Leukotriene labeled by enzyme used in enzyme immunomeasurement method

[Detailed Description of the Invention]

[Industrial Application] The present invention is enzyme immunoassay (it is hereafter written as the EIA method.). the suitable new alligator of Roy Cotrien (it is hereafter written as LT.) -- it is related with a conductor. When it states in detail, it is related with LT(s) by which the sign was carried out with the enzyme used for the EIA method. [Description of the Prior Art] In recent years, it is produced via a lipoxygenase system as one of the arachidonate cascades in the living body, The substance and LT(s) which have physiological functions, such as blood-vessel-permeability 9 Advanced effect, a bronchoconstrictor effect, and a leukocyte migration operation, are clarified, and existence of the following LT(s) is checked by the present (Proc, Na11.Acad, Sci.). USA, E 6.4275 (1979) Biochtx Biop Le ys. Rts, Gormtubn, 91.1266 (1979), proc. NatL, Acad, E3ci, USA, 77, 2014 (1980), NatILre, and 285,104 (1980) references] R:5-OH R:5-CH2 0HC and 0NHGH2COOHL T D 4 "H2 R:5-OH CHCOOHL T B 4 H2 It is becoming very important to establish Si [ as ] who argues about the relation of various diseases and LT(s) in connection with it, and the method of measuring the concentration of LT(s) in the living body correctly and simple. As a measuring method of LT(s), although the UV method and the EIA method are known, about the EIA method, it is not known at all. Therefore, LT(s) by which the sign was carried out with the enzyme used for the EIA method are not known at all. Although various methods are known as a method of on the other hand carrying out the sign of the hapten with enzyme generally, in order to measure high sensitivity and correctly, it is necessary to use a specific enzyme sign antigen with high selectivity. [Objects of the Invention] In the inside which develops that LT(s) of these artificers are specific, and the alternative EIA method, The result of having repeated examination wholeheartedly about the condensing method and the crosslinking agent especially in order to find out the thing optimal as an enzyme sign antigen of LT(s), It found out that the bridge construction condensing method using 4-(N-maleimide methyl) cyclohexane 1-carboxylic acid N-hydroxy Sukusin imide ester was most excellent in respect of singularity and selectivity, and completed the present invention. [Elements of the Invention] Namely, general [ by which the present invention is used for the EIA method ] (the inside of a formula and R are -OH(s)) H- -OH GHCOOH(IV) Quantity NH~ It comes out, a basis shown is expressed and R2 expresses enzyme. It is related with enzyme mark RLT(s) shown. When R1 expresses a basis shown by formula (II) in general formula (I), it is LT (LTE4 is expressed when it expresses a basis Ah Si and R1 are indicated to be by a formula (5) by I and TD4 when Ah Si and R express with 34 a basis shown by a formula (From).). In any case, as R1, it is desirable, but especially a desirable thing is LTC4. In a general formula (1), if enzyme shown by R2 is enzyme generally used by the EIA method, it is [ anything ] good. Although what has SH basis which does not participate in itself active is desirable as for enzyme, it may not have an 81-I basis at all. In the case of enzyme which does not have SH basis, free SH basis can be introduced by a method well learned to a person skilled in the art. As the enzyme shown by R2 used for the present invention, Although for example, malate dehydrogenase, glucose 6-phosphoric acid dehydration enzyme, glucose oxidative enzyme, Leu oxidase-ed, acetylcholine esterase, alkaline phosphatase, glucoamylase, a lysozyme, and beta-D-galactosidase are mentioned, Preferably it is beta-D-galactosidase. Yo Si manufacture of the LT(s) shown by a general formula (1) can be carried out at a method shown below. (The meaning as the above with R1 and R2 [ same ] is expressed among a formula.) A compound shown by general formula (Ro), They are Yo Si profitable To be to making Melting jl It was 4-(N-maleimide methyl) Owl hexane 1-carboxylic acid N-hydroxy Sukusin imide ester (MCAE) react to a compound shown by a general formula (From) dissolved in a phosphate buffer solution, and a suitable organic solvent (preferably tetrahydro franc). A reaction is performed for 1 hour after for 30 minutes in a source of the bottom room of inactive gas atmosphere like argon. It can refine by performing reaction wave usual post-processing. A general formula made to make a compound shown by obtained general formula (Ro) react to enzyme (it uses in a form which introduced the 9-8H set into a publicly known method when it does not have a free 18H basis.) shown by R2 with Yo Si and the object (enzyme sign LT(s) shown by To are obtained.) A reaction is performed under inactive gas atmosphere by room temperature in a phosphate buffer solution in ~ 1 hour during 30 minutes. A general formula which Fractionation reaction happiness-in-the-next-life Acid with gel chromatography, and has enzyme activity (LT(s) shown by To are recoverable.) LT(s) shown by general formula (I) of the present invention can be used as an enzyme sign antigen in a case of quantifying LTC4, LTD4, and LTE4 in the EIA method. As an EIA method, although the 1st antibody said phase method, the 2nd antibody said phase method, and a liquid phase method are known, LT(s) of the present invention can be used as a specific enzyme sign antigen with high selectivity also in which method. For enforcing the EIA method using an enzyme sign antigen shown by general formula (I), An enzyme sign antigen shown by an antiserum, a sample or standard goods, and general formula (I) which were diluted to optimal concentration is incubated under the optimum temperature, boil an antigen-antibody conjugate pipe double antibody technique etc. which were generated -- it can ask for concentration of LT(s) which an enzyme substrate is made to act on an antigen-antibody conjugate, and are made into the object from fluorescence intensity of output, after separating into a Le isolation type and a knot pattern. If it hits performing the EIA method, a living body sample pretreats if needed, and measurement is presented with it, after refining to such an extent that prevention is not done to an antigen-antibody reaction. An antiserum used for the describing [ above ] EIA method is obtained by a publicly known method. That is, an antigen is emulsified to a suitable adjuvant, for example, an in comp Lee Tong Lo India bunt, and it is obtained by carrying out sensitization to a host animal, metaphor 5 rabbit, Mol Kit, etc. They are Yo Si profitable To be to an antigen making a publicly known method carry out Yo Si condensation of LT(s) and protein (BSA), for example, cow serum albumin, as hapten. Some are indicated by ProztagLandirLz, 23 (4), and 603 (1982) as an antigen of LT(s). Although an antigen used for the present invention is not limited to these, when 1.5-difluoro 2 and 4-:nitrobenzene are used as a crosslinking agent, a good antigen with high singularity is obtained. If a substance which BSA and hapten combined via a 2.4-dinitro m-phenylene group by using an antibody as a production To shake antigen is used, an antibody to this bridge construction portion may be produced. If EIA is performed using an enzyme sign antigen prepared using the same crosslinking agent at this time, In an antigen-antibody reaction, a direction of antibody associative strength to an enzyme sign antigen appears strongly compared with associative strength of an antibody to hapten, and substitution of enzyme sign LT(s) and under-test LT(s) is not performed completely, but sensitometry may fall. Therefore, it may be more suitable to take Terrorism into consideration, if EIA has. However, depending on character of an antibody, these phenomena do not always arise, and it may be able to carry out EIA in combination of the same crosslinking method. Therefore, in order to find out optimal enzyme sign antigen, it is necessary to actually judge by performing EIA. [Example] Hereinafter, the present invention is not limited by these examples although the present invention is explained in full detail according to an example. In condensation product [general formula (I) of example LTC4 and beta-D-galactosidase, R1 expresses the basis shown by formula (II), R2 dissolves manufacturing method LTC4100microI of the compound showing beta-D-galactosidase] in substitution 1M with 0.075M phosphate buffer solution (7) R7, 2, and after-deaeration argon gas, 0.1 d of tetra-Hydroxane bath solution containing 4-(N-maleimide methyl) cyclohexane 1-carboxylic acid N-hydroxy Sukusin imide ester (MCAE) IIn9 were added here, and it was made to react for 30 minutes under [ 24C ] argon gas atmosphere. The tetrahydro franc after the end of a reaction was distilled off with argon gas, and unreacted MCAE was extracted and removed by diethylether (three 1yx). Diethylether which remains in a water layer was distilled off enough with argon gas, and the cyclohexyl maleimide derivative [compound showing the basis R1 is indicated to be by a formula (IF) in general formula (Ro)] of LTC4 was obtained. The obtained cyclohexyl maleimide derivative was dropped at 1 d of substitution under argon gas atmosphere with 0.075M phosphate buffer solution (1) R7 containing beta-D-galactosidase 1.5~, 2, and after-deaeration argon gas. This mixed-solution was stirred for 30 minutes by 24C after the end of dropping. The reaction mixture was Fractionation(ed) in Sephadex G-25 column (a registered trademark, a Pharmacia manufacture) (1, 0x50cfrL), and the title condensation product part drawings which have enzyme activity were collected. The recovery rate as an amount of enzyme was 94%. when it was referred to as 540.000, it came out and the molecular weight of beta-D-galactosidase was calculated, 418 or 3 mol of 1 mol of beta-D-galacto 7 Daese this # LTC(s) had condensed. If it calculates noting that beta-D-Ga 2 Cuccidase has 23 SH bases of isolation, it will be thought that the 80 Chi have reacted to theory top LTG4. The title condensation product had about 70% of activity of the enzyme of a basis. [Effect] The effect was checked by the EIA method (liquid phase method) for enzyme-like RLT(s) of the present invention obtained in the above-mentioned example. In this experiment, LT(s) by which the enzyme sign was carried out by five kinds of methods in addition to enzyme sign LT(s) of the present invention were performed as comparison and contrast. Opposite ff, 1 : The following enzyme sign LTC4 was obtained like enzyme sign LTG4 example which makes a crosslinking agent 1, 5-difluoro 2, and 4-dinitrobenzene, using 1.5-difluoro 2 and 4-dinitrobenzene as a crosslinking agent. Contrast 2: Enzyme sign I which makes a crosslinking agent m-maleimide benzoic acid N-hydroxy Succininimito 8 ester, TG4 The following enzyme sign LTC4 was obtained like the example, using m-maleimide benzoic acid N-human 90 Xishaksin imide ester as a crosslinking agent. Enzyme sign LTC4 condensation reaction obtained by the contrast 3 Nigel tar aldehyde method was performed with one step-by-step procedure. that is, beta-D-galactosidase 1 Da is added to 1 m of 0 and 1M phosphate buffer solutions (PH6, 8, and after-deaeration argon gas replace) which contain LTC4100pl/under shading, and loose under argon gas atmosphere -- bitter -- while mixing -- 1% guru tar Aldehy"-- 50pl was dropped. After making it react at room temperature for 2 hours, it dialyzed to the 0.01M phosphate buffer solution (PH7, 2, and after-deaeration argon gas replace) by 4c. Since a condensation reaction is random coupling, a complex is considered to be an uneven thing. contrast 4:1-ethyl 3-(3-dimethylaminopropyl) Carbo diimide "(EDC) -- enzyme sign LTC obtained by law -- < The buffer solution 0.5 m which contains EDC11v in 1 d of 0.1M phosphate buffer solutions (PH6, 8, and after-deaeration argon gas replace) containing bottom KLTGof shading4100pl was added, and it wrote with room temperature for 2 hours, and became precocious. Phosphate buffer solution 0 of the composition which contains beta-D-galactosidase 11v in reaction liquid, and 511Ll! were added, and it stirred by 4C for 24 hours. The condensation thing was refined by dialysis operation. Since LTC4 has three carboxyl groups, it is considered that the complex which carried out random coupling was obtained. Contrast 5: Tree loop chill amine 2.5 mu and iso Buchir chloro formate 1.3microl were added to tetra-Hydran 50microl containing enzyme machine [LTC4LTC4100microg obtained by the in methyl Chloroform tight method. After setting reaction volume to 100microl in a tetrahydro franc, it stirred by 10C under shading and argon gas atmosphere for 1 hour. Next, while stirring the acid anhydride reaction liquid previously obtained at a rate of 10pH in 1 minute, it was dropped at 0.5% sodium bicarbonate solution 0.5- containing beta-D-galactosidase 1 Da, and it stirred after [ 4C ] the end of dropping for 2 hours. The reaction mixed-solution was dialyzed to the phosphate buffer solution (pH 7, 2, and after-deaeration argon gas replace) of 0 and OIM. The antiserum of LTC4 was prepared as follows. LTC42 and five drops were dissolved in 0.1M phosphate buffer solution (pH 7, 2, and after-deaeration argon gas replace) Q and 5+++A!, methanol 0.7 m containing 1 *5-difluoro 2 and 4-dinitrobenzene (DFDNB) 10~ was added, and it stirred for 30 minutes by 24C. After distilling off methanol with the argon gas after the end of a reaction, extraction removal of the unreacted DFDNB was carried out by diethylether (0, three 51LlX). After distilling off diethylether which remains in solution with argon gas, borate buffer solution (PH8, 5, and after-deaeration argon gas replace) 111LI! of 0.2M containing B5aluminum0~ was added, and it settled for two days at room temperature by shading-under argon gas atmosphere conditions. It is Sephadex G-25 column (a registered trademark, a Pharmacia manufacture) (1, oxs.) under shading of after the end of a reaction, and reaction liquid. It loaded on clrL, and it was eluted with distilled water (after-deaeration argon gas replaces), parts for protein drawing were collected, and the hapten antigen made into the object was obtained. The recovery rate as protein was 95~100 Chi. dispensing a part for protein drawing to a vial as protein weight at every 11v -- the bottom of after-freeze-drying argon gas atmosphere -- one -- it saved by 80C. When it was considered as molecular weight f:54,300 of BSA, B5aluminum Mol this4 [ p8.14 mol / LTC] had joined together. The sensitization to the rabbit made the hapten antigen obtained above per 1 time of sensitization the amount of BSA(s), and did 1eta medication of it. That is, hapten antigen 1 Ya obtained above was Bathing solution(ed) to the physiological saline of 0.5 d, and it was considered as emulsion under argon gas atmosphere within the glass syringe with the incomplete Freund adjuvant of 1M. Irony medication of this emulsion 1.5 Genus was carried out at about 15 rabbit back necks. The sensitization interval was carried out every three weeks. the rise of antibody titer -- the -- it accepted by the trial blood collecting for after [ the 4 said sensitization ] ten days. Example of an experiment Six kinds of enzyme sign antigens and the antiserum which were prepared by the EIA (liquid phase method) example of LTC4 and contrast 1-5 using various enzyme sign antigens are used, LTC4 was actually measured in EIA (liquid phase method), and the comparative examination was performed about six kinds of enzyme sign antigens from the detection sensitivity in that case, and a detection range. Namely, after mixing phosphate buffer solution 100mu A containing phosphate buffer solution 100mul and the enzyme sign antigen containing a sample or standard goods, it incubated by 37iC for 1 hour. Next, dilution rabbit serum 100mul and anti-rabbit ■ycioomul were added, and it was neglected by 4c overnight. After centrifuging reaction liquid for 3 Q Q Q r7ym 10 minutes by 4C after the end of a reaction and carrying out suction removal of the top Fresh, 0.1mM reacted by 37C [ 4-Meterubergenifilen beta-Ri 1 galactoside 0.2 d"e ] for 30 minutes. After adding 0.1M Grishin buffer solution 3.0 d and stopping a reaction, it centrifuged for 10 minutes at 4C13000 rpm, and measured the fluorescence intensity of the upper slot by excitation wavelength lambda6 Thu 360Lem and fluorescence wavelength lambdaErn450rLm. The analytical curve prepared based on the measured value about standard LTC;4 is shown in Drawing 1. It became clear that the sensitivity which was most excellent in the case of the enzyme sign object using MCAE prepared in the example among six kinds of enzyme sign objects as a crosslinking agent, and the amount range of Inspection were obtained.

[Brief Description of the Drawings]

Drawing 1 is a graph which shows the analytical curve in EIA at the time of using six kinds of enzyme sign objects, and is To. -Enzyme sign object--- prepared in the 0 -2 example: The enzyme sign object-low prepared by contrast 1: Enzyme sign object-△- prepared by enzyme sign object-112 contrast 3 prepared by contrast 2: Enzyme sign object-Moe who prepared by contrast 4 : the enzyme sign object prepared by contrast 5 (three others) Drawing 1 LTCJpg/l, Ll=a