Assay for the Detection of the Phenylpiperazine Family
The current invention describes novel immunogens which are used in the production of novel antibodies with unique binding properties in that they cross-react with a variety of phenylpiperazine derivatives. These antibodies enable methods and kits to detect and/or determine phenylpiperazine derivatives (for example mCPP, TFMPP and MeOPP) in an in vitro sample which are advantageous over currently available analytical methods in terms of cost, ease of use, speed and sensitivity.
1 . A hapten of the general structure:
wherein, one of X and Y is H and the other is -(A) m -(B) n -D; wherein A is attached to the aromatic ring; wherein A is selected from —C(O)—, O, and NH; wherein m=0 or 1;
wherein B is a C 1 -C 10 , preferably a C 1 -C 6 , substituted or unsubstituted straight or branched chain alkylene moiety or arylene moiety; wherein n=0 or 1; and wherein D is selected from a carboxyl, a dithiopyridyl, a maleimidyl, a hydroxyl, a thiol, and an aldehyde moiety.
2 . An immunogen comprising a hapten of claim 1 conjugated to an antigenicity-conferring carrier material.
3 . The immunogen of claim 2 in which D of -(A) m -B-D is coupled to the antigenicity-conferring carrier material.
4 . The immunogen of claim 3 which is either 1-(3-carboxyphenyl)piperazine or 1-(4-carboxymethyletherphenyl)piperazine is coupled to the antigenicity-conferring carrier material.
5 . The immunogen of claim 2 in which the antigenicity-conferring carrier material is selected from BTG, BSA, and KLH.
6 . An antibody raised against the immunogen of claim 4 .
7 . An antibody raised against the 1-(3-carboxyphenyl)piperazine immunogen of claim 4 having specificity for 1-(3-methylphenyl)piperazine with an IC 50 value of around 1 ng/ml, which is further characterised by having cross-reactivity to 1-(3-chlorophenyl)piperazine, 1-phenylpiperazine, 1-(4-methoxyphenyl)piperazine, 1-(4-hydroxyphenyl)piperazine, 1-(4-fluorophenyl)piperazine, 1-(3-hydroxyphenyl)piperazine, 1-(3-trifluoromethylphenyl)piperazine and 1-(2-methoxyphenyl)piperazine.
8 . An antibody raised against the 1-(4-carboxymethyletherphenyl)piperazine immunogen of claim 4 having specificity for 1-(3-methylphenyl)piperazine with an IC 50 value of around 1 ng/ml, which is further characterised by having cross-reactivity to 1-(3-chlorophenyl)piperazine, 1-phenylpiperazine, 1-(4-methoxyphenyl)piperazine, 1-(4-hydroxyphenyl)piperazine, 1-(4-fluorophenylpiperazine, 1-(3-hydroxyphenyl)piperazine, and 1-(3-trifluoromethylphenyl)piperazine.
9 . A method of detecting and/or determining one or more piperazine derivatives in an in vitro sample, the method comprising contacting the sample with the antibody of claim 7 and the antibody of claim 8 , and at least one detecting agent; detecting or determining the detecting agent(s) and deducing from a calibration curve the presence of and/or amount of, the one or more piperazine derivatives in the sample.
10 . A method of detecting and/or determining one or more piperazine derivatives in an in vitro sample, the method comprising contacting the sample with the antibody of claim 7 and the antibody of claim 8 , and at least one detecting agent; detecting or determining the detecting agent(s) and deducing from a calibration curve the presence of and/or amount of, the one or more piperazine derivatives in the sample.
11 . A method of detecting or determining one or more of 1-phenylpiperazine, 1-(4-hydroxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-methylphenyl)piperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-chlorophenyl)piperazine, 1-(4-fluorophenyl)piperazine and 1-(2-methoxyphenyl)piperazine in an in vitro sample, the method comprising contacting the sample with at least one antibody selected from the antibody of claim 7 , and the antibody of claim 8 and at least one detecting agent, detecting or determining the detecting agent(s) and deducing from a calibration curve, the presence of or amount of one or more of 1-phenylpiperazine, 1-(4-hydroxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-methylphenyl)piperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-chlorophenyl)piperazine, 1-(4-fluorophenyl)piperazine and 1-(2-methoxyphenyl)piperazine.
12 . The method of claim 9 in which the sample is selected from whole blood, plasma, serum, and urine.
13 . A kit for detecting or determining one or more piperazine derivatives in a sample comprising one or more antibodies selected from the antibody of claim 7 , and the antibody of claim 8 ; and at least one detecting agent.
14 . The kit of claim 13 in which the one or more piperazine derivatives detected or determined are 1-phenylpiperazine, 1-(4-hydroxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-methylphenyl)piperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-chlorophenyl)piperazine, 1-(4-fluorophenyl)piperazine and 1-(2-methoxyphenyl)piperazine.
15 . The kit of claim 14 comprising the antibody of claim 7 and the antibody of claim 8 .