- Email: [email protected]
Labelling of drug antigens
.I. hcl. Med. Biol. Vol. Printed in Great Britain hr.
I I, No. 1, pp. 79-83,
1984
0047-0740/84 $3.00 + 0.00 Pergamon Press Ltd
Labelling of Drug Antigens B. LY and M. HEGESIPPE Office de Rayonnements Ionisants, Laboratoire Produits pour Analyses Midicales, CEN Marcoule. B.P. 171, 30205 Bagnols-sur-Ceze cedex, France
Introduction Immunological methods, and in particular radioimmunoassay, were introduced into pharmacology when it became necessary to have a tool for the determination of substances in biological fluids, with the following characteristics: rapidity, specificity, sensitivity. All these advantages were initially demonstrated in those radioimmunoassays developed for endocrinology. (‘) Since 1968 more than 100 drugs have been extensively studied and assayed using radioimmunoassay. The majority of RIA kits which are available today are prepared with radioiodinated tracers (mainly “‘I). The first assays which were developed used ‘H or 14C labelled drugs in order to ensure the antigenic identity of the tracer with the native antigen. The radioiodinated tracer was only introduced a few years later (Table I). The 3H and 14C tracers can only be produced by commercial companies and the introduction of the radioiodine tracer revolutionised RIA by making available a technique in which the method of labelling is simple, the quantification of results can be done directly using automatic counters and the specific activity can be increased. Table 2 shows the main kits used in the TDM and their origins; it can be seen that the majority of these kits originate from U.S. companies. Moreover the use of immunoassay has been extended to non isotopic methods, this being mainly in routine pharmacology, i.e. TDM. An example of this is the recent significant division between laboratories participating in the CAP controls using RIA and non RIA analyses. In Table 3 it can be seen that although digoxin is still 100% assayed using RIA, more than half of the laboratories assaying gentamycin and tobramycin choose non RIA methods. Nevertheless in pharmacological research the RIA is still the most sensitive and convenient method.
Iodine Label@
Site
In classical iodination
of proteins it is either a phenol or imidazole ring which is labelled. These groups are only rarely present in a hapten, for 79
example in bleomycin, an antitumoral drug, and antipyrin, and can be directly labelled in the normal way. Generally, the molecule requires chemical modification by the addition of an iodinable residue. The phenol-containing molecules used in this type of conjugation are listed in Fig. 1. The most commonly used residues are the TME and Bolton & Hunter reagent. Figure 2 shows the imidazole containing molecules: histamine and histidine. It is normal to conjugate the residue to the hapten before iodination with the exception of the Bolton & Hunter reagent which is iodinated before conjugation. Generally the peptide bond is carried out by the coupling reagent between carboxylic and aminic functions which can be present on either molecule. In the presence of two amino groups, the glutaraldehyde method can be used. When either the carboxylic or aminic functions are absent, a chemical modification of the molecule in a selected region is carried out to produce a carboxylic group. Figure 3 shows the usual methods for the creation of carboxylic groups on different terminal functions of haptens. If the drug hapten does not contain any terminal function in the selected part of the molecule, it is possible to synthesise a drug derivative as illustrated in Fig. 4 for Triazolam. (‘) Starting with compound A, the methyl group is replaced by hemisynthesis of a side chain terminated with a COOH group. This side chain then allows for formation of a peptidic bond with the drug and the iodinable residue. In most cases the position of the iodinable residue on the hapten is totally dependant on the strategy of the positioning of the carrier protein during the preparation of the immunogen. In other words the iodinable residue has to be placed in the same position as the carrier protein in the immunogen and consequently the iodine atom will be positioned as far as possible from the specific binding sites. An example of this type of strategy can be illustrated with diphenylhydantoin (DPH). Paxton and Coll(*J have prepared an immunogen derivative of DPH, DPH-3 w-valerate-BSA, and obtained specific antibodies to the DPH part of this derivative. Consequently it was necessary to choose DPH-3-N-ethylacetamido-TME as the conjugate for iodination. An other example
80
Around Table Substances
Table
lZSI
European companies
Digoxin
(Yd
1976 I977 1978 I979 1981 1979 1975
Substances
Diphenylhydantoin
CIS
Diag. B&hem.
Morphine
Roche
Theophyline
ChnicaI Assays RIA Diagnostics
Tobramycin
Clinical Assays
Tyramine
Bolton
and
Hunter
p. Hydroxyphenyl
Glycyl
Tyrosine
ester
0
CH,-CH,-NH,
-COCH
HO
3
Reagent
acetic
acid
56
Isotopic exchange iodination has recently been used in the preparation of A 8[‘251]tetrahydrocannabinol for the development of an assay for the main active constituent of marijuana, the A 9 tetrahydrocannabinol (A 9 THC).(“s”) The specific activity of the A 8 THC was shown to be inadequately low and subsequently the authors chose, among other
Inc.
HO
Methyl
54
Example of isotopic exchange iodination
-c-
Tyrosine
100 46 44
Apart from the method of isotopic exchange, the common methods used to iodinate hapten derivatives are: the iodine monochloride, sodium hypochloride, electrolytic, enzymatic and chloramine-T methods. The latter two are by far the most widely used methods and both are reported here.
American Diag. Clinical Assays Diagnostic Prod. NML
Methotrexate
(“J
1830 758 697
Labelling Metbods
Clinical Assays Diagnostic Prod.
Gentamicin
Non-RIA
(“,)
was reported by Tigelaar and C011(~)who prepared an antibody towards the imide part of DPH by conjugation of the protein to the phenol group and consequently the tracer was produced in the same way using a TME derivative.
Abbott Beckman Bio-Rad Clinical Assays Corning Diagnostic Prod. Damon Pantex Ventrex
Amersham
RIA No. of labs
Digoxin Gentdmicin Tobrdmycin
U.S. companies
Amersham CIS Pharmacita Farmos Diag.
3
U.S.A. CAP/TDM proficiency testmg program (Xi/g?)
Table 2 Substances
World
1
‘H/‘+Z (Yr) 1972 1973-197s 1974 197771978 1977 1974 _
Gentamicin Diphenylhydantoin Methotrexate Nortryptyline Haloperidol Cannabinol Methaqualone
the i%&ar
HO+,,,_pOH
HO -o-
0
CH2--OH-NH
I c=o I CH
Fig.
1
-CH2-NH2
Around Common
the Nuclear
Molecules
with
World
an
XI
Imidazole
Ring
CH2-CH2-NH2
Htstamtne .i”y
Hishdtne
N-qCHz-YH-COOH
NH2
Histidine
methyl
ester
N--CH2-;-COOCH, 2
N. acetyl
histidine
Fig. 2
possibilities, the substitution derivative of A THC.
method of the tosyloxy
Enzymatic method
The chloramine-T method”2’ This method can give high specific radioactivities. Carrier free Na”‘I is oxidized by chloramine-T at the optimal pH of 7.5 in presence of the hapten derivative to be labelled, and thus gives a high yield of ‘*‘I incorporation in a very short time (within 1 min). Excess chloramine-T is then reduced by the addition of sodium metabisulphite and free iodine is reduced to iodide. The labelled hapten derivative is then purified by classical chromatographic techniques of adsorption or gel filtration (See MTX example below).
Some
Classical
Chemical
Modifications
+succtnic
This method allows iodination in mild conditions using the enzyme lactoperoxidase to catalyse the oxidation of ‘25Iin the presence of small amounts of hydrogen peroxide. (13)The hapten (l-3 fig) is mixed with Nat”1 (l-2 mCi) and lactoperoxydase (50-200 ng) and the reaction is initiated by the addition of H202 (SO-200 ng) and maintained for several minutes. The lactoperoxidase catalysed reaction is stopped by the addition of cysteine or NaN, solution. The product of iodination is purified by classical chromatographic methods. Iodination using Bolton and Hunter reagent(‘4.‘5’ This method is often used in case of drugs which
of Hapten
Allowing
Conjugation
anhydride
Hapten-CH,-OH
*
Hapten--XH2-O-CtCH2)2-COOH !
to-carboxymethylhydroxylamine Hapten-C=O
-
H,N-0-CH,COOH
Haven---@-
OH
,‘l~~~~~&~~~‘,l
+ chloracetate
Hapten
Hapten-C=N-0-CHTCOOH
- Hapten+O-CH7--COOH
/ HCI
) Hapten
Cl-CH2-COOR/HCI
Hapten*
OH
+~~~?$an,“,‘,
acid
Fig. 3
- Hapten
Around
82 Exomple
of
the Nuckir
Hemisynthesis
of
World Triorolom
Derivotive
H+-?5N\
Triozolom
Cl
do not tolerate the presence of oxidizing or reducing agents. Bolton and Hunter reagent is N-succinimidyl 3-(4-hydroxyphenyl) propionate(‘6) prelabelled by the classical chloramine-T method. After purification, the N-succinimidyl group of this reagent is condensed with a free amino group of the hapten to form a conjugate in which the radioiodinated phenyl group is covalently linked, via an amide bond, to the hapten in basic conditions (pH 8-9) without any coupling reagent. It is nevertheless necessary to take some precautions to prevent the hydrolysis of this reagent in aqueous conditions.
Methotrexate has two carboxylic groups which can react with an amino group of a protein carrier or tyrosine methyl ester using a coupling reagent. Immunogen preparation””
The methyl bovin serum albumin (Me BSA) (100 mg) was dissolved in 4 mL of NaCl9% solution and to this stirred solution were added MTX (40 mg in 1 mL DMF) and EDCI (80 mg). After 24 h in the dark, 35 mL of a NaCl 9Oh solution was added into this mixture. After centrifugation the supernatant was kept and purified by ultrafiltration. This conjugate, after Lowry analysis and U.V. measurement at 305 nm was calculated to contain 20 residues/Me BSA molecule and three combinations may be obtained (Table 4).
Example of methotrexate
Methotrexate (MTX) belongs to the aminopterines family, compounds which are close to folic acid, and is a powerful inhibitor of dihydrofolate reductase (EC1.5.1.3.), an enzyme involved in cellular synthesis.“‘) Its use in cancer chemotherapy is based on this property.“”
Preparation of methotrexate-tyrosine
methylester
Tyrosine methylester (6 mg) was dissolved in 20 p L of cold water and neutralized by 40 p L of Na,Co,
Table 4
H2NTT p \/ JI >~,.ry=&_,“_~~,,,. R
2
2
NH2
Conjugates
I
RI
R2
NH-Me
OH
2
NH-Me
BSA
3
NH-Me
BSA
BSA
OH NH-Me
BSA
2
Around the Nuclear World Table 5 Compounds
~~ ~~~_~~__~ J4 n, a,
.- RI
RI
TME OH TME
TME TME OH
solution (70mg/mL). To the stirred solution was added MTX (10 mg) in 400 PL of DMF followed by I-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (8 mg) in 1OpL of water. After 1 h at roomtemperature in the dark, 100 PL of Na2C0, solution was added and NaCl was precipitated. The supernatent was then purified by silica thin layer chromatography. The plates were developed in methanol-chloroform (40/60; v/v). Three yellow bands were developed: two minorB,+R,, = 0.70, B,+R,, = 0.25 and one major B,-+ Rfi = 0.14-and, according to the polarity, these may correspond to the three combinations presented in Table 5. (See formula Table 4 but with modifications about R,R,). These bands were removed and extracted into methanol. Iodination qf methotrexate-thyrosine
methyl ester
Each of these MTX derivatives were then iodinated as follows: to 3 pg of MTX-TME (in 2OpL of 0.25 M phosphate buffer) were added 1.5 mCi Na’251, 1.5 pg of fresh chloramine-T in 10 p L of the same buffer; after 1 min 1.5 pg ofmetabisulphite in 10 PL of buffer was added and the product was purified by column silica gel chromatography using the methanol-chloroform (50/50; v/v) system. These three iodinated MTX-TME derivaties were then tested with Rabbit anti-MTX antiserum. The best binding capacity was found with the B, preparation.
Conclusion The chemical transformation
of drugs before label-
83
ling with ‘*‘I and immunogen preparation does not significantly modify the antigenic identity of the hapten. Thus a highly specific and sensitive radioimmunoassay can be developed for these haptens using this method.
References 1. Berson S. A. and Yalow R. S. Ann. N. Y. Acad. Sri. 82, 338 (1959). 2. Oliver G.’ C. Jr, Parker B. M., Brasfield D. L. and Parker C. W. 1. Cl&. Invest. 47. 1035-1042 (1968). 3. Landon J. Analyst 101, 225-243 (1976). 4. Broughton A. and Strong J. E. Clin. Chem. 22,726732 (1976). 5. Goodfriend L. L., Levine L. and Fasman G. D. Science 144, 1344 (1964). 6. Avrameas S. and Temynck T. Immunochemistry 6, 53 (1969). 7. Ko H., Royer M. E., Hester J. B. and Johnston K. T. Anal. Lett. 10, 1019-1040 (1977). 8. Paxton J. W., Rowe11F. J. and Gratcliffe J. C/in. Chim. Acfa 79, 81-92 (1977). 9. Tigelaar R. E., Rapport R. L. II, Inman J. K. and Kuoferbere H. J. C/in. Chim. Acta 43. 231-241 (1973). 10. Owens S. M., McBay A. J., Reisner H. M. and ‘PerezReyes M. Clin. Chem. 27, 619-624 (1981).
Il. Pitt C. G., Seltzman H. H., Setzer S. R. and Williams D. L. J. Labelled Compd. Radiopharm. XVII, 681-689 (1979). 12. Hunter
W. M. and Greenwood F. C. Nature 1941, 495496 (1962). 13. Marchalonis J. J. Biochem. J. 113, 229-305 (1969). 14. Bolton A. E. and Hunter W. M. J. Endocrinol. 55,3&31 (1972). 15. Bolton A. E. and Hunter W. M. Biochem. J. 133, 529-538 (1973). 16 Rudinger J. and Ruegg U. Biochem. J. 133, 538 (1973). 17 Subramanian S. and Kaufman B. T. Proc. Nat/ Acad. Sci. U.S.A. 75, 3201-3205 (1978). 18 Blever W. A. Cancer 41. 3651 (1978). 19 Claude Bouhuon F., Duprey F. and Boudene L. Clin. Chim. Acta 57, 263-267 (1974).
I I, No. 1, pp. 79-83,
1984
0047-0740/84 $3.00 + 0.00 Pergamon Press Ltd
Labelling of Drug Antigens B. LY and M. HEGESIPPE Office de Rayonnements Ionisants, Laboratoire Produits pour Analyses Midicales, CEN Marcoule. B.P. 171, 30205 Bagnols-sur-Ceze cedex, France
Introduction Immunological methods, and in particular radioimmunoassay, were introduced into pharmacology when it became necessary to have a tool for the determination of substances in biological fluids, with the following characteristics: rapidity, specificity, sensitivity. All these advantages were initially demonstrated in those radioimmunoassays developed for endocrinology. (‘) Since 1968 more than 100 drugs have been extensively studied and assayed using radioimmunoassay. The majority of RIA kits which are available today are prepared with radioiodinated tracers (mainly “‘I). The first assays which were developed used ‘H or 14C labelled drugs in order to ensure the antigenic identity of the tracer with the native antigen. The radioiodinated tracer was only introduced a few years later (Table I). The 3H and 14C tracers can only be produced by commercial companies and the introduction of the radioiodine tracer revolutionised RIA by making available a technique in which the method of labelling is simple, the quantification of results can be done directly using automatic counters and the specific activity can be increased. Table 2 shows the main kits used in the TDM and their origins; it can be seen that the majority of these kits originate from U.S. companies. Moreover the use of immunoassay has been extended to non isotopic methods, this being mainly in routine pharmacology, i.e. TDM. An example of this is the recent significant division between laboratories participating in the CAP controls using RIA and non RIA analyses. In Table 3 it can be seen that although digoxin is still 100% assayed using RIA, more than half of the laboratories assaying gentamycin and tobramycin choose non RIA methods. Nevertheless in pharmacological research the RIA is still the most sensitive and convenient method.
Iodine Label@
Site
In classical iodination
of proteins it is either a phenol or imidazole ring which is labelled. These groups are only rarely present in a hapten, for 79
example in bleomycin, an antitumoral drug, and antipyrin, and can be directly labelled in the normal way. Generally, the molecule requires chemical modification by the addition of an iodinable residue. The phenol-containing molecules used in this type of conjugation are listed in Fig. 1. The most commonly used residues are the TME and Bolton & Hunter reagent. Figure 2 shows the imidazole containing molecules: histamine and histidine. It is normal to conjugate the residue to the hapten before iodination with the exception of the Bolton & Hunter reagent which is iodinated before conjugation. Generally the peptide bond is carried out by the coupling reagent between carboxylic and aminic functions which can be present on either molecule. In the presence of two amino groups, the glutaraldehyde method can be used. When either the carboxylic or aminic functions are absent, a chemical modification of the molecule in a selected region is carried out to produce a carboxylic group. Figure 3 shows the usual methods for the creation of carboxylic groups on different terminal functions of haptens. If the drug hapten does not contain any terminal function in the selected part of the molecule, it is possible to synthesise a drug derivative as illustrated in Fig. 4 for Triazolam. (‘) Starting with compound A, the methyl group is replaced by hemisynthesis of a side chain terminated with a COOH group. This side chain then allows for formation of a peptidic bond with the drug and the iodinable residue. In most cases the position of the iodinable residue on the hapten is totally dependant on the strategy of the positioning of the carrier protein during the preparation of the immunogen. In other words the iodinable residue has to be placed in the same position as the carrier protein in the immunogen and consequently the iodine atom will be positioned as far as possible from the specific binding sites. An example of this type of strategy can be illustrated with diphenylhydantoin (DPH). Paxton and Coll(*J have prepared an immunogen derivative of DPH, DPH-3 w-valerate-BSA, and obtained specific antibodies to the DPH part of this derivative. Consequently it was necessary to choose DPH-3-N-ethylacetamido-TME as the conjugate for iodination. An other example
80
Around Table Substances
Table
lZSI
European companies
Digoxin
(Yd
1976 I977 1978 I979 1981 1979 1975
Substances
Diphenylhydantoin
CIS
Diag. B&hem.
Morphine
Roche
Theophyline
ChnicaI Assays RIA Diagnostics
Tobramycin
Clinical Assays
Tyramine
Bolton
and
Hunter
p. Hydroxyphenyl
Glycyl
Tyrosine
ester
0
CH,-CH,-NH,
-COCH
HO
3
Reagent
acetic
acid
56
Isotopic exchange iodination has recently been used in the preparation of A 8[‘251]tetrahydrocannabinol for the development of an assay for the main active constituent of marijuana, the A 9 tetrahydrocannabinol (A 9 THC).(“s”) The specific activity of the A 8 THC was shown to be inadequately low and subsequently the authors chose, among other
Inc.
HO
Methyl
54
Example of isotopic exchange iodination
-c-
Tyrosine
100 46 44
Apart from the method of isotopic exchange, the common methods used to iodinate hapten derivatives are: the iodine monochloride, sodium hypochloride, electrolytic, enzymatic and chloramine-T methods. The latter two are by far the most widely used methods and both are reported here.
American Diag. Clinical Assays Diagnostic Prod. NML
Methotrexate
(“J
1830 758 697
Labelling Metbods
Clinical Assays Diagnostic Prod.
Gentamicin
Non-RIA
(“,)
was reported by Tigelaar and C011(~)who prepared an antibody towards the imide part of DPH by conjugation of the protein to the phenol group and consequently the tracer was produced in the same way using a TME derivative.
Abbott Beckman Bio-Rad Clinical Assays Corning Diagnostic Prod. Damon Pantex Ventrex
Amersham
RIA No. of labs
Digoxin Gentdmicin Tobrdmycin
U.S. companies
Amersham CIS Pharmacita Farmos Diag.
3
U.S.A. CAP/TDM proficiency testmg program (Xi/g?)
Table 2 Substances
World
1
‘H/‘+Z (Yr) 1972 1973-197s 1974 197771978 1977 1974 _
Gentamicin Diphenylhydantoin Methotrexate Nortryptyline Haloperidol Cannabinol Methaqualone
the i%&ar
HO+,,,_pOH
HO -o-
0
CH2--OH-NH
I c=o I CH
Fig.
1
-CH2-NH2
Around Common
the Nuclear
Molecules
with
World
an
XI
Imidazole
Ring
CH2-CH2-NH2
Htstamtne .i”y
Hishdtne
N-qCHz-YH-COOH
NH2
Histidine
methyl
ester
N--CH2-;-COOCH, 2
N. acetyl
histidine
Fig. 2
possibilities, the substitution derivative of A THC.
method of the tosyloxy
Enzymatic method
The chloramine-T method”2’ This method can give high specific radioactivities. Carrier free Na”‘I is oxidized by chloramine-T at the optimal pH of 7.5 in presence of the hapten derivative to be labelled, and thus gives a high yield of ‘*‘I incorporation in a very short time (within 1 min). Excess chloramine-T is then reduced by the addition of sodium metabisulphite and free iodine is reduced to iodide. The labelled hapten derivative is then purified by classical chromatographic techniques of adsorption or gel filtration (See MTX example below).
Some
Classical
Chemical
Modifications
+succtnic
This method allows iodination in mild conditions using the enzyme lactoperoxidase to catalyse the oxidation of ‘25Iin the presence of small amounts of hydrogen peroxide. (13)The hapten (l-3 fig) is mixed with Nat”1 (l-2 mCi) and lactoperoxydase (50-200 ng) and the reaction is initiated by the addition of H202 (SO-200 ng) and maintained for several minutes. The lactoperoxidase catalysed reaction is stopped by the addition of cysteine or NaN, solution. The product of iodination is purified by classical chromatographic methods. Iodination using Bolton and Hunter reagent(‘4.‘5’ This method is often used in case of drugs which
of Hapten
Allowing
Conjugation
anhydride
Hapten-CH,-OH
*
Hapten--XH2-O-CtCH2)2-COOH !
to-carboxymethylhydroxylamine Hapten-C=O
-
H,N-0-CH,COOH
Haven---@-
OH
,‘l~~~~~&~~~‘,l
+ chloracetate
Hapten
Hapten-C=N-0-CHTCOOH
- Hapten+O-CH7--COOH
/ HCI
) Hapten
Cl-CH2-COOR/HCI
Hapten*
OH
+~~~?$an,“,‘,
acid
Fig. 3
- Hapten
Around
82 Exomple
of
the Nuckir
Hemisynthesis
of
World Triorolom
Derivotive
H+-?5N\
Triozolom
Cl
do not tolerate the presence of oxidizing or reducing agents. Bolton and Hunter reagent is N-succinimidyl 3-(4-hydroxyphenyl) propionate(‘6) prelabelled by the classical chloramine-T method. After purification, the N-succinimidyl group of this reagent is condensed with a free amino group of the hapten to form a conjugate in which the radioiodinated phenyl group is covalently linked, via an amide bond, to the hapten in basic conditions (pH 8-9) without any coupling reagent. It is nevertheless necessary to take some precautions to prevent the hydrolysis of this reagent in aqueous conditions.
Methotrexate has two carboxylic groups which can react with an amino group of a protein carrier or tyrosine methyl ester using a coupling reagent. Immunogen preparation””
The methyl bovin serum albumin (Me BSA) (100 mg) was dissolved in 4 mL of NaCl9% solution and to this stirred solution were added MTX (40 mg in 1 mL DMF) and EDCI (80 mg). After 24 h in the dark, 35 mL of a NaCl 9Oh solution was added into this mixture. After centrifugation the supernatant was kept and purified by ultrafiltration. This conjugate, after Lowry analysis and U.V. measurement at 305 nm was calculated to contain 20 residues/Me BSA molecule and three combinations may be obtained (Table 4).
Example of methotrexate
Methotrexate (MTX) belongs to the aminopterines family, compounds which are close to folic acid, and is a powerful inhibitor of dihydrofolate reductase (EC1.5.1.3.), an enzyme involved in cellular synthesis.“‘) Its use in cancer chemotherapy is based on this property.“”
Preparation of methotrexate-tyrosine
methylester
Tyrosine methylester (6 mg) was dissolved in 20 p L of cold water and neutralized by 40 p L of Na,Co,
Table 4
H2NTT p \/ JI >~,.ry=&_,“_~~,,,. R
2
2
NH2
Conjugates
I
RI
R2
NH-Me
OH
2
NH-Me
BSA
3
NH-Me
BSA
BSA
OH NH-Me
BSA
2
Around the Nuclear World Table 5 Compounds
~~ ~~~_~~__~ J4 n, a,
.- RI
RI
TME OH TME
TME TME OH
solution (70mg/mL). To the stirred solution was added MTX (10 mg) in 400 PL of DMF followed by I-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (8 mg) in 1OpL of water. After 1 h at roomtemperature in the dark, 100 PL of Na2C0, solution was added and NaCl was precipitated. The supernatent was then purified by silica thin layer chromatography. The plates were developed in methanol-chloroform (40/60; v/v). Three yellow bands were developed: two minorB,+R,, = 0.70, B,+R,, = 0.25 and one major B,-+ Rfi = 0.14-and, according to the polarity, these may correspond to the three combinations presented in Table 5. (See formula Table 4 but with modifications about R,R,). These bands were removed and extracted into methanol. Iodination qf methotrexate-thyrosine
methyl ester
Each of these MTX derivatives were then iodinated as follows: to 3 pg of MTX-TME (in 2OpL of 0.25 M phosphate buffer) were added 1.5 mCi Na’251, 1.5 pg of fresh chloramine-T in 10 p L of the same buffer; after 1 min 1.5 pg ofmetabisulphite in 10 PL of buffer was added and the product was purified by column silica gel chromatography using the methanol-chloroform (50/50; v/v) system. These three iodinated MTX-TME derivaties were then tested with Rabbit anti-MTX antiserum. The best binding capacity was found with the B, preparation.
Conclusion The chemical transformation
of drugs before label-
83
ling with ‘*‘I and immunogen preparation does not significantly modify the antigenic identity of the hapten. Thus a highly specific and sensitive radioimmunoassay can be developed for these haptens using this method.
References 1. Berson S. A. and Yalow R. S. Ann. N. Y. Acad. Sri. 82, 338 (1959). 2. Oliver G.’ C. Jr, Parker B. M., Brasfield D. L. and Parker C. W. 1. Cl&. Invest. 47. 1035-1042 (1968). 3. Landon J. Analyst 101, 225-243 (1976). 4. Broughton A. and Strong J. E. Clin. Chem. 22,726732 (1976). 5. Goodfriend L. L., Levine L. and Fasman G. D. Science 144, 1344 (1964). 6. Avrameas S. and Temynck T. Immunochemistry 6, 53 (1969). 7. Ko H., Royer M. E., Hester J. B. and Johnston K. T. Anal. Lett. 10, 1019-1040 (1977). 8. Paxton J. W., Rowe11F. J. and Gratcliffe J. C/in. Chim. Acfa 79, 81-92 (1977). 9. Tigelaar R. E., Rapport R. L. II, Inman J. K. and Kuoferbere H. J. C/in. Chim. Acta 43. 231-241 (1973). 10. Owens S. M., McBay A. J., Reisner H. M. and ‘PerezReyes M. Clin. Chem. 27, 619-624 (1981).
Il. Pitt C. G., Seltzman H. H., Setzer S. R. and Williams D. L. J. Labelled Compd. Radiopharm. XVII, 681-689 (1979). 12. Hunter
W. M. and Greenwood F. C. Nature 1941, 495496 (1962). 13. Marchalonis J. J. Biochem. J. 113, 229-305 (1969). 14. Bolton A. E. and Hunter W. M. J. Endocrinol. 55,3&31 (1972). 15. Bolton A. E. and Hunter W. M. Biochem. J. 133, 529-538 (1973). 16 Rudinger J. and Ruegg U. Biochem. J. 133, 538 (1973). 17 Subramanian S. and Kaufman B. T. Proc. Nat/ Acad. Sci. U.S.A. 75, 3201-3205 (1978). 18 Blever W. A. Cancer 41. 3651 (1978). 19 Claude Bouhuon F., Duprey F. and Boudene L. Clin. Chim. Acta 57, 263-267 (1974).