Phenethanolamine-derived haptens, immunogens, antibodies and conjugates
View Patent ↗The present invention relates to a method for preparing haptens of formulae I and II that are useful in the preparation of immunogens, antibodies and conjugates, for use in competitive immunoassays for the detection of ractopamine, isoxsuprine and ritodrine. The haptens are prepared by reacting a phenylethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E: in which, in formula I, Z 1 is a crosslinker and Z 2 is H and, in formula II, Z 1 is H and Z 2 is a crosslinker.
1. A method of preparing a hapten of Formula I
suitable for use in raising an antibody capable of binding with at least one structural epitope of a phenethanolamine, the method comprising the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with a moiety selected from the group comprising an antigenticity-conferring carrier material and a detectable labelling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
2. A method according to claim 1 , in which the amine group of the octapamine hydrochloride is temporarily protected with BOC, by reacting the octapamine hydrochloride with di-tert-butyldicarbonate before reacting the BOC-protected octapamine hydrochloride with the Z 1 X 1 , the BOC group being subsequently removed.
3. A method of preparing a hapten of Formula II
suitable for use in raising an antibody capable of binding with at least one structural epitope of a phenethanolamine, the method comprising the steps of;
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with a moiety selected from the group comprising an antigenicity-conferring material and a detectable labelling agent, and X 1 is halide; and
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
4. An immunogen comprising a hapten and an antigenicity-conferring carrier material, wherein said hapten has the formula of Formula I
wherein said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with said antigenicity-conferring material, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1,
and wherein said crosslinker links said hapten to said antigenicity-conferring carrier material at a single phenolic hydroxyl group of said hapten.
5. An immunogen comprising a hapten and an antigenicity-conferring carrier material, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said antigenicity-conferring carrier material, and X 1 is a halide;
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
and wherein said crosslinker links said hapten to said antigenicity-conferring carrier material at a single phenolic hydroxyl group of said hapten.
6. An antibody raised against an immunogen, wherein said immunogen comprises a hapten and an antigenicity-conferring carrier material, wherein said hapten has the formula of Formula I
wherein said hapten is prepared by the step of:
preparing a phenethanolamine deriavative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said antigenicity-conferring carrier material, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said antigenicity-conferring carrier material at a single phenolic hydroxyl group of said hapten; wherein said antibody specifically binds to said immunogen, said antibody being capable of binding with at least one structural epitope of a phenethanolamine, and wherein the antibody shows a cross reactivity, when compared to 100% for ractopamine, isoxsuprine and ritodrine, of less than 5% for dobutamine.
7. An antibody raised against an immumogen, wherein said immunogen comprises a hapten and an antigenicity-conferring carrier material, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said antigenicity-conferring material, and X 1 is a halide;
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said antigenicity-conferring carrier material at a single phenolic hydroxyl group of said hapten; wherein said antibody specifically binds to said immunogen, said antibody being capable of binding with at least one structural epitope of a phenethanolamine, and wherein the antibody shows a cross reactivity, when compared to 100% for ractopamine, isoxsuprine and ritodrine, of less than 5% for dobutamine.
8. A conjugate comprising a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula I
wherein said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
9. A conjugate comprising a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is functional group containing at least one hetero-atom capable of linking the R of the crosslinker with a detectable labelling agent, and X 1 is a halide;
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten tosaid detectable labelling agent as a single phenolic hydrroxyl group of said hapten.
10. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate, and at least one antibody of claim 6 ; wherein said conjugate comprises a hapten and a detectable labelling agent wherein said hapten has the formula of Formula I
and said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinking with said detectable labeling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten; and
(b) detecting or determining bound conjugate; and deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
11. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate, and at least one antibody of claim 7 ; wherein said conjugate comprises a hapten and a detectable labeling agent wherein said hapten has the formula of Formula I
and said hapten by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten; and
(b) detecting or determining bound conjugate; and deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
12. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate, and at least one antibody of claim 6 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is halide;
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten; and
(b) detecting or determining bound conjugate; and deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
13. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate and at least one antibody of claim 7 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is halide;
reacting a phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten; and
(b) detecting or determining bound conjugate; and deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
14. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate; and at least one antibody of claim 6 , wherein said conjugate comprises a hapten and a detectable labelling agent wherein said hapten has the formula of Formula I
and said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
15. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate and at least one antibody of claim 7 , wherein said conjugate comprises a hapten and a detectable labelling agent wherein said hapten has the formula of Formula I
and said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which Z 1 is a crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is a halide; and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
16. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate and at least one antibody of claim 6 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is halide;
reacting the phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
17. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate and at least one antibody of claim 7 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
and said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is a bivalent link and X 2 is a functional group containing at least on hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and X 1 is halide;
reacting the phenethanolamine derivative of formula D, with the phenylalkylcarbonyl derivative of formula E, in which Z 1 is H, Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
18. The immunogen of claim 4 or 5 , in which R is selected from the group comprising a substituted or unsubstituted, straight or branched chain, saturated or unsaturated alkylene group and a substituted or unsubstituted arylene group and X 2 is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group a vinylsulphone group, and a thiocarboxylic acid or an ester thereof.
19. An antibody raised against the immunogen of claim 18 that specifically binds to the immunogen of claim 23 , the antibody being capable of binding with at least one structural epitope of a phenethanolamine, wherein the antibody shows a cross reactivity, when compared to 100% for ractopamine, isoxsuprine and ritodrine, of less than 5% for dobutamine.
20. The conjugate of claim 8 or 9 , in which R is selected from the group comprising a substituted or unsubstituted, straight or branched chain, saturated or unsaturated alkylene group and a substituted or unsubstituted arylene group and X 2 is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group a vinylsulphone group, and a thiocarboxylic acid or an ester thereof.
21. The immunogen of claim 18 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group.
22. An antibody raised against the immunogen of claim 18 wherein R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group, the antibody being capable of binding with at least one structural epitope of a phenethanolamine, wherein the antibody shows a cross reactivity, when compared to 100% for ractopamine, isoxsuprine and ritodrine, of less than 5% for dobutamine.
23. The conjugate of claim 20 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group.
24. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate and at least one antibody of claim 22 , wherein said conjugate comprises a hapten and a detectable labeling agent, wherein said hapten has the formula of Formula I
wherein said hapten is prepared by the steps of:
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which;
Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group; and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group; which serves as a bivalent link;
X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group, a vinylsulphone group, and a thiocarboxylic acid or an ester thereof; and
X 1 is a halide;
and
reacting the phenethanolamine derivative of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1;
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten;
and
(b) detecting or determining bound conjugate; and deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
25. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate and at least one antibody of claim 22 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula I
wherein said hapten is prepared by the steps of;
preparing a phenethanolamine derivative of the formula D by reacting octopamine hydrochloride with Z 1 X 1 , in which;
Z 1 is a crosslinker, the crosslinker being —R—X 2 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group; and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group; which serves as a bivalent link;
X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group, a vinylsulphone group, and a thiocarboxylic acid or an ester thereof; and
X 1 is a halide;
and
reacting the phenethanolamine of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 2 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1;
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.
26. A method for detecting or determining a phenethanolamine in a sample, the method comprising:
(a) contacting the sample with at least one conjugate and at least one antibody of claim 22 , wherein said conjugate comprises a hapten and a detectable labeling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which
Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group; and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group; which serves as a bivalent link;
X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group, a vinylsulphone group, and a thiocarboxylic acid or an ester thereof; and
X 1 is a halide;
and
reacting the phenethanolamine of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 1 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1;
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten;
(b) detecting or determining bound conjugate; and
(c) deducing from the calibration curve the presence of, or the amount of, the phenethanolamine in the sample.
27. A kit for detecting or determining a phenethanolamine, the kit comprising at least one conjugate and at least one antibody of claim 22 , wherein said conjugate comprises a hapten and a detectable labelling agent, wherein said hapten has the formula of Formula II
wherein said hapten is prepared by the steps of:
preparing a phenylalkylcarbonyl derivative of the formula E by reacting 4-(4-hydroxyphenyl)-2-butanone with Z 2 X 1 , in which
Z 2 is a crosslinker, the crosslinker being —R—X 2 , in which R is selected from a C 1-5 substituted or unsubstituted, straight chain, saturated alkylene group; and a C 1-3 substituted or unsubstituted, straight chain, saturated alkylene group; which serves as a bivalent link;
X 2 is a functional group containing at least one hetero-atom capable of linking the R of the crosslinker with said detectable labelling agent, and is selected from the group comprising a carboxylic acid or an ester thereof; an amine; a maleimide, a halocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridyl group, a vinylsulphone group, and a thiocarboxylic acid or an ester thereof; and
X 1 is a halide;
and
reacting the phenethanolamine of formula D with a phenylalkylcarbonyl derivative of formula E, in which Z 1 is H; Y is independently H or CH 3 ; W is independently H or CH 3 ; n is independently 1 or 2; and m is independently 0 or 1;
wherein said crosslinker links said hapten to said detectable labelling agent at a single phenolic hydroxyl group of said hapten.