CN100538361C - The kit of immunoassay and use thereof - Google Patents
The kit of immunoassay and use thereof Download PDFInfo
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- CN100538361C CN100538361C CNB2003801075560A CN200380107556A CN100538361C CN 100538361 C CN100538361 C CN 100538361C CN B2003801075560 A CNB2003801075560 A CN B2003801075560A CN 200380107556 A CN200380107556 A CN 200380107556A CN 100538361 C CN100538361 C CN 100538361C
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Abstract
Use two types antibody, promptly the compatibility of target material is higher than to the first antibody of the compatibility of competition material with to the compatibility of competition material and is higher than second antibody to the compatibility of target material, sample is with these two kinds of antibody treatment.Then, the competition material in the sample at first combines with second antibody, and sample hits material to competing the ratio expansion of material like this.As a result, the target material becomes and is easy to combine with first antibody, and in turn, the reactivity of comparing the target material with the situation of only using first antibody increases.Therefore, accurately the target material in the working sample avoids being contained in the influence of the competition material in the sample simultaneously.
Description
Technical field
The present invention relates to a kind of immunoassay, this method enables target material in the accurate working sample by the feature antibody that uses two types, the invention still further relates to the kit that uses in the described method.More specifically, the present invention relates to a kind of immunoassay, wherein use two types antibody, promptly target material comparison competition material had the more first antibody of high-affinity, with competition material comparison target material is had the more second antibody of high-affinity, with these two kinds of antibody treatment samples, so the competition material in the sample at first combines with second antibody, the hit ratio of material and competition material of sample enlarges like this, make the target material become and be easy to combine with first antibody, as a result, the reactivity of comparing the target material with the situation of only using first antibody improves, so the target material can accurately be measured; The invention still further relates to the kit that in this method, uses.
Background technology
Polytype material is present in the sample that contains target material to be determined, and target material to be determined in some cases is present in the specific sample simultaneously with the competition material.For example, think in the serum most tartrate resistance acid phosphatase (TRACP) derived from the acid phosphatase of osteoclast, and think that measuring TRACP can be used as the indication of assessing function of osteoblast.Therefore, TRACP is material (the Norio Fukunaga that causes people's interest as the bone resorption mark, Toshitaka Nakamura and Toshio Matsumoto, " Osseous MetabolismMarker ", Medical Review Co.Ltd., 1995), and it is found that this kind of enzyme is except that the tool enzymatic activity, the catabolite fragment of this enzyme also is present in (J Bone MinerRes.15:1337-1345,2000 in the serum; With Clin Chem.47:74-80.2001).
In addition, by polyacrylamide gel electrophoresis the acid phosphatase in the serum is divided into from the off six bands of 0-5.This is the tartrate resistance corresponding to the 5th acid phosphatase of being with wherein, and (TRACP 5: tartrate resistance acid phosphatase 5) to be called as 5 band tartrate resistance acid phosphatases.This acid phosphatase further is divided into the sialic 5a that combines with sugar chain with high-load and contains the sialic 5b that combines with sugar chain hardly by electrophoresis.In addition, 5a is constant derived from enzyme and its blood levels of blood platelet etc., and the blood levels of having only 5b changes with the variation of bone resorption.Therefore, it is believed that 5b is the major part derived from the tartrate resistance acid phosphatase of osteoclast." clinical chemistry " (Clin.Chem.47:1497.2001) in, also recommend can be abbreviated as TRACP5b derived from the ACP of osteoclast.Therefore, in this manual, relate to derived from osteoclast and the phosphatase that is used as the ACP of bone resorption indication and be expressed as term " TRACP 5b ", and think that tartrate resistance acid phosphatase and tartrate resistance acid phosphatase 5b (TRACP 5b) derived from osteoclast are synonyms.Therefore, they are all expressed with term " TRACP 5b ".
Except that TRACP 5b, be present in the sample as ACP derived from red blood cell or derived from hematoblastic acid phosphatase.That is, when causing haemocylolysis, be contained in the sample derived from erythrocytic acid phosphatase by the collection sample.When serum was used as sample, blood platelet was destroyed in serum product blood clotting process, causes derived from hematoblastic acid phosphatase to be contained in the sample.Therefore, the conventional method of measuring the TRACP activity can not be measured osteoclast specificity T RACP5b.Improvement as these assay methods, known a kind of method, wherein the serum through comprising 5 times of dilutions is after 1 hour pre-service of 37 ℃ of cultivations, under the situation that tartrate exists, pass through to use p-nitrophenyl phosphoric acid (pNPP) as substrate, measure activity (" Nichidai-Ishi ", the 49:904-911.1990 of remaining TRACP; And Clin.Chem.33:458-462.1987).This method enables to avoid the influence derived from erythrocytic acid phosphatase, but can not get rid of the influence derived from hematoblastic acid phosphatase.In addition, as more specific activity determination method, have a kind of TRACP of utilization 5b and derived between red blood cell or the hematoblastic tartrate resistance acid phosphatase to the TRACP 5b assay method (JP-A-10-37198) of the sensitivity differences of fluorine.Yet although this method can be avoided the influence derived from red blood cell or hematoblastic tartrate resistance acid phosphatase, it can not get rid of the influence of TRACP 5a.In addition, this method need be on precision be further improved because in this method by deduct not repressed determination of activity TRACP 5b activity under the situation that fluorine exists from total tartrate resistance activity of acid phosphatase.
In method of immunity, for example use method (the J Clin Endocrinol Metab.71:442-451.1990 of people's such as Marius E, Sari L polyclonal antibody; With Clin Chem.46:1751-1754.2000) and use people's such as Jussi M.Halleen, Heather Bull monoclonal antibody method (J Bone Miner Res.13:683-687.1998; ImmunolLett.70:143-149.1999; J Bone Miner Res.14:464-469.1999; Clin Chem.45:2150-2157.1999; With Clin Chem.46:1751-1754.2000), measure activity, so the influence of TRACP 5a be can not ignore corresponding to whole the 5th band.In addition, people's such as Halleen method (designing this method with specific determination TRACP 5b) more has specificity to the activity derived from the TRACP 5b of osteoclast, but healthy human sample and suffer between patient's sample of bone resorption disease of deterioration and only show small difference.Therefore, as the susceptibility deficiency of the TRACP 5b of bone resorption mark.It is believed that the low selectivity to the TRACP 5b of the polyclonal antibody of former preparation and monoclonal antibody is the reason of above-mentioned defective.Since TRACP contains 10 times of fragments to a large amount of no enzymatic activities of organized enzyme amount in the report serum, also suppose these fragments and the competition of complete enzyme in the serum, therefore because its selectivity of disadvantageous reactive system is very low.
Even by method utilizations such as immunoassays the total antigen of the competition material of enzymatic activity, complete enzyme and enzymatic degradation product is carried out quantitatively up to now in this case.Yet the result is when epi-position is limited under the situation in monoclonal antibody, because fragment and immeasurability fragment have appearred measuring in degradation.Therefore, can infer to have for example following problem: even when measuring, do not have correlativity in the measurement result of using the plurality of reagents box to obtain with a kind of target material.In addition, also be reported under the situation of TRACP (example that provided in the past), the amount of its fragment reactive bone clinically absorbs (J.Bone Miner.Res., 15:1337-1345,2000; And Clin.Chem.47:74-80,2001).This is regarded as only being used to measure the necessary suitable examples of enzyme form alive.In this case, only the level of enzyme will be measured by removing fragment.Usually hypothesis is measured the TRACP out of true by activity determination method, because inactivation fragment and enzyme competition.And, be not used in the fragments specific antibody of purifies and separates protein fragments, therefore, do not provide the method that is used to separate in fact.
Disclosure of the Invention
It all is impossible for a long time that the TRACP that illustrates in this instructions accurately measures, and has clinical meaning although it is believed that it.Therefore, following two kinds of reasons have been considered.First kind of reason is the antibody that the TRACP 5b of indication osteoclast activity is not had high degree of specificity.Second kind of reason is to exist with the amount that surpasses organized enzyme in serum by the protein fragments that the TRACP enzymatic degradation forms.
Therefore, among the present invention, the TRACP organized enzyme had the first antibody of height binding constant value and hardly with enzyme reaction but the second antibody that combines with the inactivation degradation fragment of enzyme by exploitation, and they are used for same reactive system, only enzyme running water is flat is accurately measured, and avoids the influence of catabolite.
Consider this problem, the invention provides a kind of method of immunity, wherein at first unite and use and the first antibody of target material height reaction to be measured and the second antibody of highly reacting with competition material (for example inactivation fragment), to get rid of the influence of competition material in the reactive system, take this, special and accurately measure target material, for example organized enzyme.
That is to say, the invention provides a kind of method of immunity, wherein by the target material in the TPPA sample of two types of uses, this method comprises
Use two types antibody, the first antibody and the second antibody that promptly have following characteristic: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody is higher than compatibility to the target material to the compatibility of competition material, (iv) second antibody is higher than the compatibility of first antibody to the target material to the compatibility of competition material
Target material and competition material in the sample are combined, then with first antibody and second antibody on being adsorbed on carrier
The level of the target material of measurement combination is to measure the target material in the described sample.
In addition, the invention provides the hit kit of material of the antibody mediated immunity working sample that uses two types, comprising
Two types antibody, the first antibody and the second antibody that promptly have following characteristic: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody to the competition material compatibility be higher than to the compatibility of target material and (iv) second antibody to the competition material compatibility be higher than the compatibility of first antibody to the target material.
The accompanying drawing summary
Fig. 1 is the simple table that shows the Protein ChipSystem analysis result that uses Cyphergen Biosystems.Inc..Find 4 kinds of fragments specific reaction is with it arranged in baby's serum from chart (1) and (2) with regard to antibody Trk49.On the other hand, find almost do not have fragment specific reaction with it from chart (3) and (4) with regard to antibody Trk62.
Fig. 2 shows the result who measures the forward and backward sufferers of osteoporosis face serum of hormone therapy.Observed two peak values disappear in the treatment back before this treatment.Infer also that from this fact the fragment corresponding to these two peak values is specific to bone resorption.
Fig. 3 shows the result that the antibody Trk49 that uses same concentrations and Trk62 carry out ELISA.Antibody Trk49 only shows hypoergia.Yet, compare with the situation of only using antibody Trk62, use at the same time to have shown the reactivity that improves on the flat board of antibody Trk49 and Trk62.
Fig. 4 show by absorption antibody Trk62 to flat board and absorption antibody Trk62 or antibody Trk49 carry out the result of ELISA to the latex particle.The immune body sensitized reactant emulsion thing of the Trk62 that obtains can with the situation of flat board competition under, along with the painted minimizing of raising of the reactant emulsion substrate concentration that adds.But by contrast, with regard to the reactant emulsion thing of Trk49 sensitization, the colouring value of enzyme increases in 0.1%～0.15% experimental group.So, find when be adsorbed on antibody Trk62 on the flat board when uniting use, the immune body sensitized reactant emulsion thing of Trk49 is effective.
Implement mode of the present invention
In the method for immunity of the present invention, two types antibody, promptly first antibody and second antibody are used for working sample target material to be measured.Described first antibody and second antibody have following characteristic: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody to the competition material compatibility be higher than to the compatibility of target material and (iv) second antibody to the competition material compatibility be higher than the compatibility of first antibody to the target material.Herein, term " competition material " is meant to target material to be measured to have similar antigenic material, and has such characteristic, and promptly when preparing the antibody of target material, described competition material and target material are competed and this antibodies.For example, when the target material is protein or enzyme, the competition material comprises, for example the fragment of protein or enzyme, have in the amino acid sequence that constitutes protein or enzyme by replace, disappearance or add the variant of the amino acid sequence that amino acid residue forms and by in protein or enzyme, adding phosphate-based or changing the modified outcome that the sugar chain of protein or enzyme partly obtains.
Among the present invention, the exemplary of target material to be measured is complete enzyme.When the target material was complete enzyme, the exemplary of competition material was the material with enzymatic activity, for example the product that obtains by enzymatic degradation, i.e. enzymatic degradation product.
Each first antibody and second antibody can be any in antiserum, polyclonal antibody and the monoclonal antibody among the present invention.
Can prepare and be used for first antibody of the present invention and second antibody, method is for example to pass through with target material immune animal to be measured, as antigen, splenocyte and the myeloma cell of animal are merged with the preparation hybridoma, select to have two types monoclonal antibody of following character then, antibody as two types, promptly free hybridoma prepares the first antibody and the second antibody of various monoclonal antibodies: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody to the competition material compatibility be higher than to the compatibility of target material and (iv) second antibody to the competition material compatibility be higher than the compatibility of first antibody to the target material.Among the present invention, second antibody can be to the target material and compete the antibody that material all has compatibility.In order to obtain to satisfy two types monoclonal antibody of above-mentioned four conditions about target material and competition material, for example, following palpus satisfies: by the target material of the various monoclonal antibodies of measurement such as ELISA and the binding characteristic of competition material, and on the basis of measurement result, select to satisfy two types monoclonal antibody of above-mentioned four conditions.In addition, also can measure the target material of various monoclonal antibodies and the binding constant (" ProteinEnzyme Fundamental ExperimentalMethods " of competition material according to known method, Nankohdo Co. selects two types monoclonal antibody Ltd.) and according to measurement result.In addition, also can select two types monoclonal antibody by using various Monoclonal Antibody affinity column and measuring target material and the competition material that is adsorbed on the post.
In two types antibody each, be that first antibody and second antibody are when being antiserum or polyclonal antibody, for example following is feasible: under by the various conditions that change adjuvant or the foundation of change immune condition, with target material immune animal to be measured, like this binding characteristic of the target material of antiserum of Huo Deing or polyclonal antibody and competition material by with situation that said monoclonal antibody exists under identical method measure, and select to satisfy two types antibody of above-mentioned four conditions.
The exemplary of two types of antibody that use among the present invention is monoclonal antibodies of two types that can be used for measuring derived from the TRACP 5b of osteoclast.One of two types monoclonal antibody relevant with TRACP 5b and that obtain according to the present invention is first monoclonal antibody, the reactivity of this antibody and TRACP 5b is higher than the monoclonal antibody of report before this, and hardly with the reaction of the fragment of TRACP 5b, another be with the enzyme fragment reaction of inactivation and hardly with second monoclonal antibody of organized enzyme reaction.Utilizing this antibody of two types to make the TRACP 5b derived from osteoclast is had specific method of immunity becomes possibility, and this method is subjected to the influence of other isodynamic enzyme in TRACP 5b inactivation fragment and the blood (derived from red blood cell, blood platelet, neutrocyte or macrophage) hardly.
By the example of these monoclonal antibodies, elaboration is used for antibody of the present invention two types in further detail below.
Said monoclonal antibody can be by using the derived from human osteoclast the TRACP 5b of purifying obtain as immunogene.Although hereinafter in the method for Miao Shuing, use the antigen that from normal osteoclast, obtains to carry out immunity, not only the TRACP 5b of this antigen but also osteoclastoma etc. can be used as antigen.
Said monoclonal antibody is by hybridoma production, and described hybridoma passes through the people TRACP5b of usefulness purifying as antigen-immunized animal, and its cell and myeloma cell that can produce anti-people TRACP 5b antibody is merged acquisition.
Above-mentioned hybridoma can obtain by the following method.That is to say, in the above described manner the people TRACP 5b of Huo Deing and known adjuvant for example Fu Shi fully or Freund, aluminum hydroxide adjuvant, pertussis adjuvant etc. mix, preparation is used for the adjuvant liquid of sensitization, subcutaneous or tail vein is applied to animal (for example mouse or rat) to this liquid by the abdominal cavity, with several parts every this animal of 1-3 week immunity.Usually select in the scope of 1 μ g-100mg although be used for the amount of the antigen of sensitization, about 50 μ g are normally preferred.Though the number of times of immune operation is generally 2-7 time, the whole bag of tricks is known.Subsequently, merge with the cell with multiplication capacity (for example myeloma cell etc.) in test tube derived from the antibody produced cell of spleen etc.This antibody produced cell can obtain from mouse, nude mice or rat.
About above-mentioned fusion method, can use poly-(ethylene glycol) (PEG) to merge by the method (Nature.256,495.1975) of known Kohller and Milstein.Merging also can be by using sendai virus (Sendai virus) or being undertaken by electric fusion method.
About the method that selection from fused cell can be produced the hybridoma of the antibody that can discern people TRACP 5b, can followingly carry out described selection.That is to say, in the above-mentioned fused cell of limiting dilution, from the plastidogenetic colony of HAT nutrient culture media and HT nutrient culture media, surviving, select hybridoma.When the antibody of people TRACP 5b is contained in the nutrient solution supernatant that is inoculated into any colony that the fused cell in the 96-hole flat board etc. forms, can can produce the clone of the monoclonal antibody of people TRACP 5b by the selection of ELISA method, wherein supernatant is placed on the assay plate with the people TRACP 5b that is fixed thereon, after the reaction, secondary labels antibody is anti--mouse immuning ball protein-HRP labelled antibody and above-mentioned antibody response for example.The serve as a mark mark substance of antibody can use enzyme (for example alkaline phosphatase) except that HRP, fluorescent material, radiomaterial etc.The specific antibody of screening people TRACP5b, in contrast, can by only use BSA with it the assay plate of combination carry out above-mentioned ELISA simultaneously and finish as the ELISA of blocking agent.That is, be chosen in and have the clone who is positive and in the ELISA that only uses BSA, is negative on the flat board that people TRACP 5b is fixed thereon.
In addition, can be determined as follows simultaneously: will resist-mouse immuning ball protein antibody is inoculated in the trace inoculation plate, the nutrient solution supernatant that wherein adds fused cell reacts, the TRACP 5b that adds purifying again combines and forms immune complex with organized enzyme, then by utilize to produce the look substrate for example the pNPP enzyme of measuring such combination live.
First monoclonal antibody of using among the present invention comprises the monoclonal antibody of energy specific recognition people TRACP 5b, particularly, it and complete people TRACP 5b reaction, has low reactivity not with acid phosphatase cross reaction, and to the inactivation fragment of people TRACP 5b derived from red blood cell, blood platelet, neutrocyte or prostate and potato ACP.Its example is the monoclonal antibody of producing by the hybridoma Trk62 that is set up by the inventor.Be used for the monoclonal antibody that second monoclonal antibody of the present invention comprises energy specific recognition people TRACP 5b inactivation fragment, particularly, it hardly with active people TRACP 5b reaction, not with acid phosphatase cross reaction derived from red blood cell, blood platelet, neutrocyte or prostate and potato ACP.Its example is the monoclonal antibody of producing by the hybridoma Trk49 that is set up by the inventor.
Particularly, in order to combine with the organized enzyme specificity, the antibody that the first antibody that uses among the present invention preferably has higher compatibility to the people TRACP 5b fragment of active people TRACP 5b comparison inactivation.More preferred example is to have the binding constant of the active people TRACP 5b antibody greater than second antibody.As second antibody, suitable is has the binding constant of the TRACP 5b fragment of inactivation greater than the antibody to the binding constant of the active TRACP 5b of people of first antibody.As first antibody, for example understand the monoclonal antibody of hybridoma Trk 62 productions of setting up by the inventor.As second antibody, for example understand the monoclonal antibody of hybridoma Trk 49 productions of setting up by the inventor.Above-mentioned hybridoma Trk 49 and Trk 62 are preserved in following unit, be the biological preservation center of patent (Patented Organism Deposition Center) (IPOD), industrial technology synthetic study institute (independent administrative corporation), Chuo-dairoku, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, Japan 305-8566: hybridoma Trk 49 is in preservation preserving number on the 27th IPOD FERM BP-8249 November in 2002, and hybridoma Trk 62 is preserving number IPOD FERM BP-7890 in preservation on February 14 in 2002.
Above-mentioned each hybridoma is cultivated on the nutrient culture media of cellular incubation being generally used for, for example α-MEM, RPMI1640, ASF, S-clone etc., and monoclonal antibody can reclaim from the nutrient culture media supernatant.Following also is feasible: handle nude mice with norphytane in advance, after (hybridoma is derived from nude mice), cell causes the ascites accumulation through intraperitoneal injection in animal body, reclaim monoclonal antibody then from ascites.
About reclaiming monoclonal antibody method, can adopt conventional method from above-mentioned supernatant or ascites.Here for example understand with ammonium sulfate, sodium sulphate etc. saltout, the affinity chromatography of chromatography, ion-exchange chromatography and use albumin A.
By using monoclonal antibody of the present invention, can accurately measure TRACP 5b in the blood serum sample by the said method purifying.Set forth method of immunity of the present invention below.
For example, followingly implement method of immunity of the present invention.At first, first antibody and second antibody are adsorbed on insoluble solid support for example on the flat board, and except that above-mentioned adsorption step, this method of immunity can be undertaken by the step identical with conventional ELISA method.The sample that is used to measure at first adds the holder with absorption two types of antibody thereon.In this case, because the difference of binding constant between first antibody and the second antibody, competition material in the sample main with the second antibody generation antigen-antibody reaction that is adsorbed on the holder, the target material in the sample is main then with the first antibody that is adsorbed on the holder antigen-antibody reaction takes place advantageously.Subsequently, wash solid support in case of necessity, measure level then attached to the target material on the solid support, but the target material in the working sample like this.In this case, when the target material is that complete enzyme is for example during TRACP 5b, with correspondingly substrate solution for example pNPP (p-nitrophenyl phosphoric acid) add solid support and carry out enzyme reaction, and the level of the target material that combines with solid plate of mensuration, so accurately the target material in the working sample.
Among the present invention, the carrier of absorption first antibody can be insoluble solid support, and second antibody is adsorbable on dispersible carrier in liquid, for example latex or solubilized.In this case, the target material can followingly be measured.At first, dispersible carrier in liquid, for example latex dissolves with the second antibody sensitization or with second antibody.On the other hand, first antibody is adsorbed on the solid support.Then, the solution that will contain the second antibody of such processing adds solid support with the sample that is used to measure, the main and second antibody generation antigen-antibody reaction of the competition material in the sample like this, and the target material is main and first antibody generation antigen-antibody reaction.Subsequently, wash solid support in case of necessity, measure level then attached to the target material on the solid support, but the target material in the working sample like this.In this case, when the target material is complete enzyme such as TRACP 5b, with correspondingly substrate solution for example pNPP (p-nitrophenyl phosphoric acid) add solid support to measure, the so accurate target material in the working sample attached to the target material on the solid support.
Illustrated as mentioned, the normally insoluble solid support of carrier of absorption first antibody.Second antibody is on being adsorbed in insoluble solid support or be scattered in the solution that comprises latex etc. the back and use, or dissolving.
Insoluble solid support without limits, as long as it is the solid support that uses in the solid-phase immunoassay method of for example ELISA.Insoluble solid support comprises, for example, and the solid support made from polystyrene, polypropylene, polycarbonate, tygon, nylon, polymethacrylate etc.By physical bond, chemical bonding or affine, combine with above-mentioned any known solid support directly or indirectly according to monoclonal antibody of the present invention (antibody).The amount of antibody that is used for sensitization is usually in the scope of 1ng-100mg/ml.The sample that is used to measure people TRACP 5b adds and reacts by physical bond, chemical bonding or the affine monoclonal antibody (antibody) that is combined on the solid support.Behind the reaction certain hour, washing solid support and adding are produced the look substrate and are reacted.About substrate, can use for example pNPP of known substrate.
As can be scattered in the liquid, second antibody absorption carrier thereon, example has latex particle, magnetic-particle and lipid granule.Also can use the dispersion that in solution, obtains by with second antibody and synthetic polymer, natural polymer bondings such as (for example glucosans) and the polymer dispersed that will handle like this.In this case, the target material of measuring on the solid plate can and carry out easily by the damping fluid reagent in the second antibody adding ELISA method that can combine with the competition material.The mensuration of target material can and make the second antibody of such processing carry out easily with being adsorbed in the first antibody competition on the solid plate by second antibody and solid phase carrier bonding.In addition, the mensuration of target material can be by second antibody and synthetic polymer, natural polymer bondings such as (for example glucosans), and the antibody that will handle like this adds damping fluid reagent and carries out easily.
The kit of implementing method of immunity of the present invention comprises two types antibody, promptly above-mentioned first antibody and second antibody.As mentioned above, if desired, these antibody can be adsorbed on insoluble solid support or are scattered on the carrier (for example latex) in the liquid.If desired, this kit can further contain the reagent of the target material that is useful on mensuration and antibodies.
About the present invention, described in detail as back embodiment 1, the TRACP 5b fragment in the multiple serum is used monoclonal antibody Trk49 and the Trk62 that is produced by above-mentioned hybridoma Trk 49 and Trk62 respectively, studies by protein biochip technology.The result confirms to find that molecular weight is about the TRACP 5b fragment of 5580Da, 5795Da, 6860Da or 7075Da in the serum of baby or patients with osteoporosis, wherein bone resorption carries out actively.Therefore, find that these TRACP5b fragments can be used as the marker molecule of diagnosis bone disease (for example active bone disease of carrying out of bone resorption).Therefore, can take from these marker molecules in patient's the sample (for example serum, blood plasma or blood), or measure the amount diagnosis bone disease of these marker molecules by detection.
Can adopt all at present known methods to detect or quantitative marker molecule.Described method comprises, for example, and mass spectrometry, immunoassay, electrophoresis, liquid chromatography and gas chromatography.For mass spectrometry, for example understand the method for using laser parsing/ionization time flight mass spectrometer (LDI-TOF MS).About laser parsing/ionization time flight mass spectrometer, for example understand surperficial laser enhanced parsing/ionization time flight mass spectrometer (SELDI-TOF MS method) and matrix-auxiliary laser parsing/ionization time flight mass spectrometer (MALDI-TOF MS method).For example, when adopting SELDI-TOF MS method, can use Biosystems by Ciphergen, and the protein biosystem II mass spectrometer of Inc exploitation (Ciphergen Biosystems, Inc).This instrument is a benchmark with the protein biochip technology that comprises the combination of SELDI (surperficial laser enhanced parsing ionization) and time of-flight mass spectrometer.The particulars of this instrument are disclosed in International Patent Application WO 01/25791 A2, JP-A-2001-281222 etc.Usually, in the SELDI-TOFMS method, sample is pretreated, is adsorbed on the chip, is loaded in then on the SELDI-TOF MS mass spectrometer.When sample is serum, preferably from system, remove albumin, electrically charged the stopping of albumin that method is to use the albumin adsorbent or causes owing to ion-exchange up to chip with the buffer solution for cleaning system.The protein-chip that uses in this method is not specifically limited, as long as its adsorbable above-mentioned marker molecule.As protein-chip, chip (being also referred to as chemical chip) for example, the functional group that wherein has the protein compatibility, its modified generation hydrophobicity, ion exchange property etc., and chip (biochemical chip) has the antibody of the protein of interest matter that is fixed thereon.
About another kind of mass spectrometry, for example understand the mass spectrophotometry of using ESI method (electrospray ionization).Under the situation of using the ESI method, will be loaded into through the sample of pre-service (for example Protease Treatment) with the direct-connected mass spectrometer of separative element (for example high performance liquid chroma-tography) on usually be preferred.As method of immunity, for example understand a kind of method, wherein by preparing the polyclone or the previously known protein of monoclonal antibody measuring of marker molecule.This method of immunity comprises enzyme immunoassay (EIA method), immunoturbidimetry (TIA method), latex immunity-agglutination (LATEX method), electrochemiluminescence method, fluorescence method etc.The method of immunochromatographic method and use test paper also is effective.Usually, all these methods all are known to the person skilled in the art, and these usually known methods can be them and adopt.
In addition to the above methods, for example understand electrophoresis, liquid phase chromatography (LC), vapor-phase chromatography (GC) etc.These methods also are known to the person skilled in the art usually, and can adopt these common known methods to measure.
The present invention more specifically illustrates with reference to following preferred embodiment, and this does not also mean that and limit the scope of the invention.
(1) selection and preparation are used to prepare the antigen of monoclonal antibody
The TRACP 5b of preparation derived from human osteoclast is used as the antigen of preparation Anti-Human TRACP monoclonal antibody.A kind of purification process is described below.
After obtaining agreeing, 130g human femur head by surgical resection is frozen in the liquid nitrogen, crush with hammer, be suspended in (50mMTris-HCl, 0.3M KCl, 1mM PMSF, 1mM EDTA2Na in the 200mL damping fluid that contains protease inhibitors then, 0.1% Triton X-100,0.02% NaN
3, 1 unit/ml presses down the enzyme peptide, pH7.5), homogenate in ultrasonic refiner then.4 ℃ of stirrings of homogenate are spent the night, and 10, centrifugal 20 minutes of 000rpm, supernatant is dialysed to 10mM Tris damping fluid (pH8.2).After this, dislysate is applied to CM-Ago-Gel post (Φ 40mm * 40cm) (SIGMA), the protein of absorption increases NaCl concentration (0-0.5M NaCl) with linear gradient with the above-mentioned identical Tris buffer solution elution that contains NaCl.With substrate p-nitrophenyl phosphatase assay tartrate resistance activity of acid phosphatase, merge and contain the active part of high level.With the solution concentration that obtains like this, to containing 20mM Tris damping fluid (pH7.2) dialysis of 0.7M NaCl, dislysate is applied to Superdex 200 strains (Φ 16mm * 60cm) (AmershamPharmacia Biotech) then.In the same manner described above, measure the part mesotartaric acid resistance activity of acid phosphatase that obtains by elution, merge and comprise active part.The solution that obtains dilutes 2 times with 20mM Tris damping fluid (pH7.2) like this, be applied to HiTrap Heparin HP post (5mL) (Amersham Pharmacia Biotech), the protein of absorption improves NaCl concentration (0.35-1M NaCl) with linear gradient simultaneously with above-mentioned identical 20mM Tris damping fluid (pH7.4) wash-out that contains NaCl.Merge the part that contains high-caliber tartrate resistance activity of acid phosphatase, concentrate then and obtain the acid phosphatase of 0.4mg derived from the purifying of osteoclast.
Use A
280Determine the amount of protein.For purifying, carry out SDS-PAGE (TIFCO), silver dyeing subsequently.As a result, purity is determined by single band occurs at about 35,000 molecular weight places.Be considered to the TRACP 5b of purifying corresponding to the enzyme of single band, and be used as immunizing antigen.
(2) immunity
Tartrate resistance acid phosphatase (TRACP 5b) derived from the purifying of osteoclast is diluted to concentration 250 μ g/ml with 50mM citrate buffer solution (pH5.5), and 25 μ g (100 μ l) dilution fully mixes up to realizing emulsification with the Freund's complete adjuvant (WAKO) of 100 μ l.Give in Zhi Bei the suspending liquid peritonaeum like this with 6 ages in week of diethyl ether anesthesia the Balb/c female mice (Nippon Clear Co., Ltd.).After two weeks, the above-mentioned TRACP 5b of same amount (25 μ g/ml) mixes with incomplete Freund (WAKO).By with the identical operation of the situation of Freund's complete adjuvant, realize emulsification obtaining suspending liquid, and with suspending liquid sensitization mouse.After disposing for 2 weeks like this, carry out step same as described above.For the 4th immunity, promptly final immunity, preparation gives mouse by injection tail vein then with the TRACP 5b (25 μ g/ml) of 50mM citrate buffer solution (pH5.5) dilution.
(3) set up hybridoma
Back 3 days of final immunity under the situation with diethyl ether anesthesia,, is scattered in it in the splenocyte of preparation under aseptic condition from removing spleen with the mouse of TRACP5b sensitization through operation.Method according to Kohller and Milstein merge (Nature, 256,495,1975) by use poly-(ethylene glycol) (PEG4000) (MERK) with splenocyte and myeloma cell P3-X63-Ag8-U1 (P3U1) fusion.About fusion ratio, the quantity of splenocyte is 8 * 10
7, and the quantity of myeloma cell P3-X63-Ag8-U1 (P3U1) is 2 * 10
7Just, splenocyte and myeloma cell's fusion ratio is 4:1.Fused cell is scattered in 10%FCS (INVITROGEN) α-MEM (IRVINE) HAT) (Cosmo Bio Co., Ltd) in the nutrient culture media, be inoculated in 96-hole microtitration culture plate (Sumitomo Bakelite Co., Ltd.), then at 37 ℃ and 5% CO
2Condition under cultivate.
(4) screening
After about 2 weeks, confirm and screen the colony of growth.Carrying out method for screening is described below.
In order to produce the flat board that is used to screen, the TRACP 5b of purifying is dissolved in the 50mM citrate buffer solution in above-mentioned (1) item, and is added in the 96-hole flat board (Nunc) with the amount in 0.5 μ g/100 μ l/ hole.Make dull and stereotyped 4 ℃ to leave standstill for two nights, then with the Tris damping fluid washing that contains 0.05%Tween20 three times.Add 200 μ l, 1.5% BSA solution in each hole to suppress nonspecific reaction, make dull and stereotyped 4 ℃ of standing over night.After the flat board that makes like this washed 3 times with the Tris damping fluid that contains 0.05% Tween20,100 μ l culture supernatant were reacted in each hole, and further washing is dull and stereotyped.Then, adding a kind of second antibody (anti--mouse immuning ball protein antibody (Zymed) of HRP-mark) reacts.After the washing, the o-phenylenediamine of 100 μ l 3mg/ml produces look solution (OPD) (Nakalai) in the citric acid, and the product look substrate of HRP adds each hole and causes painted within a certain period of time.Then, the sulfuric acid of 100 μ l 1N adds each hole as stop bath, and measures the wavelength place at 492nm and measure absorbance.Find that by above-mentioned steps the clone who is positive clones by limiting dilution assay again, the supernatant of Huo Deing is checked again like this.
(5) antibody determines
Have the clone Trk49 of purifying TRACP 5b and the reactivity of Trk62 and determine, although to find their affinity differences, clone Trk49 and Trk62 and flat board reaction with high susceptibility by ELISA.As a result, select clone Trk49 and Trk62 clone as identification TRACP 5b.The antibody that produced by these clones use monoclonal antibody classification agent boxes (AmershamPharmacia Biotech) to measure, the results are shown in the following table 1 of acquisition.
Table 1: clone's feature
(6) preparation and monoclonal antibody purification
10 the week ages the Balb/c female mice (Nippon Clear Co., Ltd.) give two weeks of 0.5ml norphytane (Aldrich) after, give 1 * 10 in the peritonaeum
7Each hybridoma Trk 49 that obtains in individual above-mentioned (5) and the cell of Trk 62.After about two weeks, the operation under the situation of diethyl ether anesthesia of the ascites of accumulation is collected in the mouse peritoneal.By use progressively dilute as the ascites of sample and by in above-mentioned (4) for the result of the ELISA method confirmation of screening employing, discovery ascites contains the high concentration monoclonal antibody.This ascites is handled with 40% ammonium sulfate and to the PBS dialysis, monoclonal antibody is used protein G post (Amersham Pharmacia Biotech) purifying and confirmed by SDS-PAGE then.As a result, under the situation of two kinds of monoclonal antibody Trk49 and Trk62, when antibody is in non-reduced state, confirm to have single band at about 150,000 molecular weight places, when antibody is used mercapto-ethanol reduction, confirm to have two bands at about 50,000 and 25,000 molecular weight places.The amount of each antibody purified Trk49 and Trk62 is about 15mg or more in every mouse, that is to say that it is enough for industrial utilization.
(7) specificity analyses
In order to study the specificity of monoclonal antibody Trk49 and Trk62, use various isoforms (TRACP5b, red blood cell, blood platelet, PAP (SIGMA) and potato ACP (SIGMA)) to carry out following test.Assay method is briefly described below.
Each monoclonal antibody of using the protein G purifying left standstill 4 ℃ of this flat boards two days with in the amount adding solid plate in 2 μ g/ holes (Nunc).This flat board is with 20mM Tris (pH7.0) washing 3 times, and cleansing solution contains 0.05% Tween 20, then 200 μ L, 1.5% BSA Tris (pH7.0) is added in each hole the sealing of spending the night under 4 ℃ subsequently.The flat board of Chan Shenging washs once with cleansing solution same as described above like this, the enzyme of various isoforms is lived all adjust to 10IU/L, and each solution that 100 μ l obtain is like this added in each hole of antibody-attached flat board.Reaction was carried out 2 hours under the room temperature.After reaction is finished, dull and stereotyped with cleansing solution washing same as described above 3 times, add the substrate of 100 μ l acid phosphatases, be reflected at 37 ℃ and carried out 1 hour.The level of enzyme is painted definite from the substrate that the enzyme by antibody capture causes in each sample.After adding 50 μ l reaction-stop baths, the place measures at the 405nm optical wavelength.
As a result, find that monoclonal antibody Trk49 and Trk62 have high specificity,, do not have cross reactivity with other isoform because they only react with TRACP 5b.
(8) utilize the SELDI method to confirm fragment
Recent years, the protein fragment technology that comprises surface-enhanced laser parsing ionization (SELDI) and time of-flight mass spectrometer combination is by Ciphergen Biosystems Inc., and USA develops, and has begun clinical practice, for example, be used to detect new tumor marker.The inventor attempts by using described technology and utilizing the monoclonal antibody that is produced by hybridoma Trk49 that sets up and Trk62 to catch TRACP 5b fragment in the serum in practice.
1) test of use PS20 chip
At first, the inventor by use PS20 protein-chip array (Protein ChipArrays) (Ciphergen Biosystems, Inc), TRACP 5b or its fragment of search and antibody Trk49 and Trk62 reaction in serum.This PS20 chip refers to antibody-in conjunction with chip and has following feature: the reactive material in the sample at first with the antibodies that is combined on the metal surface, and dissociate and in vacuum, fly from antibody with laser with the material of antibody response, measure the molecular weight of this material thus.The experimental working technique of protein-chip array is briefly described below.At first, (SankoJunyaku Co., Ltd.), each antibody that is produced by their uses protein G post purifying (Amersham Pharmacia) to cultivate the hybridoma Trk49 be adapted to synthetic media S-clone and Trk62.The concentration of each antibody is adjusted to 500 μ g/mL, and the solution that 2 μ l obtain like this adds the PS20 chip.Reaction is after 1 hour under the room temperature, the blocking-up damping fluid (1M monoethanolamine PBS pH8.0) that adds 5 μ l (WAKO), reaction was at room temperature carried out 10 minutes.After the blocking-up, lavation buffer solution (the 0.5% Triton 100PBS) room temperature washing of chip usefulness 8mL 5 minutes.Repeated washing twice like this, and this chip is with the soft rinsing of PBS then.Wipe near the zone of reaction spot gently, and in 4 samples each is got 3 μ l add chip, the serum of sufferers of osteoporosis face after the serum that these 4 samples are the healthy volunteer, 12 years old or following child's serum, the loose patient's of hormone replacement therapy osteoid serum and the hormone replacement therapy.After reaction was finished, sample absorbed with Kimwipe (Jujo Kimberley), and chip is used 8mL lavation buffer solution (0.5% Triton 100PBS) room temperature washing 5 minutes.Repeated washing twice like this, and this chip is with the soft rinsing of PBS then.Wipe water droplet with Kimwipe (Jujo Kimberley) from chip, and the saturated sinapic acid of 0.5 μ l (Ciphergen Biosystems Inc)/50% acetonitrile (Wako)/0.5% TFA (Wako) solution adds twice.The protein-chip array that produces ProteinBiologySystem II mass spectrometer deciphering like this (Ciphergen Biosystems, Inc).
2) result who uses the PS20 chip to test
Determination data is shown among Fig. 1 and Fig. 2.In this data layout, abscissa axis refers to the molecular weight of protein from each sample that the PS20 protein-chip separates, and axis of ordinates refers to be reflected at the peak height that above-mentioned molecular weight place reaches the analyte amount of wave detector.In this case, the unit of axis of ordinates is subjective unit (AU).In above-mentioned test, the loose patient's of child's serum and hormone replacement therapy osteoid serum is the active serum of bone resorption in process.As test findings, only when using antibody Trk49 to experimentize, in peak and near the peak the 6860Da (Fig. 1, (2) and Fig. 2, (5)) observed under the situation of above-mentioned two test group near the 5795Da.In process, do not observe these peaks (Fig. 1, (1) and (3)) under the HAS's of bone resorption gentleness the situation.Under the situation of the child's serum that shows peak value, except that described peak, go back the peak of nearly 5580 Da of susceptible of proof and the peak (Fig. 1, (2)) of nearly 7075Da.After the HRT treatment, the peak disappearance (Fig. 2, (6)) of nearly 5795Da of sufferers of osteoporosis face and nearly 6860Da.It is to have 20 or following error at measurment usually that word " closely " is used to express reason corresponding to the molecular weight at each peak.In addition, when using antibody Trk62 to experimentize, do not find specific fragment (Fig. 1, (3) and (4)) in the serum that bone resorption enlivens in the process.As experimental result, the inventor successfully finds the quite new TRACP5b fragment of several types, and they are unknown in the past.In fact, this fragment is at about 5795Da, about 5580Da, about 6860Da and the detected protein of about 7075Da or its catabolite.These fragments are new fully materials and are the labels that the energy reactive bone absorbs or falls ill.The amount of binding fragment and the binding constant of each antibody are closely related.Answer reactive special attention of antagonist Trk62.Antibody Trk62 reacts with the fragment of binding antibody Trk49 hardly, has proved that antibody Trk62 has the specificity of height to organized enzyme.On the contrary, antibody Trk49 and fragment are reacted finely.Infer from these facts: be used for monoclonal antibody of the present invention two types, be antibody Trk49 and Trk62, respectively fragment and enzyme are had the specificity of height, so among the present invention, TRACP 5b can use two kinds of antibody by extremely sensitive ground of enzyme immunoassay specific assay.
(9) binding constant of measurement monoclonal antibody
Carry out following test and be used for determining the binding constant of each antigen.About determining method, according to " protein enzyme infrastest method (ProteinEnzyme FundamentalExperimental Methods) ", Nankohdo Co., Ltd. measures.Prepare two types of antigenic solutions, the i.e. TRACP 5b of (A) purifying and (B) by using the TRACP 5b fragment (potpourri of TRACP 5b fragment of Trk49 affinity column purifying, it contain at least have about 5580Da, 5795Da respectively, the TRACP 5b fragment of 6860Da and 7075Da molecular weight), be the citrate buffer solution of 5 μ g/mL 20mM to obtain concentration.Each hole of 96-hole flat board (Nunc) is with each antigenic solution sensitization of 50 μ L.After 4 ℃ of standing over night, wash 3 times with the 20mM Tris damping fluid that 200 μ L contain 0.05% Tween 20 in each dull and stereotyped hole, and 100 μ L sealers (20mM Tris damping fluid contains 1.5% BSA and 10% sucrose) are added each hole, 4 ℃ of standing over night subsequently.The 20mMTris damping fluid washing 3 times of 0.05% Tween 20 is contained in each dull and stereotyped hole once more with 200 μ L, be that each antibody-solutions of 20 μ L of 12 concentration (0,0.1,0.15,0.20,0.25,0.3,0.35,0.4,0.45,0.5,0.75,1.0 μ g/mL) acquisition adds in each hole by each antibody of stepwise dilution.Be reflected at and carried out under each condition 2 hours: (i) 37 ℃ and (ii) 4 ℃.Behind such first set reaction, wash 3 times with the 20mM Tris damping fluid that 200 μ L contain 0.05%Tween 20 in each dull and stereotyped hole, and the second antibody of 1000 times of dilutions of 50 μ L is (anti--mouse I gG-HRP labelled antibody: as Zymed) to add each hole.Be reflected at 37 ℃ and carried out 2 hours, wash 3 times with the 20mM Tris damping fluid that 200 μ L contain 0.05%Tween20 in each dull and stereotyped hole.Then, it is painted to cause that the OPD solution of 100 μ L 3mg/mL adds each hole.Sulfuric acid solution termination with 100 μ L 0.1N is painted, subsequently in 492nm place colorimetric.
From the result of such acquisition, by the A of every 1M concentration under the condition (i)
492, the amount of the monoclonal antibody of at first definite combination.For example, owing to A when the amount of antibody on the TRACP 5b flat board is 0.05 μ g
492Be 0.250, measure when the molecule of antibody and do at 146,000 o'clock, in conjunction with the amount of antibody can followingly determine:
0.25/{0.05/(0.146×10
-9)}＝0.731×10
-9 (1)
Then, (ii) the concentration (PL) of the antibody of each antibody concentration combination is definite from the value that obtains down in condition (i) down for condition.
The A of [PL]=condition under (ii)
492The A of/every 1M concentration
492(2)
Then, determine the concentration [PF] of free antibodies from the antibody concentration that adds.
Antibody concentration-[PL] (3) of [PF]=adding
[PL]/[PF] does curve map to the concentration of binding antibody, (nK) determines binding constant by slope of a curve.
Ka=-slope/n
Be shown in the following table 2 as two kinds of monoclonal antibody Trk49 of found that of measuring by said method and binding constant (ka) value of Trk62.
Table 2: the binding constant of monoclonal antibody
During result's implication, find reactivity in having understanded above-mentioned reactive system, promptly compatibility is as follows:
The reactivity of Trk49 and fragment〉reactivity of Trk62 and enzyme〉reactivity of Trk492 and enzyme〉reactivity of Trk62 and fragment
Therefore, find the following fact: because the reactivity of monoclonal antibody Trk49 and TRACP 5b fragment is higher than the reactivity with TRACP 5b itself, and, should reactivity when the binding constant of TRACP 5b being compared higher with monoclonal antibody Trk62, therefore monoclonal antibody Trk49 at first optionally combines with TRACP 5b fragment in the reactive system, more advantageously combines with TRACP 5b in the reactive system that monoclonal antibody Trk62 can reduce in the amount of competition fragment.This result has supported to use the result of the test of protein-chip.
(10) test of measuring by ELISA
Therefore, produce the flat board of type in following 5: Trk49 1 μ g/ hole/100 μ L, Trk49 2 μ g/ holes/100 μ L, Trk62 1 μ g/ hole/100 μ L, Trk62 2 μ g/ holes/100 μ L and Trk49 1 μ g+Trk62 1 μ g)/hole/100 μ L.Except that following change: the volume 100 μ L of sensitised antibodies solution, the volume 100 μ L of confining liquid, the volume 100 μ L of sample, the method for producing flat board is identical with the specific production method of mensuration monoclonal antibody in above-mentioned (7).About first set reaction, combining anteserum (the TRACP5b fragment that comprises the molecular weight that has about 5580Da, 5795Da, 6860Da or 7075Da at least respectively) with high TRACP 5b level adds the antibody that is adsorbed on the flat board as sample, and antigen-antibody reaction is at room temperature shaken and carried out.Then, carry out washing step, the synthetic substrate solution of 100 μ L is added in each hole, carry out enzymatic reaction in 1 hour under 37 ℃.Reaction stops through adding 0.2N NaOH solution, and colorimetric is measured enzyme whereby and lived subsequently.What obtain the results are shown among Fig. 3.
Based on its binding constant, suppose with enzyme to have hypoergia, and enzyme had low affinity therefore do not have necessary susceptibility to measuring with antibody Trk49 that fragment has a high response.On the other hand, based on binding constant, infer the antibody Trk62 demonstration good linear relationship that has high response with enzyme according to antibody Trk62.In addition, have at flat board under the situation of antibody Trk49 and antibody Trk62 potpourri, obtain longer linearity and improved susceptibility.Even since when the amount of each antibody when 1 μ g is increased to 2 μ g, linear and susceptibility is not almost improved, supposition is as the amount of each antibody that relates in reacting, 1 μ g/ hole is enough basically.That is, infer that antibody Trk49 combines with fragment on dominance ground in fact in the reactive system because be improved according to the theoretical linear and susceptibility based on the binding constant test, antibody Trk62 combines with complete enzyme then.
(11) the fragment absorption process of use latex reagent
Use and to have found that specificity in fact absorbs the monoclonal antibody Trk49 of fragment and monoclonal Trk62 in contrast, with each body sensitized emulsion in these two kinds of monoclonal antibodies to prepare latex reagent.Being prepared as follows of latex reagent carried out.
The latex suspension of 2mL 1% is mixed the potpourri stir about of acquisition 1 hour with the antibody Trk49 of 2mL 0.1mg/mL and each solution of Trk62.After centrifugal, precipitation is suspended in 1% the BSA solution, and with suspending liquid stir about 1 hour again.Again after centrifugal, precipitation is suspended in the PBS solution to obtain latex reagent.The dilution of this latex reagent is 8 concentration, promptly 0.15% and from 0.1% by 7 concentration of 2 times of dilutions (0.1,0.05,0.025,0.0125,0.00625,0.00313 and 0.00156%) acquisition, prepare 8 kinds of latex reagents altogether.Operation in the ELISA method is described below.
At first, getting each each 50 μ L in these latex reagents adds in each hole of the flat board of having inoculated antibody Trk62 (2 μ g/ hole).Then, the 100 μ L combining anteserums with high-level TRACP 5b (8U/ml) that will obtain from the patient who obtains its agreement add each hole, and the vibration reaction is 1 hour under the room temperature.After reaction was finished, this flat board washed 3 times with the 20mM Tris damping fluid (pH7.0) that contains 0.05% Tween20, and to wherein adding synthetic substrate solution.Cultivate after 1 hour for 37 ℃, the reacting terminating solution of 50 μ L (0.2N NaOH solution) adds each hole, measures at the 405/490nm place subsequently.What obtain the results are shown among Fig. 4.Add in the experiment at Trk62-body sensitized emulsion reagent, cause and dull and stereotyped competition of going up monoclonal antibody, so that with the painted decline of increase enzyme of latex reagent concentration.Yet, under the situation of antibody Trk49, in the test of adding 0.1～0.15% latex reagent, obtain the enzyme colouring value that significantly improves.This fact for example, shows not only the flat board with antibody sensitization simultaneously, and be used to the carrier that adsorbs for example the absorption process of latex etc. be effective for the absorption of competing fragment.In addition, use that to replace the latex with antibody Trk49 sensitization also be effective by antibody Trk49 being dissolved in the solution that obtains in the solution.
Industrial usability
Elaborate as mentioned, immunoassay of the present invention has height by using with the target material Spend reactive first antibody and with the competition material for example the inactivation fragment of target material have highly anti-The combination of the SA of answering property, make special and Accurate Measurement target material to be measured for example organized enzyme become May, to avoid the impact of competition material in the reaction system.
Claims (6)
1. one kind wherein contains the hit immunoassay of material of sample with the competition material of target material by the TPPA of using two types, and described method comprises
Use two types antibody, the first antibody and the second antibody that promptly have following characteristic: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody is higher than compatibility to the target material to the compatibility of competition material, (iv) second antibody is higher than the compatibility of first antibody to the target material to the compatibility of competition material, wherein compete material and be incorporated into second antibody earlier, target material in the sample enlarges with the ratio of competition material like this, to avoid competing the influence of material to combining between target material and the first antibody
Target material and competition material in the sample are combined with first antibody and second antibody, wherein said first antibody and second antibody are adsorbed on the carrier, or first antibody is adsorbed on the carrier, and second antibody is adsorbed on the carrier that is scattered in the solution or is dissolved in the solution, then
The level of the target material of measurement combination is to measure the target material in the described sample, and the measurement of the target levels of substance that its material that hits is complete enzyme and combination is that the enzymatic activity of described complete enzyme is measured, and competition material wherein is the material with described enzymatic activity.
2. immunoassay according to claim 1, wherein said competition material is the enzymatic degradation product.
3. immunoassay according to claim 1, wherein said complete enzyme is tartrate resistance acid phosphatase 5b (TRACP5b), wherein said first antibody is the monoclonal antibody of being produced by hybridoma Trk62, described hybridoma Trk62 was in preservation on February 14 in 2002, and preserving number is IPOD FERM BP-7890; Described second antibody is the monoclonal antibody of being produced by Trk49, and described hybridoma Trk49 was in preservation on November 27 in 2002, and preserving number is IPOD FERMBP-8249.
4. immunoassay according to claim 1, wherein said carrier are the insoluble solid holders.
5. immunoassay according to claim 1, the carrier that wherein adsorbs first antibody is a solid support, and second antibody is adsorbed on the carrier that is scattered in the solution or is dissolved.
6. one kind by using the kit of the target material in two types the antibody mediated immunity working sample, and it comprises
Two types antibody, the first antibody and the second antibody that promptly have following characteristic: (i) first antibody has the compatibility to target material and competition material, (ii) first antibody is higher than competing the compatibility of material the compatibility of target material, (iii) second antibody is higher than compatibility to the target material to the compatibility of competition material, (iv) second antibody is higher than the compatibility of first antibody to the target material to the compatibility of competition material, wherein compete material and be incorporated into second antibody earlier, target material in the sample enlarges with the ratio of competition material then, to avoid competing the influence of material to combining between target material and the first antibody
Target material and competition material in the sample are combined with first antibody and second antibody, wherein said first antibody and second antibody are adsorbed on the carrier, or first antibody is adsorbed on the carrier, second antibody is adsorbed on the carrier that is scattered in the solution or is dissolved in the solution, the measurement of the target levels of substance that its material that hits is complete enzyme and combination is that the enzymatic activity of described complete enzyme is measured, and the competition material is the material with described enzymatic activity.
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