CN1189618A - Immunoassay method - Google Patents

Abstract

The present invention provides an immunogen, antibodies, kits and methods of using the same to measure diacyl hydrazine compounds. The methods are easy to use, inexpensive and provide suitable cross-activity and sensitivity to enable use under FIFRA guidelines.

Description

Method of immunity

The present invention relates to be used to detect the method for immunity of diacyl hydrazide compound.

The method of measuring the diacyl hydrazide compound is known.High pressure liquid chromatography (HPLC) for example, gas-liquid chromatograph (GLC), GC-mass spectrometry and HPLC-mass spectrometry are a few methods that exists at present.But there are some shortcomings in these methods.These methods generally need costliness and complicated instrument.In addition, the preparation of sample and analysis are very long, need be to the chemist and the technician of these method special trainings.In addition, these existing technical methods can not be same step rapid screening analog or metabolin or both.In addition, before detecting, generally need to separate individual compound.So, measure as need these methods be impracticable when field test or the substantive test for rapid low consumption.

The special role of diacyl hydrazide compound is as pesticide, specifically is as quickening caterpillar decortication compound (MAC).The same with all potential harmful chemicals, these pesticides are subjected to the control of government department.In the process that obtains the control approval, according to federal insecticide, fungicide and rat poison regulations (FIFRA) registration rule, detailed rules and regulations O requires need be to residue analysis, and this analysis requires these uses are had the assay method of adequate sensitivity.In addition, assay method must have cross reactivity with the main metabolites of diacyl hydrazide pesticide so that be applicable to the FIFRA registration rule, metabolism that detailed rules and regulations N requires and environmental impact research.For the usage of monitoring pesticide may also need these mensuration.

So, needing a kind of diacyl hydrazide assay method, it has enough sensitivity and cross reaction sexual satisfaction FIFRA rule, and the energy rapid screening is used simple and inexpensive.

The inventor has developed a kind of immunochemistry and has measured, and it is enough sensitive, has enough cross reactivities and grants use with the requirement of satisfying the FIFRA rule.In addition, assay method uses simple and inexpensive.

Aspect first, provide an immunogene of the present invention, having comprised: with the compound of the following general formula of carrier mass coupling R wherein, R ¹, R ², R ³And R ⁴Be hydrogen, (C separately separately ₁-C ₆) (the C of alkyl and replacement ₁-C ₆) alkyl, (C ₂-C ₆) (the C of alkenyl and replacement ₂-C ₆) alkenyl, (C ₁-C ₆) alkoxy, (C ₁-C ₆) carboxyalkyl, and halogen atom.

Aspect second of the present invention, the method for measuring the diacyl hydrazide or derivatives thereof is provided, comprise that step (A) provides the sample of the diacyl hydrazide that contains unknown quantity; (B) when having immobilization diacyl hydrazide antigen, sample is contacted with the antibody of diacyl hydrazide antigen-binding affinity so that form combination and unconjugated antibody-antigenic compound with having; (C) unconjugated antibody-antigenic compound is separated with the antigen-antibody complex of combination; (D) with detectable label mark binding antibody compound; (E) but make label that change detected take place; (F) diacyl hydrazide in the working sample.

Aspect the 3rd of the present invention, the method for measuring the diacyl hydrazide or derivatives thereof is provided, comprise that step (A) provides the sample of the diacyl hydrazide that contains unknown quantity; (B) sample is contacted so that form immobilized antibody-antigenic compound with having with the immobilized antibody of diacyl hydrazide antigen-binding affinity; (C) the diacyl hydrazide antigen that will have a detectable label substance markers contacts the antibody-antigenic compound that forms mark with not compound immobilized antibody; (D) but make mark that change detected take place; (E) diacyl hydrazide in the working sample.

Aspect the 4th of the present invention, the kit of measuring diacyl hydrazide is provided, comprise that (A) has the antibody with the diacyl hydrazide binding affinity; (B) can with the mark of antibodies; But can produce the substrate of change detected when (C) having label.

Figure (1) has described a protein elution process of DEAE ion exchange column.

Figure (2) has described the inhibition curve of RH2703.

Figure (3) has described the inhibition curve of RH2651.

Figure (4) has described the inhibition curve of RH5992.

Figure (5) has described the inhibition curve of RH0345.

Figure (6) has described the inhibition curve of RH2485.

Figure (7) has described the external perimysium reference curve that obtains in PBST and matrix blank sample.

Figure (8) has described the linear regression line that calculates of wild cabbage sample 20139-01.

Figure (9) has described the linear regression line that calculates of wild cabbage sample 20139-02.

Figure (10) has described the linear regression line that calculates of wild cabbage sample 20139-04.

Figure (11) has described the linear regression line that calculates of wild cabbage sample 20139-05.

Figure (12) has described the linear regression line that calculates of pedotheque 151.

Figure (13) has described the linear regression line that calculates of pedotheque 157.

Figure (14) has described the linear regression line that calculates of pedotheque 175.

Figure (15) has described the linear regression line that calculates of pedotheque 187.

Figure (16) a and b have described one and have directly measured curve and direct bioassay standard curve.

As used herein, term " antigen " is understood that to stimulate any material of antibody generation. In a similar fashion, term " diacyl hydrazide antigen " is understood that to stimulate any diacyl hydrazide compound of antibody generation, and the antibody that wherein produces has the binding affinity with diacyl hydrazide and its derivative. Term " immunogene " is understood to include any material for induce immune response simultaneously. Immunogene generally is made up of carrier molecule and another component (haptens).

As used herein, term " mensuration " is understood to include qualitative analysis,, identifies the existence of analyte in the sample that is, and quantitative analysis, that is, and and the amount of analyte in the working sample. It also is understood to include the term of the preparation of standard and calibration curve, and such preparation is well known in the art.

As used herein, term " diacyl hydrazide compound " is understood to be in its scope and comprises diacyl hydrazide and metabolin thereof, synthetic analogues, environment degradable thing and the photochemical degradating thing of origin thus.

As used herein, term " IC₅₀" be defined as the concentration to half needed inhibitor that the light absorption value that records reduces to light absorption value when not having inhibitor to exist. This determine by drawing residual activity percentage (%) relatively the curve of inhibitor concentration finish, wherein the percentage of residual activity (%) by

% activity=(A₀-A _i/A ₀) * 100 provide, wherein A₀The light absorption value that records when not having inhibitor to exist and A_iThat inhibitor concentration is i^thThe time light absorption value that records. Thereby suppressing percentage is provided by relational expression 100-(% activity).

Cross reactivity percentage is by equation:

% cross reactivity=(IC _50.5992/ IC _{50. analog}) * 100 obtain, wherein IC _50.5992Be the IC of the RH5992 that as above determines ₅₀Concentration and IC _{50. analog}Be analog promptly, RH2703, the IC of RH2651 and RH0345 ₅₀Concentration.

The sensitivity of representing with ng is the concentration of the inhibitor determined from the regretional analysis of the standard deviation of replicate analysis normal concentration, as equation:

S=(Y _Int/ m) * and V is described, and wherein S is the sensitivity of representing with ng, Y _IntBe the Y-intercept from the tropic of the standard deviation of replicate analysis of light absorption value unit representation, m be every ng/ml light absorption value unit the tropic slope and V is the volume that is added to the sample of droplet flat board, unit/ml.

Recovery per cent is by relational expression:

%R=(C ₀/ C _aCalculate) * 100, wherein C ₀Be to observe concentration that (mensuration) arrive and C _aBe actual (maximum) concentration.

As above narrate, be provided for preparing the immunogene of diacyl hydrazide antibody, usually, immunogene comprises haptens and carrier molecule.

Haptens is the diacyl hydrazide or derivatives thereof normally.In one embodiment, haptens is the benzoyl hydrazine, and this benzoyl hydrazine compound is the compound of top general formula (1).

Suitable (C ₁-C ₆) example of alkyl includes, but not limited to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, just-and amyl group, isopentyl, neopentyl, n-hexyl, isohesyl, or the like.Suitable (C ₁-C ₆) example of substituted alkyl includes, but not limited to use hydroxyl, halogen atom, (C ₁-C ₄) methyl that replaces of alkoxy and nitro, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, n-pentyl, isopentyl, neopentyl, n-hexyl, or the like.

Suitable (C ₂-C ₆) example of alkenyl includes, but not limited to vinyl, positive propenyl, isopropenyl, the n-butene base, isobutenyl, uncle's butenyl group, positive pentenyl, isopentene group, new pentenyl, the n-hexylene base, or the like.Suitable (C ₂-C ₆) example of substituted alkenyl base includes, but not limited to use hydroxyl, halogen atom, or the vinyl that replaces of nitryl group, positive propenyl, isopropenyl, the n-butene base, isobutenyl, uncle's butenyl group, positive pentenyl, isopentene group, new pentenyl, the n-hexylene base, or the like.

Suitable (C ₁-C ₆) example of alkoxy includes, but not limited to methoxyl, ethoxy, positive propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isoamoxy, neopentyl oxygen and just own oxygen base.

Suitable (C ₁-C ₆) example of carboxyalkyl includes, but not limited to carboxyl (COOH), ethyloic (CH ₂COOH), carboxyethyl (CH ₂CH ₂COOH) and carboxylic propyl group (CH ₂CH ₂CH ₂COOH).

The example of suitable halogen atom includes, but not limited to Cl-, Br-, F-, and I-.

In preferred embodiments, R is-CH ₂COOH, R ¹And R ²Respectively be methyl R ³And R ⁴Respectively be hydrogen.In a more preferred embodiment, R is-COOH R ¹And R ²Respectively be methyl R ³And R ⁴Respectively be hydrogen.

Carrier mass can be a protein, such as but not limited to, bovine serum albumin(BSA), ovalbumin, or keyhole limpet hemocyanin; Polysaccharide, such as but not limited to, glucosan, agarose, agar or cellulose; Or synthetic polymkeric substance or multipolymer, such as but not limited to, polyacrolein, polyamide, polyacrylamide, poly-butyric ester, polyureas, polyureas acid amides, or polystyrene.

In preferred embodiments, carrier mass is a carrier protein, more preferably bovine serum albumin(BSA) (BSA) or keyhole limpet hemocyanin (KLH), most preferably keyhole limpet hemocyanin.

Immunogene of the present invention can be used to prepare anti-diacyl hydrazide compound antibody.Immunogene is imported suitable animal and collects separation and antibody purification.The antibody that obtains is polyclonal antibody, has high binding affinity and cross reactivity with diacyl hydrazide compound and its derivant, particularly benzoyl hydrazine with its derivant.

Another kind of optionally method utilizes the hybridoma technology of knowing in this area that immunogene is used to prepare monoclonal antibody.

In one embodiment, the immunogenic antibody that has prepared the general formula (1) of anti-and carrier mass coupling.In preferred embodiments, prepared the immunogenic antibody of the general formula (1) of anti-and carrier mass coupling, R is-CH in the general formula (1) ₃COOH, R ¹And R ²Be methyl, R ³And R ⁴Be hydrogen.In a more preferred embodiment, prepared the immunogenic antibody of the general formula (1) of anti-and carrier mass coupling, wherein R is-COOH, R ¹And R ²Be methyl, R ³And R ⁴Be hydrogen.

As above narration the invention provides the method for measuring the diacyl hydrazide or derivatives thereof, and at first, the method comprising the steps of (A) provides the sample of the diacyl hydrazide that contains unknown quantity.

Sample generally is any material that contains diacyl hydrazide.Suitable example comprises air, water, soil, and biological agents.In one embodiment, sample can be air sample or the sample that originates from air sample.In another embodiment, sample can be water sample or the sample that originates from water sample.Still also have in another embodiment, sample can be pedotheque or the sample that originates from pedotheque.

In one embodiment, sample can be biological agents or contain the sample of biological agents or originate from the sample of biological agents.Suitable biological agents example comprises and is not limited to vegetable material; Biological fluid such as blood, serum, blood plasma, lymph liquid, gastric lavage liquid, bile, liquor coneae; Biological tissue such as plant and animal are organized and microbiological specimens such as bacterium and virus.

In preferred embodiments, sample is soil or vegetable material or the sample that originates from this.

The diacyl hydrazide compound as described in the top general formula 1 with and derivant.These derivants can be, but are not limited to, metabolin, and synthetic analogues, the environment degradable thing originates from this photochemical degradation thing, and useful especially diacyl hydrazide is the benzoyl hydrazine according to general formula 1, is described in the following table 1:

The benzoyl hydrazine	????R	????R 1	????R 2	????R 3	????R 4
						??RH5992	??-CH 2CH 3	??-CH 3	??-CH 3	????H	????H
??RH2485	????H	??-CH 3	??-CH 3	????CH 3	????-OCH 3
						??RH2703	??-CH 2COOH	??-CH 3	??-CH 3	????H	????H
??RH2651	??-COOH	??-CH 3	??-CH 3	????H	????H
						??RH0345	??-Cl	????H	????H	????H	????H

In the presence of immobilization diacyl hydrazide antigen, the sample of step (A) contacts with having with the antibody of the high-affinity of diacyl hydrazide in the step (B).

Antibody as mentioned above, the antibody that obtains from the immunogene that contains with the RH2651 of KLH coupling is preferred.

Fixing diacyl hydrazide antigen can be the above-mentioned any diacyl hydrazide with above-mentioned any carrier molecule coupling.In preferred embodiments, immobilized antigen is RH2651 or the RH2703 with carrier protein couplet, more preferably with the RH2703 of BSA coupling.The combination of antigen and carrier molecule is called envelope antigen.

Envelope antigen generally is fixed on the solid support with surface of submissively adhering to this antigen.Suitable example includes, but are not limited to the droplet flat board; Membrane material such as cellulose nitrate, cellulose, cellulose acetate, polycarbonate, or the like; Test tube; Particle such as globule, column material, or the like; And have can be in conjunction with the deriving surface of the land of adhering to the antigen on it.General envelope antigen is applied to solid support and adsorbs thereon.

In one embodiment, envelope antigen is adsorbed on the droplet flat board.

In step (B), exist between free antigen (that is the benzoyl hydrazine of unknown quantity in the sample) and fixing (that is, in conjunction with) antigen having the competition of the binding site on the antibody with the diacyl hydrazide high-affinity.The result forms combination and unconjugated antigen-antibody complex.In step (C), in conjunction with compound with do not separate in conjunction with compound.Separation can be any method known in the art, as is not limited to gravity separation, and magnetic separates, and cleans and remove unconjugated compound.

In case separate with unconjugated antigen-antibody complex, use the compound of detectable label substance markers combination in the step (D).Mark can be to be fixed in conjugated antigen-antibody complex and detectable any material.Label can directly detect or by with the substrate reactions indirect detection.Suitable example includes, but are not limited to enzyme, color dye, fluorescent material, chemiluminescent material, bioluminescent material, and radioactive isotope.In preferred embodiments, detectable mark is an enzyme.Usually, enzyme and the material coupling that has with binding antibody-antigenic compound binding affinity.Label can be antibody-antigen with combining of compound, protein-ligand, or the combination of avidin (streptavidin)-biotin.In preferred embodiments, in conjunction with being antibody-antigen combination, wherein mark is incorporated into and has binding antibody-antibody of antigenic compound binding affinity.Such combination causes typical case well known in the art " interlayer " configuration.The antibody that combines with mark can be polyclone or monoclonal.In addition, can use the antibody fragment such as the Fab fragment of any above-mentioned antibody that contains the antibodies district.Certainly, will depend on type with the antibody of antigen coupling with the distinct antibodies of label coupling.That is exactly that selected have binding affinity with antibody and sessile antibody-antigenic compound the label coupling.

For example, in preferred embodiments, among the present invention, in rabbit, produce antibody with the antigen coupling.Therefore, the antibody with the mark coupling is anti--rabbit antibody that binding affinity is arranged with rabbit antibody.

In case mark behind immobilized antibody-antigenic compound, in step (E), on label, produce the variation that can measure.This variation that can measure can be produced by ultraviolet-visible, fluorescence, wave radiation, or the substrate of importing and label reaction produces the variation that can measure.Will be understood that can measure variation can be that mark is intrinsic, for example, mark can be a radioactive isotope, and wherein radioactivity as gamma-rays, can produce so that measure radioactivity by the importing means simply.

In one embodiment, wherein can measure variation is to contact with substrate by the compound with mark to realize.Such substrate is well known in the art and depends on used type.In preferred embodiments, label is that enzyme and substrate are to produce the compound that can measure variation with enzyme reaction.In a more preferred embodiment, enzyme is a phosphatase and substrate is a phosphate cpd.The general generation of the interaction of enzyme and substrate has the compound that can measure characteristic.For example compound is a fluorescence, has strong light absorption value, or the like.

At last, after step (E) generation can be measured variation, measure diacyl hydrazide in step (F).Such assay method is methods known in the art, for example, ultraviolet-visible pectrophotometer, fluorometry, the γ counting, or the like.

It is generally acknowledged that the assay method of narrating above is indirect determination.Simultaneously, should understand also having directly of being included in the scope of the present invention measures.At first, direct method is included in the sample that step (A) provides the diacyl hydrazide that contains unknown quantity.Sample and diacyl hydrazide are as mentioned above.

The sample of step (A) forms immobilized antibody-diacyl hydrazide compound with having to contact with the immobilized antibody of the binding affinity of diacyl hydrazide in step (B).Antibody as mentioned above, the antibody that RH2651 produces is preferred.

Antibody generally is fixed on the solid support with surface of submissively adhering to this antibody.Suitable example includes, but are not limited to the droplet flat board; Membrane material such as cellulose nitrate, cellulose, cellulose acetate, polycarbonate, or the like; Test tube; Particle such as globule, column material, or the like; And have can be in conjunction with the deriving surface of the land of adhering to the antibody on it.In preferred embodiments, as above about the description of envelope antigen, antibody is the coated antibody that is fixed on the solid support.

In the step (B), the binding site that free antigen (that is the benzoyl hydrazine of unknown quantity in the sample) has been filled the immobilization polyclonal antibody forms immobilized antibody-antigenic compound.In the step (C) with the diacyl hydrazide antigen of detectable label substance markers with do not contact with the site of immobilized antibody of benzoyl hydrazine combination in the sample.Thereby labelled antigen will be filled the residue binding site of antibody.So that immobilized antibody and mark and unlabelled antigen coupling.Diacyl hydrazide antigen and mark are as mentioned above.But in preferred embodiments, antigen is RH2651 and label is an enzyme, preferably horseradish peroxidase (HRP).

At last, in step (D), realizing to measure variation on the label and mensuration diacyl hydrazide in step (E).The mensuration that can measure the generation of variation and diacyl hydrazide is as above described to indirect determination.

As herein described to diacyl hydrazide such as RH5992 and its derivant RH2703, RH2651, the enough sensitivities of the mensuration of RH0345 and RH2485 can be used for the field residue research of FIFRA register request.In addition, this assay method has enough analogs different with structure such as the cross reactivity of RH0345, therefore be sure of that this mensuration will be applicable to the N-tert-butyl group-N of replacement, all types of N '-benzoyl hydrazine pesticide.

Except the sensitivity (ppb) measured and it were common to the pesticide type that RH5992 represents, biggest advantage may be the laboratory efficient that increases.For example, a verified analyst can easily finish 20 samples every day.This appraisal comprises that specimen preparation and result calculate.If work in 1 day 10 hours, the work that each sample only need consume 30 minutes.

Other purposes of antibody of the present invention comprises and antibody being combined with solid support so that the residue in the concentrating sample matrix, thus can with the fluorescence labeling mark they and be used for fabric study to identify the position of pesticide residues thing at CC.This information provides the important information about the mode of action of the toxicity of special diacyl hydrazide compound and metabolism or mechanism.Simultaneously, the direct immunization assay method can be incorporated into " soaking branch " test of analyzing in the field and check the type of using.

Below abbreviation be used in the following examples and the other parts of this instructions.BCA Bicin choninic acid BSA bovine serum albumin(BSA) DCC dicyclohexyl carbodiimide DEA diethanol amine DMF dimethyl formamide HRP horseradish peroxidase KLH keyhole limpet hemocyanin NHS N-hydroxy-succinamide PBS PBS PBST PBS and 0.01% polysorbas20 PNPP p-nitrophenyl phosphoric acid

Embodiment 1 immunogene is synthetic

In 5ml pears type flask, add 21umole haptens RH2651, and be dissolved in 200ulDMF (Fisher Scientific).In another 4ml test tube, 16mgDCC (EM Sciences) and 9.1mgNHS (Sigma Chemical) are dissolved in 200ulDMF.DCC and NHS transferred to contain in the haptenic flask, reactant is stir about 2 hours at room temperature.The transfer reaction thing continues to spend the night 4 ℃ of stirrings to the cold house.

Formulation vehicle albumen again in the 5.4ml deionized water, KLH (Imject ^R, when PierceChemical Co. prepares again 20mg protein (pH7.2) is dissolved in PBS).Shift haptens to the little sedimentator (the little sedimentator of 235B type, Fisher Scientific) of activation and removed replacement urea precipitation in centrifugal 5 minutes.Shift supernatant to the carrier protein solution of preparation again and room temperature continuous stirring 4 hours.

With 14000rpm the proteins react potpourri was removed protein precipitation in centrifugal 5 minutes.The supernatant of whole volumes is added to Swift ^RPolyacrylamide desalts that post (Pierce Chemical Co.) is gone up and with 0.01M PBS (pH7.3) wash-out, with the collection of a 3ml part.Be added to collection these stream parts in back on the post fully at sample.Get 10 3ml stream parts and measure the light absorption value of the every stream part in 280nm place.Merge and contain immunogenic stream part and measure protein concentration (referring to BCA protein determination reagent explanation-Pierce Chemical Co.) with the BCA method.

Haptens/protein bound ratio is determined as follows.Set up the light absorption value typical curve of the KLH of the 0ug/ml-1200ug/ml of 254nm place scope.The protein concentration of the immunogene stream that the BCA method is recorded according to typical curve part is transformed into the light absorption value of 254nm.Set up the RH2703 of concentration range 26260nmole/ml and RH2651 that concentration range is 6.5-210nmole light absorption value typical curve at the 254nm place.

Measure the 254nm immunogenic light absorption value (A in place _Conj), deduct the light absorption value (A of the KLH of 254nm place that calculates _KLH) obtain the light absorption value (A that haptens produces _Hap).Change this value (A from typical curve _Hap) become haptenic concentration (nmole/ml).

From haptenic calculating concentration (nmole/ml) remove in immunogenic mensuration concentration calculate haptens to the mole of protein in conjunction with ratio.The KLH mean molecular weight that uses is 6.7 * 10 ⁶Dalton.Mole is removed the average that obtains hapten molecule among the KLH of per 10,000 atomic mass units in 670 in conjunction with ratio.The results are shown in Table 2.

2 other immunogenic synthesizing of embodiment

Method according to embodiment 1 prepares immunogene, but used haptens is RH2703.The result is illustrated in the table 2.

Embodiment 3 envelope antigens are synthetic

Prepare envelope antigen (RH2703 and RH2651) according to the immunogenic method of embodiment 1 preparation, that different is BSA (Imject ^R, Pierce Chemical Co.) and replace KLH as carrier protein.Being used to calculate mole BSA mean molecular weight in conjunction with ratio is 68,000 dalton.With mole in conjunction with ratio remove obtain in 6.8 per 10, the hapten molecule average of 000amu BSA.The result of the envelope antigen of preparation is illustrated in the table 2.

Table 2

Sample	Protein μ mol/ml	Haptens μ mol/ml	Haptens/protein	????N (1)
					??RH2651-KLH	?2.8×10 -4	????0.437	????1560	????2
??RH2703-KLH	?2.9×10 -4	????0.943	????3250	????5
					??RH2651-BSA	????0.03	????1.5	????50	????7
??RH2703-BSA	????0.04	????2.2	????50	????8

⁽¹⁾N is the hapten molecule number of the protein of per 10,000 atomic mass units.

The production of embodiment 4 antibody

From 2.5ml immunogene (being formulated as the product of the embodiment 2 of 1mg/ml among 0.01M PBS again), 25mg Mycobacterium tuberculosis suspending liquid and 2.5ml Freund prepare elementary inoculum.With the potpourri homogenization up to thickness and cream color.

Utilize New Zealand white rabbit (5) to produce antibody.Rabbit is the female of the no disease of weight between the 4.75-5.5 pound.At first will resist Bordetella pertussis (to be prepared in the 0.6ml salt solution and to suspend 6 * 10 ¹⁰The Bordetella pertussis inoculum of cell) intramuscular inoculation rabbit and blood sampling provide background to tire.

The dosage of the hypodermic elementary inoculum of each rabbit is 1.0ml.The immunity inoculation that inoculation rabbit and acceptance in per 21 days are strengthened.After 8 days, collect antibody for the rabbit bloodletting the 4th booster shots.After another time cycle, begin booster shots again and after the 4th booster shots, do the production bloodletting again.

Embodiment 5 measures rabbit anti-serum

(PBS is formulated as the about 10ug/ml of initial concentration again with envelope antigen in pH7.2) to be cushioned liquid at bag.The 1st and 2 holes that are listed as of 96 hole droplet flat boards are preserved for control sample.The envelope antigen that at first in the hole of the 3rd row, adds the 200ul initial concentration.The hole of 4-12 row is contained the 100ul bag and is cushioned liquid.Shift 100ul envelope antigen part in the hole of the 3rd row and be cushioned liquid to the Kong Zhongyu bag of the 4th row and mix, by this way, be cushioned liquid with 1: 2 serial dilution envelope antigen at the Kong Zhongyong of each row that obtain subsequently bag.Dilution proceeds to the 11st row, and wherein envelope antigen has carried out 1: 256 dilution.Abandon last 100ul.Only contain the bag be cushioned liquid do not have antigen the 12nd row the hole as negative control.Hole A1 and A2 are the air blank, do not accept reagent.Hole B1 and B2 with the envelope antigen bag by and be the background hole.Hole C1 and C2 are used as positive control by KLH (about 10ug/ml) bag that the 100ul bag is cushioned in the liquid.Remaining hole D1-H2 is untreated.Seal the flat board of such preparation with sealing strip, be capped parcel, be positioned in the refrigerator, be incubated overnight at 4 ℃ with plastic packets.

Spend the night behind the incubation, from refrigerator, take out dull and stereotyped and turned letter.With in 300ul BSA confining liquid (1%BSA, pH7.2 is by dissolving 1.0g BSA preparation in the 100mlPBS) blind hole (except A1 and A2) not in conjunction with binding site.This solution is incubation 10 minutes at least at room temperature.

With confining liquid dilution in 1: 1000 specimen, in A row, begin 200ul is added to the hole from the A3 hole.The 100ul confining liquid is contained in B3-H3 row's hole.The specimen (100ul) that shifts the A round is to B round and mixing fully.1: 2 serial dilution sample is to G3 row's hole (1: 64000) and abandon last 100ul by this way.It is negative control that antibody is not contained in H3 row's hole.Pre-serum incubation background hole B1 and B2 with the dilution in 1: 500 of 100ul confining liquid.1: 1000 dilution incubation positive control hole C1 and C2 with specimen.With the specimen incubation dull and stereotyped about 1 hour.

Behind the incubation, remove specimen and use the washing lotion hole flushing.With damping fluid flushing port 4-5 time.When the 3rd or the 4th washing.Allow damping fluid to be retained in the hole, before abandoning, soaked 3-5 minute.Again prepare the goat anti-rabbit igg that is connected with alkaline phosphatase (Pierce Chemical Co.) and with confining liquid dilution 1: 5000 with the glycerine of 1ml 50%.In each hole, add this solution of 100ul and room temperature incubation flat board at least 1 hour.The anti-rabbit antibody that flush away is excessive adds 100ulPNPP solution (5mgPNPP is dissolved in 8ml deionized water and 2ml DEA damping fluid) in each hole.At the room temperature incubation dull and stereotyped 30 minutes.Add 50ul 2N NaOH then and stop enzyme reaction.Measure the light absorption value in each hole, 410nm place with DynatechMR5000 droplet plate reader.The result is illustrated in the table 3.

Table 3

Immunogene	Rabbit #	Test blood sample #1	Test blood sample #2	Produce blood sample #1	Produce blood sample #2
						?RH2651-KLH	????1331	??1/32000	??1/64000	??1/64000	????1/128000
?RH2651-KLH	????1332	??1/32000	??1/128000	??1/64000	????1/128000
						?RH2651-KLH	????1333	??1/32000	Not blood sampling 2	??1/64000	????1/128000
?RH2651-KLH	????1334	??1/32000	??1/64000	??1/128000	????1/128000
						?RH2651-KLH	????1335	??1/32000	??1/32000	??1/128000	????1/128000
?RH2703-KLH	????1336	??1/16000	??1/64000	??1/64000	????1/128000
						?RH2703-KLH	????1337	??1/16000	??1/64000	??1/64000	????1/128000
?RH2703-KLH	????1388	??1/16000	??1/64000	??1/64000	????1/128000
						?RH2703-KLH	????1399	??1/32000	??1/64000	??1/64000	????1/128000
?RH2703-KLH	????1340	????nd 1	??1/64000	??1/64000	????1/128000

1 does not measure antibody titer.The ear scar of 2 these animals has stoped and has obtained test blood.

Embodiment 6 separates and antibody purification

Merge the immune serum of collecting in the injection process of embodiment 3 and use the BCA method to measure total protein, discovery is 66mg/ml.Measure the volume of immune serum and be divided into the identical aliquot of two each 90ml.The deionized water dilution of two volumes of an aliquot.Dilute the liquor capacity that obtains successively with isopyknic 4M sulfuric acid amine and obtain 2M sulfuric acid amine aqueous solution at last.Spend the night at stirring at room suspending liquid.By collecting protein precipitation in centrifugal 30 minutes at 10000g-av..Protein precipitation is dissolved in the 0.02M sodium phosphate buffer (pH8.0) of minimum volume, and this damping fluid is by the preparation of dilution 0.1M sodium phosphate buffer.

In 2L 0.02M sodium phosphate buffer (pH8),, in 4L,, in 4L, at room temperature dialysed 4 hours at last then 4 ℃ of dialysed overnight the protein solution of room temperature dialysis preparation again 4 hours.

(Inc.) independently aliquot is from the antiserum ion-exchange purification IgG of merging with two for DE52, Whatman, and this cellulose humid volume is 150ml, has filled the high bed of about 26cm in the post of 2.6cm (i.d.) to utilize the DEAE cellulose.Sodium phosphate buffer (pH8.0) balance columns with 0.02M.The protein capacity of post is about 1.5g (every ml humid volume 10mg).The dialysis protein solution pump of whole volumes to post, is begun wash-out with the sodium phosphate buffer pH8.0 of 0.02M.Adjust flow velocity by about 5ml/ minute, every 5ml collects stream part.The 150ml eluent of beginning contains any protein that does not keep.

Behind the 150ml of beginning, the linear gradient volumetric molar concentration varies continuously to 0.3M.Gradient forms the chamber two gradients and forms, and 250ml 0.02M damping fluid is contained in first chamber, and 250ml 0.3M damping fluid is contained in second chamber.Ensuing 500ml eluent is collected with stream part of 5ml one pipe in first chamber of continuous stirring in the elution process.Detect the protein eluent by the light absorption value of measuring 280nm, light absorption value is drawn to elution volume (collection tube #) obtains chromatogram.

Measure the IgG of the aliquot of each pipe by the indirect method of this paper narration.Merge the part that contains IgG, reduce volume with stirring cell concentration device with Amicon filter paper (molecular weight repels the limit-10,000 dalton).Measure the protein concentration of the IgG that the concentrates stream part that merges with the BCA method of standard.The sodium azide that in protein solution, adds aseptic filtration and-20 ℃ of storages.Protein elution curve from the DEAE ion exchange column is described in Fig. 1.The total protein that records from the light absorption value of 280nm is represented in open loop.Every antibody titer that flows part that on behalf of the indirect determination of 410nm place, closed loop record.Assembling section stream part 51-80 is further purified with the protein affinity chromatography.

With the affine Pak of a-protein ^Post and immunity are pure ^R(immunity is pure for the IgG damping fluid ^IgG purification kit, Pierce Chemical Co.) IgG (1mg/ml) of affinity purification DEAE purifying.Place a-protein post and damping fluid to room temperature, wash post with 5ml binding buffer liquid.Dilution DEAE IgG purification obtained protein concentration 6mg/ml in 1: 9, and the 1ml aliquot is added on the post.Wash post with other 15ml binding buffer liquid.

Pure with the 5ml immunity ^The IgG elution buffer carries out the wash-out of IgG, obtains 1ml stream part.Measure the light absorption value of the every part in 280nm place, (the 20mM sodium phosphate, cellulose column desalts to the stream part (stream part 3) with remarkable light absorption value before the 100mM NaCl, the 5ml that pH7.4) regulated with using 10ml 0.02M PBS in advance.Utilize the branch wash-outs such as 0.02M PBS of 10 1ml, collect first-class part of 1ml.Measure the light absorption value of each stream part total protein with the BCA method.

The IgG of 1: 5 diluted protein matter A affinitive layer purification obtains protein concentration 1mg/ml.From the 9mg/ml IgG concentration that the average total serum protein of 66mg/ml obtains estimating, the IgG recovery of estimation is 67%.

The optimization of embodiment 7 indirect determinations

Utilize RH2703-BSA as envelope antigen.There is constant a-protein IgG purification (10ul 20ug/ml solution, the 100ng/ hole) time, measure (referring to people such as Voller by pattern, Bull of World Health Organization, obtain the concentration optimization of readable reaction (410nm of 0.3-0.5 light absorption value unit) needs when 53,35 (1976)) making the antigen (RH2703) that has minimum.Acquisition makes the concentration optimization of a-protein IgG purification to the readable reaction of the RH5992 of minimum when having constant envelope antigen (RH2703-BSA, 100ul 10ug/ml solution).

Antibody concentration the best is about 20ug/ml (100ng/ micropore) and envelope antigen is best is 25ng/ml or 2.5ng/ micropore.When converting mole foundation to and supposing that haptens/protein bound ratio is 55, the haptens molal quantity is above three times of the IgG that adds.

RH2703 when having determined to exist the RH2703-BSA envelope antigen of optium concentration, RH2651, RH5992, RH0345 and RH2485 are to the inhibition percentage of protein IgG.At test substances 0ng/ml never to 7 each test substances of concentration determination of 1000ng/ml scope.The test of each concentration repeats 7 times.Antigen negative and negative antibody hole negative control is provided and the anti-KLH of KLH/ hole as positive control.

Being cushioned liquid with the bag that contains 0.1% polysorbas20 makes the serial dilution of test substances storage liquid and obtains 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml test concentrations.Bag is cushioned liquid w/0.1% polysorbas20 as the 0ng/ml test concentrations.In room temperature with the test substances (each 190ul) of 10ul a-protein IgG (20ul/ml) incubation test concentrations 1 hour.At incubation week after date, shift 100ul test substances-IgG potpourri to suitable droplet hole.The droplet hole with the RH2703-BSA of optium concentration bag by and seal the excess protein binding site with the 1%BSA solution that the bag of 0.3ml is cushioned in the liquid.At room temperature dull and stereotyped 1 hour of incubation again.

Behind second time incubation, washing is dull and stereotyped, add with the goat of alkaline phosphatase coupling anti--rabbit immunoglobulin is (with 1ml 50% glycerine reconstruct 0.6mg, subsequently with confining liquid dilution 1: 5000-Pierce Chemical Co.).As incubation as described in the embodiment 5, washing is dull and stereotyped, adds the PNPP substrate.Do not add the NaOH stop buffer, before reading 410nm place light absorption value, allow dull and stereotyped colour developing 2 hours.Suppress curve description at Fig. 2-6.Special antibody purification characteristic is illustrated in the table 4.

Determine IC as mentioned above ₅₀Use Microsoft Excel The index curve that Analysis ToolPak (Microsoft, copyright 1993) sets is analyzed the relation of active percentage to inhibitor concentration.In order to calculate IC ₅₀, when y equals 50, promptly keep 50% active or 50% when suppressing, by indicial equation y=bm ⁿX obtains x.Use Microsoft Excel ^c, Ver5.0 chartography (Microsoft, copyright 1993) is charted.

As mentioned above from IC ₅₀Calculate cross reaction percentage.Use Microsoft Excel , Ver5.0 chartography (Microsoft, copyright 1993) makes the figure of the average light absorption value of 7 replicate analysis that obtain from 7 inhibitor concentration.The linear dynamics scope is presented at the linear segment of the curve that obtains at the figure of the light absorption value of 410nm from various inhibitor concentration.Meter sensitivity as mentioned above.

The limit that definition detects is three times a mensuration background noise, for the estimated background noise, has calculated the standard deviation of the replicate analysis of each inhibitor concentration, carries out the minimum quadratic method of regretional analysis.The y-intercept of the tropic is used as the background absorption that noise produces.Take advantage of background absorption with 3, the slope of the typical curve that obtains with the range of linearity is divided by, and converts inhibitor concentration to.The concentration that definition obtains like this is the determination limit of inhibitor.

Table 4

Detection material	IC50，ng/ml	Sensitivity ng	Detection limit ng/ml	Cross reactivity %
					??RH5992	????0.61	????0.07	????0.66	????100
??RH2703	????1.13	????0.19	????1.7	????54
					??RH2651	????1.59	????0.22	????1.9	????38
??RH0345	????6.5	????0.08	????0.75	????9
					??RH2458	????0.05	????0.08	????1.8	????NA

Embodiment 8-19 indirect determination

RH2703-BSA envelope antigen (10ug/ml) bag with the 100ul/ hole is obtained every hole package amount 1ug by the droplet flat board.Spend the night at 4 ℃ of incubation flat boards.The BSA solution that is cushioned in the liquid with 1% the bag in 300ul/ hole seals unconjugated protein binding site before use.

Get the aliquot of about 1g wild cabbage and pedotheque.With wild cabbage sample #03 (1.0191g) and pedotheque #49 (1.0180g) in contrast and with the RH5992 spike of 50ng or 500ng so that measure the recovery.Sample and used sample size are shown in the following table 6.

Sample aliquot is placed 25ml pyramid type centrifuge tube and with 50 or the RH5992 spike recovery sample of 500ng, this RH5992 prepares with PBST dilution RH5992 storage standard liquid (1mg/ml in the acetonitrile).

By using sound wave clasmatosis device (Sonicator ^Heat Systems, the Inc.W-225R type has the little end probe of H-1) handled 3 minutes at 80% power sound wave, with 5ml PBST aliquot extraction sample.With 4000g Centrifugical extraction potpourri 5 minutes, decant the PBST layer.Obtain 1: 100 dilution (wild cabbage sample) and 1: 500 dilution (pedotheque) of primary sample extract with the aliquot of every kind of sample of PBST dilution.

The aliquot (180ul) of dilute sample and every kind of RH5992 titer of 10ul (2,10,20,100,200 and 2000ng/ml) and IgG (10ul) mixing are obtained having the RH5992 final concentration be respectively 0.1,0.5,1,5,10 and the sample of 100ng/ml.Sample mixes in the borosilicate acid esters glass tube of 12 * 8 96 well format of arranging fully.Pipe corresponding to the negative antibody hole contains the 10ul damping fluid.Do not have IgG to add and contain RH5992 among the PBST of 100ppb corresponding to the pipe in antigen negative hole.Pipe corresponding to the KLH positive control is empty.

Incubation week after date at 1 hour, transfer 100ul spike sample is to the droplet flat board and continued incubation 1 hour.

Behind the incubation, washing is dull and stereotyped and add the goat anti-rabbit igg of 100ul and alkaline phosphatase coupling and room temperature incubation 1 hour, behind the incubation, washing was dull and stereotyped again, adding 100ul PNPP substrate as mentioned above.Continue also to measure in 1-2 hour the light absorption value at 410nm place with Dynatech MR5000 microplate with the substrate incubation.The mean value of the light absorption value that calculating antigen negative hole records is also removed it automatically from instrument connection, remove the light absorption value that the non-specific bond of antibody produces by this way.

Add residual level in the standard measure sample with standard.Use Microsoft Excel , the Ver5.0 chartography obtains the figure of the light absorption value inhibitor concentration that adds relatively reciprocal from the average inverse of the repeat samples light absorption value that deducted non-specific bond.Use Microsoft Excel Analysis ToolPak is with the data substitution equation y=mx+b that obtains.In this relational expression, y is the light absorption value inverse that records of experiment, and b is a constant, and m is that the slope that calculates and x are the concentration of the inhibitor that adds.Calculate each data point with respect to standard error, remove irrelevant value at last from the relevant calculation value of linear equation.

When y equals unconstrained dose of light absorption value inverse that records, separate linear equation, obtain sample concentration for X.Multiply by corresponding dilution gfactor and convert the result to total ng number.Remove with the sample quality of getting and to obtain end product ng/g in total ng number.

Set up the typical curve of quantitative sample residues with the external perimysium reference method.Use Microsoft Excel Analysis ToolPak carries out index curve and sets and linear regression analysis.Calculate recovery per cent as mentioned above.

Analyzing peak value with external perimysium reference method and internal standard method (addition) is the recovery sample of 50ng/g and 500ng/g.The result of recovery per cent is illustrated in the parenthesis in the table 5.The canonical plotting that obtains in PBST and matrix blank sample (wild cabbage sample #03 and pedotheque #49) is illustrated among Fig. 7.

Table 5

Sample	Internal standard Cng/g	External perimysium reference Cng/g
			Wild cabbage R 1	????52(104％)	????53(106％)
Wild cabbage R 2	????471(94％)	????426(85％)
			Soil R 1	????55(110％)	????55(110％)
Soil R 2	????511(102％)	????514(103％)

Result from the internal standard analysis of wild cabbage and pedotheque is illustrated in the table 6.Analyze the stability of two wild cabbages and soil extract thing sample estimates matrix after one week again.The result is illustrated in the parenthesis.The calculating linear regression line of each sample is illustrated in Fig. 8-15.

Table 6

Sample ID	????Cug/g	Sample ID	????Cug/g
				Wild cabbage		Soil
????20139-01	????1.75(1.41)	????92-0017-151	????41.4
				????20139-02	????2.41	????92-0017-157	????20.0
????20139-04	????5.92	????92-0017-175	????18.4
				????20139-05	????13.2(12.9)	????92-0017-187	????28

Synthesizing of embodiment 20 labelled antigens

The RH2651 of 4.6mg (10.5umole) amount is placed the pears type flask of 5ml and is dissolved in 100ml DMF.In other test tube, 4.6mg NHS and 8.8ul DCC respectively are dissolved in 100ulDMF.NHS and DCC added RH2651 and about one hour in stirring at room.Continuing stirring at 4 ℃ spends the night.Then 10mg HRP is dissolved in 2.7ml 0.01M PBS, pH7.2.The centrifugal RH2651/NHS/DCC of the little hydro-extractor E of Beckman (Beckman instrument) three times.Supernatant is dropwise added HRP and stirring.In stirring at room potpourri 4 hours, in little hydro-extractor centrifugal 3 minutes then.The Swift that crosses with the PBS balance post (Pierce Chemical Co.) that desalts desalts to the supernatant that contains coupling protein matter.Collect stream part with the PBS elute protein and with 3ml/ part.The definite stream part of containing protein conjugate of light absorption value that merges 280nm is also determined protein concentration with BCA protein determination (PierceChemical Co.).

Following proof RH2651 combines with HRP's.10ug/ml 2651-HRP with 100ul wraps by the microplate hole and 4 ℃ of store overnight.Behind the turned letter flat board, with the BSA blind hole among 1% the PBS.After the sealing, 1: 2 serial dilution (with beginning in 1: 500) that adds the 2651IgG of the a-protein purifying for preparing according to embodiment 6 in the hole is so that survey RH2651.Behind 1 hour room temperature incubation, wash flat board with 0.01% polysorbas20 PBS.Add the goat anti-rabbit igg of alkali phosphatase enzyme mark and at room temperature incubation 1 hour again.Wash flat board and add right-nitrobenzene phosphoric acid substrate.15 minutes colour developing after dates are with the light absorption value at Dynatech MR5000 microplate mensuration 410nm place.

The assessment of RH2651 and horseradish peroxidase has been confirmed 1: 32000 ²⁶⁵¹The anti-1ug 2651-HRP of IgG tires.This has shown the coupling reaction success.

Embodiment 21 coated antibody optimizations

With ²⁶⁵¹The IgG bag is by microplate.In each hole, add 100ul 10ug/ml since the 1st row ²⁶⁵¹IgG also makes 1: 2 serial dilution on flat board.Seal plate also is stored in 4 ℃ up to preparing use.Behind the turned letter, with 0.01% be dissolved in that polysorbas20 among the PBS is washed flat board and in room temperature with 1% the BSA sealing 1 hour among the PBS of being dissolved in.After the sealing, turned letter is dull and stereotyped and add 150ulPBS in each hole.Begin to add 50ul 2651-HRP dilution and continue edge dilution in dull and stereotyped 1: 2 from 10ug/ml along dull and stereotyped then.Behind 1 hour room temperature incubation, wash flat board as the front and in each hole, add 150ul 1-Step ^Slow-TMB (from the peroxidase substrate of Pierce Chemical Co.).After colour developing in 1 hour, add 150ul 1N H ₂SO ₄Cessation reaction is measured the light absorption value of each hole at the 450nm place with the DynatechMR5000 microplate.The hole that contrast comprises the hole that does not have envelope antigen and do not have 2651-HRP to add.Determine coated antibody ²⁶⁵¹IgG concentration is that 0.625ug/ml and 2561-HRP concentration are that 1.25ug/ml is for using 1-Step ^The Slow-TMB substrate is an optimum concentration.

Embodiment 22 directly measures

Each uses 100ul 0.625ug/ml ²⁶⁵¹The IgG bag is by the hole of microplate.Staying a row does not wrap and is compared.Keeping flat board at 4 ℃ spends the night.Washed dull and stereotyped and sealed at least 30 minutes with 0.01% the polysorbas20 among the PBS of being dissolved in second day with 1% the BSA that is dissolved among the PBS.Prepare RH5992 from the storage liquid of 1mg/ml, RH2651, the titer of RH2703 and RH2485.The titer of RH0345 is prepared into 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, and 100ng/ml.Behind the turned letter confining liquid, in suitable hole, add the 150ul titer.Add 150ulPBS and do zero contrast., in each hole, add 50ul 1.25ug/ml2651-HRP and rock dull and stereotyped mixed content thing after 45 minutes at the room temperature incubation.Row that contain antibody and titer do not accept 2651-HRP but 50ul PBS as negative control., wash flat board as the front and in each hole, add 150ul 2651-HRP after 45 minutes at the room temperature incubation, rock dull and stereotyped mixed content thing.Row that contain antibody and titer do not accept 2651-HRP but 50ul PBS as negative control.At the room temperature incubation after 45 minutes, resemble that flat board is washed in the front and add 150ul Step 1 in each hole ^SlowTMB.After at room temperature 2 hours, in each hole, add 150ul 1N H ₂SO ₄, read the light absorption value at 450nm place with the DynatechMR5000 microplate.

Figure 16 a has shown the typical curve of determination experiment.The linear segment of visible curve is between 0.01ng/ml and 10ng/ml.Figure 16 b utilizes positive slope of light absorption value generation reciprocal to show the typical typical curve of setting-out line scope.Detection limit for the calculating of RH5992 is that the sensitivity that 0.096ng/ml calculates is 0.005ng.Table 7 has provided the data of RH5992 and analog.

Table 7

Test substances	IC 50ng/ml	Sensitivity ng	Determination limit ng/ml	Cross reactivity, % RH2651 RH5992
					??RH5992	????3.64	????0.005	????0.106	????32????100
??RH2703	????2.10	????0.002	????0.031	????56????173
					??RH2651	????1.17	????0.001	????0.013	???100????311
??RH0345	???16.94	????0.255	????5.10	???6.9???21.5
					??RH2485	????4.64	????0.024	????0.482	????25???78.4

Claims (17)

1. immunogene comprises: with the compound of the following general formula of carrier mass coupling R wherein, R ¹, R ², R ³, and R ⁴Be hydrogen independently separately, (C ₁-C ₆) (the C of alkyl and replacement ₁-C ₆) alkyl, (C ₂-C ₆) (the C of alkenyl and replacement ₂-C ₆) alkenyl, (C ₁-C ₆) alkoxy, (C ₁-C ₆) carboxyalkyl.

2. immunogene according to claim 1, wherein carrier mass is selected from and comprises protein, polysaccharide, the group of synthetic polymer or multipolymer.

3. immunogene according to claim 1, wherein R is-COOH, R ¹And R ²Be methyl, R ³And R ⁴Be hydrogen.

4. immunogene according to claim 1, wherein R is-CH ₂COOH, R ¹And R ²Be methyl, R ³And R ⁴Be hydrogen.

5. the antibody that produces of the immunogene of claim 1.

6. the antibody that produces of the immunogene of claim 3.

7. the antibody that produces of the immunogene of claim 4.

8. measure the method for diacyl hydrazide or derivatives thereof, comprise step:

(A) provide the sample of the diacyl hydrazide that contains unknown quantity;

When (B) having immobilized diacyl hydrazide antigen sample is contacted so that forms combination and binding antibody-antigenic compound not with having with the antibody of the binding affinity of diacyl hydrazide antigen;

(C) antibody-antigenic compound of separating and combining and unconjugated antibody-antigenic compound;

(D) with detectable label mark binding antibody compound;

(E) on mark, produce the variation that to measure; With

(F) diacyl hydrazide in the working sample.

9. method according to claim 8, wherein detectable label is selected from enzyme, color dye, fluorescent material, chemiluminescent substance, bioluminescence material, and radioactive isotope.

10. method according to claim 8, wherein can measure mark is the enzyme-antibody coupling matter of energy binding antibody.

11. measure the method for diacyl hydrazide or derivatives thereof, comprise step:

(A) provide the sample of the diacyl hydrazide that contains unknown quantity;

(B) sample is contacted so that form immobilized antibody-antigenic compound with having with the immobilized antibody of diacyl hydrazide antigen-binding affinity;

(C) will contact the antibody-antigenic compound of formation mark with the diacyl hydrazide antigen that can measure the label mark with not compound sessile antibody;

(D) on mark, produce the variation that to measure; With

(E) diacyl hydrazide in the working sample.

12. method according to claim 11 wherein can be measured mark and be selected from enzyme, color dye, fluorescent material, chemiluminescent substance, bioluminescence material, and radioactive isotope.

13. according to the described method of claim 11, wherein mark is the enzyme-antigen conjugates of energy binding antibody.

14. measure the kit of diacyl hydrazide, comprising:

(A) has the antibody of the affinity that combines with diacyl hydrazide;

(B) can be incorporated into the label of antibody; With

Can produce the substrate that to measure variation when (C) having label.

15. kit according to claim 14, it further contains diacyl hydrazide antigen.

16. kit according to claim 14, wherein label is affinity and the enzyme a kind of antibody coupling that has with antibodies.

17. kit according to claim 14, wherein label is and has and antibody binding affinity and the enzyme coupling of diacyl hydrazide antigen.

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