EP0082156A4 - 8-anilino naphthalene-1-sulphonic acid analogues. - Google Patents
8-anilino naphthalene-1-sulphonic acid analogues.Info
- Publication number
- EP0082156A4 EP0082156A4 EP19820901830 EP82901830A EP0082156A4 EP 0082156 A4 EP0082156 A4 EP 0082156A4 EP 19820901830 EP19820901830 EP 19820901830 EP 82901830 A EP82901830 A EP 82901830A EP 0082156 A4 EP0082156 A4 EP 0082156A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- compound
- ans
- carbon atoms
- strong acid
- anion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- FWEOQOXTVHGIFQ-UHFFFAOYSA-N 8-anilinonaphthalene-1-sulfonic acid Chemical class C=12C(S(=O)(=O)O)=CC=CC2=CC=CC=1NC1=CC=CC=C1 FWEOQOXTVHGIFQ-UHFFFAOYSA-N 0.000 title abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 35
- 150000001875 compounds Chemical class 0.000 claims abstract description 27
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 238000003018 immunoassay Methods 0.000 claims abstract description 20
- 230000008569 process Effects 0.000 claims abstract description 17
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 16
- 239000002253 acid Substances 0.000 claims abstract description 14
- 150000001450 anions Chemical class 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 125000001931 aliphatic group Chemical group 0.000 claims abstract description 5
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 4
- 229910052727 yttrium Inorganic materials 0.000 claims abstract description 4
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 3
- 229910052760 oxygen Inorganic materials 0.000 claims abstract 3
- 230000027455 binding Effects 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 230000000155 isotopic effect Effects 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000003127 radioimmunoassay Methods 0.000 claims description 7
- 125000004209 (C1-C8) alkyl group Chemical group 0.000 claims description 6
- 108010017384 Blood Proteins Proteins 0.000 claims description 5
- 102000004506 Blood Proteins Human genes 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 230000009870 specific binding Effects 0.000 claims description 4
- 239000002168 alkylating agent Substances 0.000 claims description 3
- 229940100198 alkylating agent Drugs 0.000 claims description 3
- -1 sulphate anion Chemical class 0.000 claims description 3
- 102000014914 Carrier Proteins Human genes 0.000 claims description 2
- 239000000427 antigen Substances 0.000 claims description 2
- 102000036639 antigens Human genes 0.000 claims description 2
- 108091007433 antigens Proteins 0.000 claims description 2
- 108091008324 binding proteins Proteins 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 238000003556 assay Methods 0.000 description 30
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 20
- 229940034208 thyroxine Drugs 0.000 description 19
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 16
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 235000011149 sulphuric acid Nutrition 0.000 description 6
- 239000001117 sulphuric acid Substances 0.000 description 6
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 6
- 229940035722 triiodothyronine Drugs 0.000 description 6
- 230000006872 improvement Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 230000021615 conjugation Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229960005156 digoxin Drugs 0.000 description 3
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 3
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000704 physical effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229960000278 theophylline Drugs 0.000 description 3
- 108010044091 Globulins Proteins 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010071690 Prealbumin Proteins 0.000 description 2
- 102000007584 Prealbumin Human genes 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108010074605 gamma-Globulins Proteins 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 239000005495 thyroid hormone Substances 0.000 description 2
- 229940036555 thyroid hormone Drugs 0.000 description 2
- QAEDZJGFFMLHHQ-UHFFFAOYSA-N trifluoroacetic anhydride Chemical compound FC(F)(F)C(=O)OC(=O)C(F)(F)F QAEDZJGFFMLHHQ-UHFFFAOYSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- CXOFVDLJLONNDW-UHFFFAOYSA-N Phenytoin Chemical compound N1C(=O)NC(=O)C1(C=1C=CC=CC=1)C1=CC=CC=C1 CXOFVDLJLONNDW-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 1
- 239000012346 acetyl chloride Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001348 alkyl chlorides Chemical class 0.000 description 1
- 150000001350 alkyl halides Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 229940064790 dilantin Drugs 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- FWMUJAIKEJWSSY-UHFFFAOYSA-N sulfur dichloride Chemical compound ClSCl FWMUJAIKEJWSSY-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
Definitions
- THIS INVENTION relates to new analogues of 8-anilino naphthalene -1- sulphonic acid, processes for their preparation, and their use in enzyme i ⁇ munoassay techniques.
- the known organic chemical 8-anilino naphthalene-1-sulphonic acid (ANS) has been widely used as an additive to reagents used in the technique known as radio-immuno assay to prevent interferences resulting from the binding of serum protein constituents to the drug or hor mone ligand/ligand-tracer which binding interferes with the specific binding of such ligand/ligand-tracer to the specific binding antibody used in the proceedure.
- proceedures such as the radio-immuno assay for thyroid hormones such as thyroxine e.g. Chopra, I.J. 'A radioimmunoassay for the measurement of thyroxine in unextracted serum'. J. Clin.
- the use of ANS provides a specific benefit to the assays in providing marked improvements in the speed of the assay as well as in the specificity of the assay. The reason for this improvement has been extensively studied as for example Cheng S. et al. Biochemistry 16 (1977) p3707 in which ANS binding to pre-albumin was studied; and Nilsson S.F. and Peterson P.A.
- This invention relates to a chemical modification of the molecular structure of ANS to form an analogue such that the analogue retains the protein binding characteristics of ANS but does not possess the light absorbance or fluorescence properties, or the instability to light characteristic of ANS which inhibits its use with non-isotopic immunoassay techniques.
- ANS The structure of ANS is as shown in figure 1 and it would appear to be the peculiar nature of the configuration of the three aromatic rings that confers on the molecule the ability to be tightly bound to thyroid binding globulin, pre-albumin, and also the binding to albumin.
- the sulphonate group causes the molecule to be water soluble. From consideration of the structure it would appear that the lone pair of electrons present on the anilino nitrogen will allow electron conjugation to occur throughout the three aromatic rings. It is usual for such conjugation to be associated with shifts in light absorbance to wavelengths greater than 300 nm, and this conjugation would also appear to be associated with the known fluorescent characteristics of ANS,
- the structure of the ANS analogues may have the following formula as shown in figure 2.
- R 1 may be H or an alkyl group of from U8 carbon atoms
- Y may be H or an aliphatic of from 1-8 carbon atoms
- n is 1 or 2
- m is 1 or 2
- A is the anion of a strong acid.
- R 1 is lower alkyl of 1-4 carbon atoms and is more preferably methyl or ethyl. Most preferably however R 1 is H.
- Y is lower alkyl of 1-4 carbon atoms and is more preferably methyl or ethyl.
- a preferred compound of the invention is where R 1 is H, R 2 is -COCH 3 and A is sulphate thereby providing the compound N-acetyl-8- anilinium-naphthalene 1-sulphonic acid sulphate, (hereinafter referred to as NA-ANS).
- A is an anion of a strong acid and may include chloride or nitrate but is most preferably sulphate.
- a compound of Figure 2 wherein R 2 is H and R 1 is H may be prepared by reaction of ANS with a strong acid under appropriate conditions. Host preferably the acid in sulphuric acid and the reaction occurs at room temperature.
- acetic anhydride trifluoroacetic anhydride, acetyl chloride or a thioacylating agent of 1-8 carbon atoms (e.g.a sulphenyl chloride or thioanhydrides) in the presence of a strong acid which is most preferably anhydrous sulphuric acid at room temperature.
- a strong acid which is most preferably anhydrous sulphuric acid at room temperature.
- a suitable alkylating agent is alkyl halide (eg alkyl chloride).
- the synthesis of such an analogue, N-acetyl-8-anilinium- naphthalene-1-sulphonic acid sulphate, (NA-ANS) will be detailed and this analogue will be shown to retain the protein binding characteristics of ANS while the above noted light and fluorescent detrimental properties of ANS are significantly altered.
- NA-ANS is able to be successfully utilised in non-isotopic immunoassays in a manner analogous to the use of ANS itself in radio-irtnunoassays and such uses will be detailed. Synthesis of NA-ANS
- NA-ANS in non-isotopic immunoassays was demonstrated by way of example by its use in non-homongenous enzyme immunoassays for thyroxine, triiodo-thyronine, digoxin and theophylline. It will be evident to those skilled in the art that such use and observed benefits are not exclusive to the particular non-isotopic immunoassay procedure described and utilised herein but are equally applicable to other enzyme and non-enzyme non-isotopic immunoassay procedures.
- b-galactosidase thyroxine, serum thyroxine, and anti-thyroxine antibody framents were incubated in buffer for thirty minutes at room temperature, solid phase precipitating antibody added, and the mixture incubated for a further 30 minutes. The mixture is then centrifuged at 2000 rpm for 5 minutes on a bench centrifuge and the supernatant assayed for residual enzyme activity.
- Reagents 1. b-galactosidase thyroxine solution containing 200 nM moles thyroxine and 130 nM moles protein. 2. Anti-thyroxine antibody.
- Fab derivatives of anti thyroxine gamma globulin sufficient to give 60% binding of b-galactosidase thyroxine in assay. 3. Solid phase precipitating antibody. Sepharose-anti Fab antibody diluted in buffer to give 100% binding of Fab in the assay.
- the procedure of the assay is as follows and all steps are carried out at room temperature. Duplicate assays were carried out on all samples.
- (a) Make a dilution of enzyme thyroxine by taking 1 part of enzyme and 134 parts of buffer. Pipette in order into a '0.25' ml conical autoanalyser cup 200uL of the diluted enzyme-thyroxine, 20uL of sample or calibrator, and 50uL of the diluted Fab antibody. Also prepare a 'total* enzyme activity tube using 200uL of enzyme ligand in buffer, 20 uL of a calibrator, with 50 uL of buffer, and treat similarly to the other tubes.
- Triiodothyronine Assay An assay for triiodothyronine was similarly established using methods similar to that described above with the exception that the buffer used was borate 0.05M pH 8.6 and the use of appropriate Fab fragments directed against triiodothyronine and a b-galactosidase triiodothyronine derivative. In an analogous manner to the above similar binding curves in the presence and absence of NA-ANS were observed.
- the subject invention provides the benefits to non-istopic immunoassays similar to that observed with ANS in isotopic immunoassays and allows for improved, simple and rapid assays by its inclusion in the assay.
- the previous limitations on such assays by the chemical and physical properties of ANS detrimental to such assays have been circumscribed by the altered properties observ ed with the alterations to the ANS molecule.
- the invention also includes within its scope a process for the determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance and a protein capable of binding said Tnndable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein characterized in that said reaction additionally includes as a reaction component a compound as defined in Figure 1 wherein said compound binds to serum proteins present in the reaction system thereby inhibiting binding of said bindable substance to said serum proteins and thus improving the specificity of said process for determination of said component.
- the abovementioned process is more applicable to non isotopic immunoassay procedures such as enzyme immunoassays which may be homogeneous or non-homogeneous.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
New analogues of 8-anilino naphthalene-1-sulphonic acid, processes for their preparation, and their use in enzyme immunoassay techniques. In particular the invention refers to compounds of the formula <IMAGE> where R1 is H or an aliphatic group of from 1-8 carbon atoms, R2 is H or C(Y)=X where X is O or S, Y is H or an aliphatic group of 1-8 carbon atoms, n is 1 or 2, m is 1 or 2 and A is an anion of a strong acid.
Description
"8-ANILINO NAPHTHALENE -1- SULPHONIC ACID ANALOGUES"
THIS INVENTION relates to new analogues of 8-anilino naphthalene -1- sulphonic acid, processes for their preparation, and their use in enzyme iπmunoassay techniques.
The known organic chemical 8-anilino naphthalene-1-sulphonic acid (ANS) has been widely used as an additive to reagents used in the technique known as radio-immuno assay to prevent interferences resulting from the binding of serum protein constituents to the drug or hor mone ligand/ligand-tracer which binding interferes with the specific binding of such ligand/ligand-tracer to the specific binding antibody used in the proceedure. Such use is exemplified in proceedures such as the radio-immuno assay for thyroid hormones such as thyroxine e.g. Chopra, I.J. 'A radioimmunoassay for the measurement of thyroxine in unextracted serum'. J. Clin. Endocrinol 34 (1972) p938; and also in the assay for triiodothyronine e.g. Sekadde CB. et al. 'Rapid Radioimmunoassay of Triiodothyronine'. Clin.Chem. 19 (1973) pl016. In such cases the use of ANS provides a specific benefit to the assays in providing marked improvements in the speed of the assay as well as in the specificity of the assay. The reason for this improvement has been extensively studied as for example Cheng S. et al. Biochemistry 16 (1977) p3707 in which ANS binding to pre-albumin was studied; and Nilsson S.F. and Peterson P.A. J.Biol.Chem. 250 (1975) 8543 in which ANS binding to thyroid binding globulin was studied. The binding of ANS to other proteins has also been extensively studied as for example Stryer L. J.Mol.Biol 13 (1965) p.482 and Steiner R.F. et al J.Biol.Chem. 241 (1966) p560., and in addition to its use with the assay of thyroid hormones ANS has been used as an assay constituent in radio-immuno assays for drugs such as for example with a commercial disoxin radio-immunoassay 'Disoxin I 125 IMUSAY' Abbott Laboratories North Chicago 111. USA. It is noted that by including ANS in the assay marked improvement in the specificity of the assay results. The improvement would appear to be due to binding of ANS to serum albumin wherein such binding prevents disoxin otherwise being so bound.
Hore recently developed immunoassay techniques such as enzyme imrnunoassays both homogeneous and non-homogeneous, and fluorescent immunoassays would appear also to benefit from the use of ANS but its use in such assays is hampered by the physical properties of ANS which is unstable in the presence of light, absorbs light strongly in the region of 340-420 nm, and shows natural fluoresence which is further enhanced when combined with proteins. Thus instead of using the simplified and rapid techniques possible when ANS is used in a thyroxine assay for example, these techniques utilise more cumbersome procedures to allow such assays to function.
This invention relates to a chemical modification of the molecular structure of ANS to form an analogue such that the analogue retains the protein binding characteristics of ANS but does not possess the light absorbance or fluorescence properties, or the instability to light characteristic of ANS which inhibits its use with non-isotopic immunoassay techniques.
The structure of ANS is as shown in figure 1 and it would appear to be the peculiar nature of the configuration of the three aromatic rings that confers on the molecule the ability to be tightly bound to thyroid binding globulin, pre-albumin, and also the binding to albumin.
The sulphonate group causes the molecule to be water soluble. From consideration of the structure it would appear that the lone pair of electrons present on the anilino nitrogen will allow electron conjugation to occur throughout the three aromatic rings. It is usual for such conjugation to be associated with shifts in light absorbance to wavelengths
greater than 300 nm, and this conjugation would also appear to be associated with the known fluorescent characteristics of ANS,
It is the object of this invention to create analogues of ANS by chemical substitution on the anilino nitrogen to involve the lone pair of electrons and thus block conjugation of the anilino aromatic ring to the naphthalene ring system. The structure of the ANS analogues may have the following formula as shown in figure 2.
In Figure 2, R1 may be H or an alkyl group of from U8 carbon atoms, R2 may be H or C(Y)=X where X may be 0 or S, Y may be H or an aliphatic of from 1-8 carbon atoms, n is 1 or 2, m is 1 or 2 and A is the anion of a strong acid.
Preferably R1 is lower alkyl of 1-4 carbon atoms and is more preferably methyl or ethyl. Most preferably however R1 is H.
Preferably Y is lower alkyl of 1-4 carbon atoms and is more preferably methyl or ethyl.
A preferred compound of the invention is where R1 is H, R2 is -COCH3 and A is sulphate thereby providing the compound N-acetyl-8- anilinium-naphthalene 1-sulphonic acid sulphate, (hereinafter referred to as NA-ANS).
A is an anion of a strong acid and may include chloride or nitrate but is most preferably sulphate.
A compound of Figure 2 wherein R2 is H and R1 is H may be prepared by reaction of ANS with a strong acid under appropriate conditions. Host preferably the acid in sulphuric acid and the reaction occurs at room temperature. A compound of Figure 2 wherein R2 is H and R1 is C(Y)=X my be prepared by reaction of ANS with an acylating agent of 1-8 carbon atoms (e.g. acetic anhydride, trifluoroacetic anhydride, acetyl chloride or a thioacylating agent of 1-8 carbon atoms (e.g.a sulphenyl chloride or thioanhydrides) in the presence of a strong acid which is most preferably anhydrous sulphuric acid at room temperature.
A compound of Figure 2 wherein R2 is C1-8 alkyl and R1 is C(Y)=X may be prepared by reacting ANS with a suitable alkylating agent of 1 to 8 carbon atoms in the presence of a strong acid such as sulphuric acid at room temperature. A suitable alkylating agent is alkyl halide (eg alkyl chloride).
A compound of Figure 2 wherein R1 is C1-8 alkyl and R2 is C(Y)=X may be prepared by reacting the product of the process of the above paragraph initially with a base and then an acylating agent or thioacylating agent as described above. The synthesis of such an analogue, N-acetyl-8-anilinium- naphthalene-1-sulphonic acid sulphate, (NA-ANS), will be detailed and this analogue will be shown to retain the protein binding characteristics of ANS while the above noted light and fluorescent detrimental properties of ANS are significantly altered. Thus NA-ANS is able to be successfully utilised in non-isotopic immunoassays in a manner analogous to the use of ANS itself in radio-irtnunoassays and such uses will be detailed. Synthesis of NA-ANS
To a suspension of 6g. of ANS (ammonium salt, Sigma Chemical Co. practical grade) and 60 mls. of acetic anhydride was added dropwise with swirling 30 drops of concentrated sulphuric acid, An immediate change of colour frorc green to purple was evident. After 10 minutes 300 mls. of ethyl acetate was added to the fixture and after a further 30 minutes the NA-ANS product was filtered, washed with ethyl acetate and dried in vacuo to give 5g. of a purple powder of aelting point 172-176 degrees (decomposes).
Physical Properties
Infrared spectrum -.KBr disc:- 3450,3150,1615,1595,1495,1270, 1170,1120,1050,1030,830,765,735,698,675,620,615,580,545 and 510 cm-1.
Mass spectrum - m/e 216 (100%), 281 (20%), 282 (4%).
UV-Visible spectrum - lambda max. 290 nm., extinction coeffnt. 7800 in 10 mM phosphate buffered saline.
Fluorescent spectrum - excitation 352 nm., emission 442 nm. in 10 mM phosphate buffered saline.
NMR spectrum - proton and 13C spectra observed were consistent with the NA-ANS structure. Verification of Structure.
Luts H.A. reported in J.Org.Chem. 33 (1968) p, 4528 the synthesis of pyridinium N-Acetyl-8-anilino-napthalene-1-sulphonate. This substance was synthesised following the method detailed and was converted into NA-ANS by treatment with both acetic anhydride - concentrated sulphuric acid as above and also by treatment with concentrated sulphuric acid in ethyl acetate as solvent. NA-ANS was also able to be converted into pyridinium N-acetyl-8-anil ino-naphtha!ene-1-sulphonate by treatment with pyridine at room temperature. Use of NA-ANS in non-isotopic immunoassays
The use of NA-ANS in non-isotopic immunoassays was demonstrated by way of example by its use in non-homongenous enzyme immunoassays for thyroxine, triiodo-thyronine, digoxin and theophylline. It will be evident to those skilled in the art that such use and observed benefits are not exclusive to the particular non-isotopic immunoassay procedure described and utilised herein but are equally applicable to other enzyme and non-enzyme non-isotopic immunoassay procedures. A. Total Serum Thyroxine enzyme immunoassay
Our patent application PCT/AU80/00065 and the references cited therein describe the manufacture of antibodies and antibody derivatives together with the manufacture of enzyme-ligand derivatives for a series of different non-homogeneous enzyme immunoassays and such procedures were followed for these experiments. b-Galactosidase-thyroxine was synthesised by using a thyroxine maleimide derivative using a method modified from those described by Ishikawa E. et al in 'Enzyme Labelled Immunoassay of
Hormones and Drugs: Ed, Pal S,B, Walter de Gruyter Berlin New York (1978) page 43,
Principle; b-galactosidase thyroxine, serum thyroxine, and anti-thyroxine antibody framents (Fab) were incubated in buffer for thirty minutes at room temperature, solid phase precipitating antibody added, and the mixture incubated for a further 30 minutes. The mixture is then centrifuged at 2000 rpm for 5 minutes on a bench centrifuge and the supernatant assayed for residual enzyme activity. Reagents: 1. b-galactosidase thyroxine solution containing 200 nM moles thyroxine and 130 nM moles protein. 2. Anti-thyroxine antibody. Fab derivatives of anti thyroxine gamma globulin sufficient to give 60% binding of b-galactosidase thyroxine in assay. 3. Solid phase precipitating antibody. Sepharose-anti Fab antibody diluted in buffer to give 100% binding of Fab in the assay.
4. Enzyme substrate solution, on-nitro phenyl b-galactoside 1.2 mg/ml phoshate buffered saline. 5. Buffer solution. 0.01 M phosphate pH 7.40.15 M NaCl containing 4 mg/ml bovine serum albumin, bovine gamma glo bulin 4 rag/ml, gelatin fragments 7.5 mg/ml, 0.3 W oleic acid, and 0.79 mg/ml of NA-ANS. 6. Thyroxine standards. Human serum containing 10,40,80,130,180,240 nM of thyroxine. Method:
The procedure of the assay is as follows and all steps are carried out at room temperature. Duplicate assays were carried out on all samples. (a) Make a dilution of enzyme thyroxine by taking 1 part of enzyme and 134 parts of buffer. Pipette in order into a '0.25' ml conical autoanalyser cup 200uL of the diluted enzyme-thyroxine, 20uL of sample or calibrator, and 50uL of the diluted Fab antibody. Also prepare a 'total* enzyme activity tube using 200uL of enzyme
ligand in buffer, 20 uL of a calibrator, with 50 uL of buffer, and treat similarly to the other tubes.
(b) Incubate for 30 minutes
(c) Add 50 uL of Sepharose anti Fab, cap the tubes, and then incubate for a further 30 minutes with gentle continuous inversion to keep the antibody in suspension.
(d) Centrifuge on a bench centrifuge for 5 minutes at 1000g. with the caps in place.
(e) Assay for enzyme content on a centrifugal analyser by sampling the supernatant directly from the sample cup.
Follow the gain in absorbance over 5 minutes at 405 nm.
(f) Calculate the mean of the duplicate enzyme rates of the unknown specimens and calculate the percent bound for each standard or unknown sample as follows: %bound = 100 - (100xobs.enz.rate/total enz.rate)
(g) Derive. a binding curve by plotting %bound Vs concentration of calibrator.
(h) Read the concentration of the test samples from the curve. A typical curve resulting from use of the assay is shown in figure 3, and also shown is a curve resulting from following the above method with the sole alteration of omitting NA-ANS from the buffer solution. It is to be seen that in the absence of NA-ANS a practical total serum thyroxine assay is not achieved. It is apparent however that the much reduced binding curve seen in the absence of NA-ANS corresponds to the calibration curve and it is probable that this curve is a measure of free thyroxine in serum as opposed to the total thyroxine assay resulting from use of NA-ANS. B. Total Triiodothyronine Assay An assay for triiodothyronine was similarly established using methods similar to that described above with the exception that the buffer used was borate 0.05M pH 8.6 and the use of appropriate Fab fragments directed against triiodothyronine and a b-galactosidase triiodothyronine derivative. In an analogous manner to the above similar binding curves in the presence and absence of NA-ANS were observed.
C. Serum Digoxin Assay
An assay for serum digoxin was reported in our previous patent application PCT/AU80/00065. It has been found that the inclusion of NA-ANS in the assay results in an improvement in the accuracy and precision of the assay as compared to the results observed in the absence of NA-ANS.
D. Serum Theophylline Assay
In a similar manner it has been observed that an enzyme immunoassay for theophylline fashioned in a manner similar to that reported in the above patent application for dilantin was also aided with respect to accuracy and precision by the inclusion of NA-ANS in the buffer solution.
It is evident from the above results that the subject invention provides the benefits to non-istopic immunoassays similar to that observed with ANS in isotopic immunoassays and allows for improved, simple and rapid assays by its inclusion in the assay. The previous limitations on such assays by the chemical and physical properties of ANS detrimental to such assays have been circumscribed by the altered properties observ ed with the alterations to the ANS molecule.
Thus, the invention also includes within its scope a process for the determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance and a protein capable of binding said Tnndable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein characterized in that said reaction additionally includes as a reaction component a compound as defined in Figure 1 wherein said compound binds to serum proteins present in the reaction system thereby inhibiting binding of said bindable substance to said serum proteins and thus improving the specificity of said process for determination of said component. The abovementioned process is more applicable to non isotopic immunoassay procedures such as enzyme immunoassays which may be homogeneous or non-homogeneous.
Claims
THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A Compound of the formula
wherein R1 is H or aliphatic of 1-8 carbon atoms, R2 is H or C(Y)=X where X is 0 or S, Y is H or aliphatic of 1-8 carbon atoms, n is 1 or 2, m is 1 or 2 and A is an anion of a strong acid.
2. A compound of claim 1 where R1 is lower alkyl of 1-4 carbon atoms.
3. A compound of claim 2 where R1 is methyl or ethyl.
4. A compound of claim 1 where R1 is H.
5. A compound of any one of claims 1-4 wherein X is O and Y is lower alkyl of 1-4 carbon atoms.
6. A compound of claim 5 where Y is methyl or ethyl.
7. A compound of claim 1 where Y is H and X is O.
8. A compound of claim 1 where Y is methyl, X is O and R1 is H.
9. A compound of claim 1 where A is sulphate anion and n is 2.
10. N-acetyl-8-anilinium-naphthalene-1-sulphonic acid sulphate.
11. A process for the preparation of a compound having the formula
where R is C1-8 alkyl, A is the anion of a strong acid, n is
1 or 2 and m is 1 or 2 including the step of reacting ANS with an alkylating agent of 1-8 carbon atoms such as a C1-8 alkyl helide in the presence of a strong acid to form said compound. 12. A process for the preparation of a compound of the formula
where n is 1 or 2, m is 1 or 2 and A is the anion of a strong acid including the step of reacting ANS with a strong acid to form said compound.
13. A process for the preparation of a compound of formula
where R is C1-8 alkyl and R3 is C(Y)=X where Y is H or C1-8 alkyl, X is O or S, n is 1 or 2, m is 1 or 2, and A is the anion of a strong acid including the step of reacting the compound prepared by the process of claim 11 with an acylating agent or thioacylating agent having 1 to 8 carbon atoms after initial reaction with a base.
14, A process for the preparation of a compound of the formula
where R3, n, m and A are as defined in claim 12 including the step of reacting ANS with an acylating agent or thioacylating agent having 1 to 8 carbon atoms in the presence of a strong acid.
15. A process for the determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance and a protein capable of binding said bindable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein characterized in that said reaction additionally includes as a reaction component a compound as defined in claim 1 wherein said compound binds to serum proteins present in the reaction system thereby inhibiting binding of said bindable substance to said serum proteins and thus improving the specificity of said process for determination of said component.
16. A process as claimed in claim 15 wherein said component is as defined in claim 10.
17. A process as claimed in claim 15 or 16 which is utilized for radio immunoassays.
18. A process as claimed in claim 15 or 16 which is utilized for non- isotopic immunoassays both homogeneous and non-homogeneous.
19. A process as claimed in claim 15 or 16 which is utilized for enzyme immunoassays both homogeneous and non-homogeneous.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU9477/81 | 1981-06-26 | ||
| AUPE947781 | 1981-06-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0082156A1 EP0082156A1 (en) | 1983-06-29 |
| EP0082156A4 true EP0082156A4 (en) | 1983-12-01 |
Family
ID=3769112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19820901830 Withdrawn EP0082156A4 (en) | 1981-06-26 | 1982-06-18 | 8-anilino naphthalene-1-sulphonic acid analogues. |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0082156A4 (en) |
| JP (1) | JPS58501073A (en) |
| WO (1) | WO1983000147A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5102786A (en) * | 1987-05-21 | 1992-04-07 | Pb Diagnostic Systems, Inc. | Biological diagnostic assay system |
| US5474907A (en) * | 1994-03-25 | 1995-12-12 | Eastman Kodak Company | Multilayer analytical element for salicylate assay |
| EP0880704B1 (en) * | 1996-07-18 | 2003-09-24 | Dade Behring Marburg GmbH | Reagents for assays for mycophenolic acid |
| EP0880705B1 (en) | 1996-07-18 | 2003-06-18 | Dade Behring Marburg GmbH | Reagents for assays for mycophenolic acid |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2535337C2 (en) * | 1975-08-07 | 1985-02-07 | Bayer Ag, 5090 Leverkusen | Process for the preparation of 1-amino-naphthalene-7-sulfonic acid |
| CH593248A5 (en) * | 1976-01-30 | 1977-11-30 | Ciba Geigy Ag |
-
1982
- 1982-06-18 EP EP19820901830 patent/EP0082156A4/en not_active Withdrawn
- 1982-06-18 WO PCT/AU1982/000097 patent/WO1983000147A1/en not_active Ceased
- 1982-06-18 JP JP57501860A patent/JPS58501073A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| BIOCHEMISTRY, vol. 16, no. 16, 1977 * |
| See also references of WO8300147A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0082156A1 (en) | 1983-06-29 |
| JPS58501073A (en) | 1983-07-07 |
| WO1983000147A1 (en) | 1983-01-20 |
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