3 hatch finish after on vortex DL instrument abundant mixing reading on Luminex100.

8.4 testing result sees the following form:

The position	Sample	β-HCG	CA199	f-PSA	t-PSA	NSE	CA125	CA153	CA242	AFP	CEA	Total incident
													1	20ul-0	51	68	85.5	77	64	64	79	69	33.5	47	1286
2	20ul-1	130	231	88	112	124	48	343	103	75	107	1243
													3	20ul-2	300.5	435	338	1006	639.5	329.5	442	160	418.5	244.5	1282
4	20ul-3	2256	678.5	2812	7041.5	1313	751	336	469.5	2417	540	1268
													5	20ul-4	5087.5	822	8409	18835	2091	1112.5	281	678	3553	850	1359
6	1	78.5	196	104	81.5	560	91.5	123	75.5	115.5	102	1258
													7	2	109	117	91	239.5	662	78.5	90.5	95	113	80	1177
8	3	71.5	56	105	61	182	65.5	86	68	82	97	1344
													9	4	70	90	109	97	140	76	126	67	59	53	1229
10	5	91	44	120.5	401.5	67	84.5	116	49	35	60	1216
													11	6	77.5	73.5	177	95	499	299	121	133	2187	99.5	1279
12	7	76.5	88	4397	10944	421.5	71	186.5	67	174	50	1355
													13	8	39.5	59	90.5	56.5	456	74.5	136.5	71.5	60.5	21	1253
14	9	77	67.5	91.5	76	353	56.5	102	57	86	77	1250

Annotate: bold Italic partly is the standard items testing result in the form, and other are the pattern detection result.

The result shows, can obtain the quantitative measurement result of a plurality of tumor markerses simultaneously with the inventive method, for example has a large amount of tumor markers t-PSA in No. 7 sample, provides complementary reference index thereby can be clinical diagnosis.

Embodiment 2

The indication of human serum sample HAMA reaction and to the detection of alpha-fetoprotein in the serum (AFP)

The mouse IgG that at the antibody of HAMA reaction is purifying is available from by Biodesign company, anti-and two anti-the AFP standard items are available from Biodesign company available from the Shanghai The 2nd Army Medical College at AFP, and Beads is available from Luminex company.

1. prepare

1.1 anti-a removal in advance with AFP antigen contains amino micromolecule and foreign protein (dialysis or mistake chromatographic column), measures its concentration.

1.2 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-hydroxysulfosuccini-mide (NHS), standby (protection against the tide).

1.3 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-(3-dimethylainopropyl)-N-ethyl-carbodiimide (EDC), standby (protection against the tide).

2. microballoon (beads) activation

2.1 get respectively No. 33 and No. 46 beads stoste vortex DL instrument suspendibles 20 seconds, pipette 200 μ Lbeads and (be equivalent to 2.5 * 10 ⁶Beads) in two 1.5ml polypropylene centrifuge tubes.

2.2 centrifugal 2 minutes of 15000rpm (being provided with 3 minutes) removes supernatant.

2.3 add 100 μ L distilled water, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm removes supernatant.

2.4 repeat 2.3.

2.5 in two pipes, add 80 μ L 0.1mol/L phosphate buffers (PBS) respectively, pH6.2, vortex DL instrument suspendible 20 seconds.

2.6 (NHSS) is diluted to 50mg/ml with distilled water.(now with the current)

Be added in two kinds of beads solution vortex DL instrument suspendible 20 seconds 2.7 get 10 μ L 50mg/ml (NHSS) respectively.

2.8 EDC is diluted to 50mg/ml with distilled water.(now with the current)

Be added in two kinds of beads solution vortex DL instrument suspendible 20 seconds 2.9 get 10 μ L 50mg/ml EDC respectively.

2.10 lucifuge, 37 ℃ were hatched 20 minutes.

2.11 centrifugal 2 minutes of 15000rpm removes supernatant.

2.12 add 250 μ L 50mmol/L 2-(N-morpholino) ethanes μ Lfonic acid (MES) pH5.0 respectively.

2.13 centrifugal 2 minutes of 15000rpm removes supernatant.

2.14 repeat 2.12,2.13, descend the step experiment at once.

3. crosslinked, sealing and store

3.1 getting the mouse IgG that 20 μ g have handled well joins among No. 46 beads that activated; Get AFP one anti-the joining among No. 33 beads that activated that 20 μ g have handled well.Vortex DL instrument suspendible 20 seconds.

3.2 lucifuge, 37 ℃ the reaction 2 hours, per 15 minutes mixings are once.

3.3 add 1ml PBS-TBN respectively, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant (bSA of the phosphate buffer that consists of 10mmol/L pH7.4 of PBS-TBN, 0.02% polysorbas20,1mg/ml and 0.05% Sodium azide).

3.4 add 500 μ L PBS-TBN respectively, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant.

3.5 add 500 μ L PBS-TBN respectively, vortex DL instrument suspendible 20 seconds.

3.6 the concentration with two kinds of beads of microscopic counting is respectively:

No. 33: 1 * 10 ⁶Individual/ml, No. 46: 1 * 10 ⁶Individual/ml.

3.7 2-8 ℃ keeps in Dark Place.

4. two anti-biotin (Biotin) marks

4.1 two anti-pre-service

Two contain Sodium azide, glycocoll etc. in anti-contains amino micromolecule and other micromolecule	1 * PBS with pH7.4 fully dialyses.
		Two contain big molecule such as bovine serum albumin(BSA) in anti-	Protein A post or other pillar purifying.
Two anti-concentration calibrations	Its OD280 of spectrophotometric instrumentation (1OD280 is equivalent to the 0.7mg/ml monoclonal antibody).Finally with 1 * PBS, pH7.4 schedules 2mg/ml (concentrate and use the centrifugal post of Pall company's desalination) with concentration.

4.2 biotin labeling reaction

Get above-mentioned pretreated AFP two anti-10 μ L, add the 1mg/ml NHSS-Biotin DMSO solution of 25 μ L, mixing, 4 ℃ of refrigerator lucifuges were reacted 2 hours.Dialysed overnight is standby.

5. the preparation of antigen standard items (B liquid)

Phosphate buffer compound concentration with pH7.4 is the AFP standard items liquid of 0ng/ml, 5ng/ml, 20ng/ml, 200ng/ml, 500ng/ml.

6.A the preparation of liquid

Get an amount of mark respectively one anti-microballoon join for No. 33 and No. 46 in the phosphate buffer of 1ml pH7.4, make in the solution every kind to contain 10000 microballoons approximately.

7. the preparation of two anti--PE (C liquid)

It is anti-to get the good biotinylation AFP of mark two, adds in the pH7.4 phosphate buffer, and two anti-final concentrations are 5 μ g/ml, and the PE total concentration that adds the streptavidin mark simultaneously is 60 μ g/ml, two anti--the PE cumulative volume is 5ml, 4 ℃ keep in Dark Place standby.

8.HAMA the detection of AFP in the indication of reaction and the human serum

8.1 collect 5 parts of 5 parts of 2ml/ parts of human serum sample of reacting with no HAMA of human serum sample that the HAMA reaction is arranged, 5000rpm * 5min gets supernatant, and is standby.

8.2 add each 5 μ L of B liquid of 5 variable concentrations in 5 reacting holes of 96 hole reaction plates successively, every hole adds 25 μ L A liquid and 25 μ L C liquid, mixing more respectively; Select 10 holes else and add 10 kinds of above-mentioned human serum sample 5 μ L/ holes and 25 μ L/ hole A liquid and 25 μ L/ hole C liquid, mixing respectively; In 37 ℃ of lucifuge incubator reactions 40 minutes.

8.3 hatch finish after on vortex DL instrument abundant mixing, reading on Luminex100.

8.4 testing result is as follows:

Laboratory test results

	Sample or concentration known	Detect microballoon (33#)	HAMA indicates microballoon (46#)
				Standard items	0ng/ml 5ng/ml 20ng/ml 200ng/ml 500ng/ml	56.7 250.8 768.8 4829.5 7439.5	90 101 110 96 95
Sample	Sample 1 sample 2 samples 3 samples 4 samples 5 samples 6 samples 7 samples 8 samples 9 samples 10	2845.0 2875.0 2349.0 3215.0 3014.5 115.0 3478.0 257.0 6473.0 578.5	29364.5 29687 25476.5 32452.5 31484 145 123 114.5 134 94.5

8.5 experimental result is judged

The average MIF of 46# microballoon standard items HAMA feminine gender is 113, and the MIF of sample 1 to 5 all is higher than more than 10 times of HAMA feminine gender, and therefore indicating the sample of sample 1 to 5 is the HAMA serum sample.And the MIF of sample 6 to 10 is less than 1.5 * 113, and then judgement sample 6 to 10 is the HAMA feminine gender.

Embodiment 3

5 kinds of tumor markers parallel detections (band HAMA indication)

Basically according to the step of embodiment 1 and 2, with 5 kinds of tumor markers parallel detections of HD-HOOK indication, difference is to select for use SCCA, CA125, CA15-3, CEA, β-HCG as antigen to be detected.Use a kind of detection microballoon and HAMA indication microballoon at each, and 5 kinds of coding fluorescences that the detection microballoon is different with having of 5 kinds of indication microballoons.

In addition, the compound concentration of hybrid antigen standard (B liquid) is as follows:

TM	STD1	STD2	STD3	STD4	STD5	STD6
							SCCA	0ng/ml	2ng/ml	20ng/ml	200ng/ml	1000ng/ml	2000ng/ml
CA125	0U/ml	40U/ml	200U/ml	600U/ml	1200U/ml	2400U/ml
							CA15-3	0U/ml	1U/ml	10U/ml	100U/ml	400U/ml	800U/ml
CEA	0ng/ml	2ng/ml	20ng/ml	200ng/ml	1200ng/ml	2400ng/ml
							β-HCG	0ng/ml	2ng/ml	20ng/ml	200ng/ml	1000ng/ml	2000ng/ml

Phosphate buffer with pH7.4 is prepared B liquid by last table.

In addition, the preparation of A liquid, C liquid and H liquid is with embodiment 1 and 2.

Microballoon with preparation detects patients serum's tumor in digestive tract mark content, and method is with embodiment 2.Testing result sees the following form:

	SCCA	CA125	CA15-3	CEA	β-HCG	Hama indicates microballoon
							Std1	85	78	95	75	76	84
Std2	198	155	354	214	225	94
							Std3	534	457	945	546	635	105
Std4	1245	1145	2785	1345	2140	125
							Std5	3489	2875	5345	3575	4575	114
Std6	7985	5978	9475	7540	8745	135
							Sample 1	3245	1257	4575	2475	1350	125
Sample 2	104	785	157	650	900	134
							Sample 3	752	74	978	75	1240	116
Sample 4	92	68	125	87	70	105
							Sample 5	85	95	6475	3215	96	95
Sample 6	24573	450	27851	23565	27452	26485
							Sample 7	30215	27955	28450	23451	29785	27850
Sample 8	29451	26785	26450	27459	23875	28950
							Sample 9	16470	10054	19075	15742	18642	147820
Sample 10	21453	22078	25409	20985	22456	21765

Experimental result is judged: sample 1-5 is the HAMA negative sample, and sample 6-10 is the HAMA positive sample.

Embodiment 4

The quantitative detection of alpha-fetoprotein (AFP) in the indication of human serum sample HD-HOOK effect and the serum

One anti-and two anti-ly provide at AFP by the Shanghai The 2nd Army Medical College, the pure product of AFP antigen are provided by Biodesign, and microballoon is provided by Luminex company, and conventional reagent is commercially available product.

1. prepare

1.1 anti-a removal in advance with AFP antigen contains amino micromolecule and foreign protein (dialysis or mistake chromatographic column), measures its concentration.

1.2 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-hydroxysulfosuccini-mide (NHS), standby (protection against the tide).

1.3 in the 1.5ml polypropylene centrifuge tube, accurately take by weighing 5mg left and right sides N-(3-dimethylainopropyl)-N-ethyl-carbodiimide (EDC), standby (protection against the tide).

2. microballoon activation

2.1 get respectively No. 33 and No. 51 microballoons (Luminex company) stoste vortex DL instrument suspendible 20 seconds, pipette 200 μ L microballoons and (be equivalent to 2.5 * 10 ⁶Microballoon) in two 1.5ml polypropylene centrifuge tube.

2.2 centrifugal 2 minutes of 15000rpm (being provided with 3 minutes) removes supernatant.

2.3 add 100 μ L distilled water, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm removes supernatant.

2.4 repeat 2.3.

2.5 in two pipes, add 80 μ L 0.1mol/L phosphate buffers (PBS) respectively, pH6.2, vortex DL instrument suspendible 20 seconds.

2.6 (NHSS) is diluted to 50mg/ml with distilled water.(now with the current)

Be added in two kinds of microspheres solution vortex DL instrument suspendible 20 seconds 2.7 get 10 μ L 50mg/ml (NHSS) respectively.

2.8 EDC is diluted to 50mg/ml with distilled water.(now with the current)

Be added in two kinds of microspheres solution vortex DL instrument suspendible 20 seconds 2.9 get 10 μ L 50mg/ml EDC respectively.

2.10 lucifuge, 37 ℃ were hatched 20 minutes.

2.11 centrifugal 2 minutes of 15000rpm removes supernatant.

2.12 add 250 μ L 50mmol/L 2-[N-Morpholino respectively] ethanesulfonic acid (MES) pH5.0.

2.13 centrifugal 2 minutes of 15000rpm removes supernatant.

2.14 repeat 2.12,2.13, descend the step experiment at once.

3. crosslinked, sealing and store

3.1 get AFP one anti-the joining in No. 33 microballoons that activated that 20 μ g have handled well; Getting the AFP antigen that 20 μ g have handled well joins in No. 51 microballoons that activated.Vortex DL instrument suspendible 20 seconds.

3.2 lucifuge, 37 ℃ the reaction 2 hours, per 15 minutes mixings are once.

3.3 add 1ml PBS-TBN respectively, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant (bSA of the phosphate buffer that consists of 10mmol/L pH7.4 of PBS-TBN, 0.02% polysorbas20,1mg/ml and 0.05% Sodium azide).

3.4 add 500 μ L PBS-TBN respectively, vortex DL instrument suspendible 20 seconds, centrifugal 2 minutes of 15000rpm.Remove supernatant.

3.5 add 500 μ L PBS-TBN respectively, vortex DL instrument suspendible 20 seconds.

3.6 the concentration with two kinds of microballoons of microscopic counting is respectively:

No. 33: 1 * 10 ⁶Individual/ml, No. 51: 1 * 10 ⁶Individual/ml.

3.7 2-8 ℃ keeps in Dark Place.

4. two anti-biotin labelings

4.1 two anti-pre-service

Two contain Sodium azide, glycocoll etc. in anti-contains amino micromolecule and other micromolecule	1 * PBS with pH7.4 fully dialyses.
		Two contain big molecule such as bovine serum albumin(BSA) in anti-	Protein A post or other pillar purifying.
Two anti-concentration calibrations	Its OD280 of spectrophotometric instrumentation (1OD280 is equivalent to the 0.7mg/ml monoclonal antibody).Finally with 1 * PBS, pH7.4 schedules 2mg/ml (concentrate and use the centrifugal post of Pall company's desalination) with concentration.

4.2 biotin labeling reaction

Get above-mentioned pretreated AFP two anti-10 μ L, add the 1mg/ml biotin DMSO solution of 25 μ L, mixing, 4 ℃ of refrigerator lucifuges were reacted 2 hours.Dialysed overnight is standby.

5. the preparation of antigen standard items (B liquid)

Phosphate buffer compound concentration with pH7.4 is the AFP standard items liquid of 0ng/ml, 5ng/ml, 20ng/ml, 200ng/ml, 500ng/ml.

6.A the preparation of liquid

Get above-mentioned mark one No. 33 anti-microballoons join in the phosphate buffer of 1ml pH7.4, make to contain 10000 microballoons in the solution approximately.

7. the preparation of two anti--PE (C liquid)

It is anti-to get the good biotin AFP of above-mentioned mark two, adds in the pH7.4 phosphate buffer, and two anti-final concentrations are 5 μ g/ml, and the PE total concentration that adds the streptavidin mark simultaneously is 60 μ g/ml, two anti--the PE cumulative volume is 5ml, 4 ℃ keep in Dark Place standby.

8.H the preparation of liquid

Get above-mentioned mark No. 51 microballoons of AFP antigen join in the phosphate buffer of 1ml pH7.4, make to contain 10000 microballoons in the 1ml solution approximately.

9.HD-HOOK the detection of AFP in the indication of effect and the human serum

9.1 5 parts in the human serum sample of 5 parts in the human serum sample of the high value＞20000ng/ml of collection AFP detection and AFP detection high value＜1000ng/ml, 2ml/ part, 5000rpm * 5min gets supernatant, and is standby.

9.2 add each 5 μ L of B liquid of 5 variable concentrations in 5 reacting holes of 96 hole reaction plates successively, every hole adds 25 μ L A liquid and 25 μ L C liquid, mixing more respectively; Select 10 holes else and add 10 kinds of above-mentioned human serum sample 5 μ L/ holes and 25 μ L/ hole A liquid and 25 μ L/ hole C liquid, mixing respectively; In 37 ℃ of lucifuge incubator reaction 30mins, add the H liquid in 5 μ L/ holes then, mixing; In 37 ℃ of lucifuge incubator reaction 10mins.

9.3 hatch finish after on vortex DL instrument abundant mixing, reading on Luminex100.

9.4 testing result is as follows:

Laboratory test results
					Sample or concentration known	Detect microballoon (33#)	HD-HOOK indicates microballoon (51#)
Standard items	0ng/ml 5ng/ml 20ng/ml 200ng/ml 500ng/ml	56.7 250.8 768.8 4829.5 7439.5	11205.5 11304 11098 11196 11245
				Test sample	Sample 1 sample 2 samples 3 samples 4 samples 5 samples 6 samples 7 samples 8 samples 9 samples 10	7945 5678.5 6745.5 3725 9875 115.0 3478.0 257.0 6473.0 578.5	10020 154 230 145 285 12456 12378 13795 11245 11659.5

9.5 experimental result is judged

The mean value of 51# microballoon (HD-HOOK indicates microballoon) fluorescence signal numerical value when synantigen standard items concentration not is 11209.7, and 2SD is 151.0, so the normal value of indication microballoon is 11360.7; Indication microballoon fluorescence signal value all is lower than 90% (10224.63) of indication microballoon normal value in the reactive system of sample 1 to 5, therefore indicates the serum sample of sample 1 to 5 that the HD-HOOK effect is arranged.Wherein sample 1 has slight HOOK effect, causes reading on the low side, and sample 2-4 has significant HOOK effect (be lower than normal value 10%), causes reading seriously on the low side, causes false negative.And sample 6 to 10 is HD-HOOK feminine gender (promptly the numerical value of Ce Dinging is believable, can get rid of the false negative that the HD-HOOK effect causes).

Embodiment 5

5 kinds of tumor markers parallel detections (band HD-HOOK indication)

Basically according to the step of embodiment 1 and 4, with 5 kinds of tumor markers parallel detections of HD-HOOK indication, difference is to select for use SCCA, CA125, CA15-3, CEA, β-HCG as antigen to be detected.Use a kind of detection microballoon and HD-HOOK indication microballoon at each, and 5 kinds of coding fluorescences that the detection microballoon is different with having of 5 kinds of indication microballoons.

In addition, the compound concentration of hybrid antigen standard (B liquid) is as follows:

TM	STD0	STD1	STD2	STD3	STD4	STD5
							SCCA	0ng/ml	2ng/ml	20ng/ml	200ng/ml	1000ng/ml	2000ng/ml
CA125	0U/ml	40U/ml	200U/ml	600U/ml	1200U/ml	2400U/ml
							CA15-3	0U/ml	1U/ml	10U/ml	100U/ml	400U/ml	800U/ml
CEA	0ng/ml	2ng/ml	20ng/ml	200ng/ml	l200ng/ml	2400ng/ml
							β-HCG	0ng/ml	2ng/ml	20ng/ml	200ng/ml	1000ng/ml	2000ng/ml

Phosphate buffer with pH7.4 is prepared B liquid by last table.

In addition, the preparation of A liquid, C liquid and H liquid is with embodiment 1 and 2.

8.3 the microballoon with preparation detects patients serum's tumor in digestive tract mark content, method is with embodiment 2.

Testing result sees the following form:

Sample	β-HCG	β-HCG-HOOK indication microballoon fluorescent value	CA153	CA153-HOOK indication microballoon fluorescent value	CA125	CA125-HOOK indication microballoon fluorescent value	SCCA	SCCA-HOOK indication microballoon fluorescent value	CEA	CEA-HOOK indication microballoon fluorescent value
											STD0	51	11110	79	12457	64	5421	31.5	7543	47	9534
STD1	130	11045	343	11789	128	4978	145	7478	107	9745
											STD2	300.5	9876	442	13121	329.5	5246	410.5	7369	244.5	9324
STD3	2256	11132	2145	12346	751	5347	2400	7274	2147	9218
											STD4	5087.5	11095	6257	11975	1112.5	5124	3535	7345	5495	9635
STD5	9546.5	11115	12375	12678	5325	5074	7365	7510	9372.5	9415
											The normal fluorescent value of HOOK indication microballoon	11 896.0		13356.33		5537.13		7631.7		9872.82
Sample 1	81	12431	598	12475	125	5142	97	7542	411	9547
											Sample 2	5748	11456	35	13456	116.5	5147	37	7568	1419	9684
Sample 3	68.5	11345	1271	12695	113.5	5263	4570	7648	150	9387
											Sample 4	66	12785	3915	15437	61	5347	64	7489	955	9574
Sample 5	47.5	13456	102	14289	2154	5289	59	7398	1784	9471
											Sample 6	7421	11873	10471	15432	164.5	5478	120	7345	7459	7216
Sample 7	124	13452	98	13487	4712	3621	5741	5487	421	9350
											Sample 8	5321	5483	7962	6425	458	1425	75	7456	58	9415
Sample 9	75	14782	59	13756	982	1578	645	1459	6579	5215
											Sample 10	8035	7425	245	1457	3545	5374	3785	2478	1463	987

Testing result is judged: bold Italic partly is the standard items testing result in the form, and other are the pattern detection result, and the numerical value of band underscore shows that sample has the HOOK effect.

Embodiment 6

5 kinds of tumor markers parallel detections (band HD-HOOK indication and HAMA indication)

Repeat the process of embodiment 3 and 5, difference is to use simultaneously No. 31 detection microballoons (33#), No. 46 HAMA indication microballoons, No. 51 HD-HOOK to indicate microballoon that 20 samples of embodiment 3 and 5 are carried out parallel detection.

The result shows that the detection of each microballoon does not have the phase mutual interference, can detect the sample with HAMA reaction simultaneously exactly and have HD-HOOK effect sample.

Embodiment 7

Detection kit

No. 33 detection microballoons and No. 46 HAMA indication microballoons of preparing among the embodiment 1-2 are loaded on respectively in the container, make the kit that to indicate HAMA to react.

In addition, also No. 51 HD-HOOK indication microballoons of preparation among the embodiment 4 can be loaded in another container, so that indication HD-HOOK effect, thereby make the detection kit of indicating the HD-HOOK effect simultaneously.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1, the immune sandwich detection method of target antigen in a kind of test sample is characterized in that, may further comprise the steps:

(a) with sample, detect microballoon, heterophile antibody disturb indication microballoon and mark the second antibody of detectable signal mix, form a reaction system,

Wherein said detection microballoon is the binary complex of the first antibody-microballoon shown in the I formula,

anti ₁X-bead (I)

In the formula, " X " represents target antigen, " anti ₁X " represent first antibody at described target antigen " X ", " bead " represents microballoon, the combination between "-" expression first antibody and the microballoon,

And described second antibody and first antibody can be incorporated into the different epi-positions of target antigen simultaneously,

Thereby form " second antibody-antigen-first antibody-microballoon " tetraplex;

Wherein, it is the binary complex of the different preferendum interference indicant-microballoon shown in the III formula that wherein said heterophile antibody disturbs the indication microballoon,

Z-bead″ (III)

In the formula, the different preferendum of " Z " expression is disturbed indicant, " bead " " expression can with " bead " the different microballoons of difference mutually, the different preferendum of "-" expression is disturbed the combination between indicant and the microballoon,

Thereby in sample, exist under the situation of heterophile antibody, form " second antibody-heterophile antibody-different preferendum is disturbed indicant-microballoon " tetraplex;

(b) detectable signal on the microballoon in " second antibody-antigen-first antibody-microballoon " described in detection reaction system tetraplex, and with standard value or typical curve relatively, thereby whether and/or quantity the existence of determining target antigen in the reaction system;

And the detectable signal in " second antibody-heterophile antibody-different preferendum is disturbed indicant-microballoon " described in detection reaction system tetraplex on the microballoon, obtain the signal value that heterophile antibody disturbs the indication microballoon, and the heterophile antibody when being as good as preferendum antibody interferes with effect disturbs indication microballoon signal value (normal value) relatively, when heterophile antibody disturbs indication microballoon measured value to disturb 1.5 times of indication microballoon normal values greater than heterophile antibody, judge that then the measurement result of target antigen is unreliable; If heterophile antibody disturbs indication microballoon measured value to be less than or equal to heterophile antibody when disturbing 1.5 times of indication microballoon normal values, then judge to be as good as the preferendum antibody interferes with in the detected sample.

2. the method for claim 1 is characterized in that, in step (a) with (b), also comprises step:

(b1) HD-HOOK is indicated microballoon join in the system of step (a), wherein HD-HOOK indication microballoon is the binary complex of the target antigen-microballoon shown in the formula II,

X-bead’ (II)

In the formula, " bead ' " expression can with " bead " and " bead " " the different microballoons of difference mutually, "-" represents the combination between target antigen X and the microballoon,

Thereby having in the presence of the second antibody of detectable signal, forming " second antibody-antigen-microballoon " ternary complex; With

(b2) detectable signal on the microballoon in " second antibody-antigen-microballoon " ternary complex in the detection reaction system, obtain the signal value of HD-HOOK indication microballoon, and the HD-HOOK indication microballoon signal value (normal value) during with no HD-HOOK effect relatively, when HD-HOOK indication microballoon measured value is indicated the microballoon normal value less than HD-HOOK, judge that then the measurement result of target antigen is unreliable; If when HD-HOOK indication microballoon measured value is indicated the microballoon normal value more than or equal to HD-HOOK, judge that then the concentration of target antigen is in measurable range in the sample.

3. the method for claim 1 is characterized in that, first antibody, different preferendum disturb the combination between indicant or target antigen and the microballoon that covalent bond or aglucon reaction or non-specific adsorption are arranged.

4. the method for claim 1, it is characterized in that, when described heterophile antibody disturbs indication microballoon measured value to disturb 2 times of indication microballoon normal values greater than heterophile antibody, judge that then the measurement result of target antigen is unreliable, wherein said normal value is following definite:

(a ') concentration is known and target antigen standard items series that be in measurement range is mixed with described detection microballoon, the described second antibody that has detectable signal respectively, form a reaction system, thereby form " second antibody-antigen-first antibody-microballoon " tetraplex of different target antigen standard items concentration;

(b ') disturbs the indication microballoon to join in the system of step (a ') described heterophile antibody;

(c ') detects described heterophile antibody and disturbs detectable signal on the indication microballoon, with P+2SD is indication microballoon normal value, when P represented different target antigen standard items concentration in the formula, heterophile antibody disturbed the mean value of indication microballoon signal value, and 2SD is 2 times a standard deviation.

5. method as claimed in claim 2, it is characterized in that, in step (b2), when HD-HOOK indicates microballoon measured value≤0.9 * HD-HOOK to indicate the microballoon normal value, judge that then there is the HD-HOOK effect in sample, and described HD-HOOK indication microballoon normal value is in order to method is definite down:

(a ') concentration is known and target antigen standard items series that be in measurement range is mixed with described detection microballoon, the described second antibody that has detectable signal respectively, form a reaction system, thereby form " second antibody-antigen-first antibody-microballoon " tetraplex of different target antigen standard items concentration;

(b ') joins described HD-HOOK indication microballoon in the system of step (a '), thereby having in the presence of the second antibody of detectable signal " second antibody-antigen-microballoon " ternary complex of formation different target antigen standard items concentration;

(c ') detects the detectable signal on the microballoon in the described ternary complex, with P+2SD is HD-HOOK indication microballoon normal value, when P represented different target antigen standard items concentration in the formula, the mean value of microballoon signal value in the ternary complex, 2SD were the standard deviation of 2 times microballoon signal value.

6. the method for claim 1 is characterized in that, target antigen quantity is the 1-1000 kind.

7. the method for claim 1 is characterized in that, described antigen is protein.

8. method as claimed in claim 2 is characterized in that, described various microballoon bead, bead ' and bead " are the microballoons with different fluorescence.

9. kit that is used to detect target antigen is characterized in that it comprises: container, and be loaded on following material in the container respectively:

(a) detect microballoon, wherein said detection microballoon is the binary complex of the first antibody-microballoon shown in the I formula,

anti ₁X-bead (I)

In the formula, " X " represents target antigen, " anti ₁X " represent first antibody at described target antigen " X ", " bead " represents microballoon, the combination between "-" expression first antibody and the microballoon,

(b) have the second antibody of detectable signal, wherein, described second antibody and first antibody can be incorporated into the different epi-positions of target antigen simultaneously,

(c) heterophile antibody disturbs the indication microballoon, and it is the binary complex of the different preferendum interference indicant-microballoon shown in the III formula that wherein said heterophile antibody disturbs the indication microballoon,

Z-bead″ (III)

In the formula, the different preferendum of " Z " expression is disturbed indicant, described different preferendum disturbs indicant to be selected from: mouse antibodies, rat antibody, chicken source antibody, rabbit source antibody, sheep source antibody, horse source antibody, ox source antibody or rheumatoid factor, " bead " " expression can with " bead " the mutual different microballoons of difference, the different preferendum of "-" expression is disturbed the combination between indicant and the microballoon.

10. kit as claimed in claim 9 is characterized in that, also comprises

(d) HD-HOOK indication microballoon, described HD-HOOK indication microballoon is the binary complex of the target antigen-microballoon shown in the formula II,

X-bead’ (II)

In the formula, " bead ' " expression can with " bead ", " bead " " the mutual different microballoons of difference, the combination between "-" expression target antigen X and the microballoon.