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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• 1982
  • 1982-06-18 WO PCT/AU1982/000097 patent/WO1983000147A1/en not_active Application Discontinuation
  • 1982-06-18 EP EP19820901830 patent/EP0082156A4/de not_active Withdrawn
  • 1982-06-18 JP JP50186082A patent/JPS58501073A/ja active Pending

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