The production and evaluation of antibodies for enzyme immunoassay of AZTTP

Article abstract of DOI:10.1081/NCN-100002084

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.

Full text of DOI:10.1081/NCN-100002084

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THE PRODUCTION AND EVALUATION OF ANTIBODIES
FOR ENZYME IMMUNOASSAY OF AZTTP
a
a
Fatima Akeb , Christophe Creminon , Jacques Grassi , Roger Guedj & Danièle Duval
a
Université de Nice-Sophia Antipolis , Laboratoire de Chimie Bio-Organique, ESA CNRS
6001, Faculté des Sciences, Parc Valrose, Nice Cedex 2, 06108, France
Published online: 17 Aug 2006.
To cite this article: Fatima Akeb , Christophe Creminon , Jacques Grassi , Roger Guedj & Danièle Duval (2001) THE
PRODUCTION AND EVALUATION OF ANTIBODIES FOR ENZYME IMMUNOASSAY OF AZTTP, Nucleosides, Nucleotides and Nucleic
Acids, 20:3, 243-250, DOI: 10.1081/NCN-100002084
To link to this article: http://dx.doi.org/10.1081/NCN-100002084
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(3), 243–250 (2001)
THE PRODUCTION AND EVALUATION
OF ANTIBODIES FOR ENZYME
IMMUNOASSAY OF AZTTP
1
2
2
Fatima Akeb, Christophe Creminon, Jacques Grassi,
1
2,∗
Roger Guedj, and Dani e` le Duval
1Laboratoire de Chimie Bio-Organique, ESA CNRS 6001, Facult e´ des
Sciences, Universit e´ de Nice-Sophia Antipolis, Parc Valrose, 06108
Nice Cedex 2, France
2
Service de Pharmacologie et d’Immunologie, Direction des Sciences
du Vivant, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France
ABSTRACT
We describe the development of the first enzyme immunoassay for quantifying
AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were
raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin
(KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity
to AZTTP. One of the antisera produced was specific for AZTTP.
INTRODUCTION
Nucleosides analogs such AZT, ddC, ddI, d4T, 3TC, are currently used in
treatment of AIDS. ddN are converted, successively, into the mono-(ddNMP), di-
(ddNDP) and tri-phosphorylated nucleotides (ddNTPs) by the appropriate cellular
kinases (1). The active species that inhibit the HIV reverse transcriptase are ddNTPs.
The pharmacokinetics of ddNs shows large inter individual differences (2). In addi-
tion, plasma concentrations of ddNs are not related to intracellular concentrations
ꢀ
of the corresponding 5 -triphosphates (2–5). It is therefore important to define
the extent of ddN metabolism and the intracellular concentration of the different
metabolites. Different techniques have been described for the quantification of ddN
(6,7) and of their metabolites (8–11) but none of them can be used ideally for routine
243
Copyright ꢁC 2001 by Marcel Dekker, Inc.
www.dekker.com

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44
AKEB ET AL.
monitoring of intracellular AZTTP in patients and to define optimum drug dosages.
With respect to AZT, for example, the RIA procedure used so far enables only the
determination of the total concentration of AZT metabolites (12) except when it is
used in combination with HPLC or SPE (13–16) (Solid Phase Extraction). HPLC
assay allows the determination of each of these species, but its sensitivity is marginal
as compared to RIA (17,18). The sensitivity of HPLC assay can be improved by
coupling with a RIA test (19–21), but this method as well as SPE/RIA requires prior
separation of AZT metabolites, treatment with phosphatases and quantification of
the resultant AZT with a RIA. HPLC with tandem spectrometry constitutes an inter-
esting method for the determination of intracellular level of AZTTP with a limit of
detection of 4 fmol/ml (22) but this procedure remains time consuming and costly
for routine monitoring. An alternative and attractive method is an immunoassay
based on competitive enzyme linked immunoassay (ELISA). This paper describes
a specific AZTTP ELISA.
RESULTS AND DISCUSSION
Anti-AZTTP antibodies were produced in rabbits using a keyhole limpet
hemocyanin (KLH)-AZTTP conjugate as immunogen 2. Conjugation of AZTTP
ꢀ
ꢀ
to KLH can be achieved either via the terminal 5 -phosphate or via the base of
the nucleotide (23). We previously reported that 5 -O-hemisuccinate ddN can be
used to raise anti-ddN antibodies (24). So, we chose to use the immunogen 2 pre-
pared by coupling the phosphate γ moiety of AZTTP to amine groups of KLH.
AZT was triphosphorylated in a “one pot” reaction following a protocol devel-
oped for phosphorylation of purine nucleoside (25). Briefly, AZT was dissolved in
◦
PO(OEt) and treated at −10 C with 3 eq of POCl resulting in the formation of an
3
3
activated dichlorophosphate which was transformed to AZTTP using an excess of
(
Bu NH) H P O . The triphosphate 1 was isolated by anion exchange chromatog-
3 2 2 2 7
raphy on DEAE-Sephadex A-25 column with a linear gradient of TEA buffer and
purified by HPLC as triethylammonium salt with a 30% yield (Scheme 1) (26).
Immunogen 2 was prepared by coupling AZTTP to KLH through activation of
the nucleotide by EDC. This conjugate was isolated after dialysis against a 0.01 M
Tris-HCl buffer. Tracer 3 was prepared by coupling AZTTP to acetylcholinesterase
(AchE) using the conventional carbodiimide method (27) and isolated by size ex-
clusion chromatography on Biogel A-1.5 M column with EIA buffer.
The conjugate 2 was emulsified in Freund’s adjuvant and was injected sub-
cutaneously in three rabbits. Blood was harvested at regular time intervals for
monitoring AZTTP antibodies development.
Conventional competitive immunoassays using unpurified rabbit antisera as
first antibodies and AZTTP-AChE conjugate as tracer were performed under the
conditions described for other haptens (28). Briefly, the different antisera were in-
cubated after performing serial dilutions in EIA buffer with AZTP-AChE conjugate
in 96 well immunoplate coated with mouse monoclonal antirabbit IgG antibodies.

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ENZYME IMMUNOASSAY OF AZTTP
245
O
N
CH3
HN
O
O
O
O
1
)
POCl3/PO(OEt)3
+
−
O
AZT
NH(Et)3
O
P
O
P
O
P
O
2
) H4P2O7N(But)3/PO(OEt)3
OH
OH
OH
5
'- O-triphosphate-AZT 1
N3
KLH-NH2
O
AChE-NH2
EDC/H2O
pH 7.5
O
N
CH3
CH3
HN
O
HN
O
O
P
O
O
O
O
O
O
N
O
KLH-NH
O
P
O
P
O
AChE-NH
P
O
P
O
P
O
OH
OH
OH
OH
OH
OH
N3
N3
immunogen 2
tracer 3
Scheme 1. Synthesis of immunogen and tracer of AZTTP.
4
After the final booster, anti-AZTTP antibodies were detectable at 1/10 dilution,
thus demonstrating the immunogenic potency of AZTTP-KLH immunogen.
Sensitivity and the specificity of the immunoassay were evaluated at the di-
4
lution 2/10 . The sensitivity of the assay, which is defined by the dose of AZTTP
which reduces the binding of AZTTP-AChE conjugate tracer by 50%, was evalu-
ated in presence of serial dilutions of AZTTP in range of 0.2 nM-2 µM. Figure 1
4
shows a typical standard curve for AZTTP with the best bleeding at 1/10 dilution.
The sensitivity was 0.8 nM and the limit of detection (LOD) was around 0.2 nM.
The sensitivity of this assay has to be improved to determine intracellular AZTTP
concentrations in HIV-infected patients which are very low (0.01–0.6 nM) (16).
The specificity of AZTTP antibodies was analyzed by testing the capacity of
AZT, AZTMP and AZTDP to inhibit the binding of the AZTTP-AChE conjugate
tracer. Results are presented in Table 1. Two antibodies (AS1 and AS3) recognized
AZTTP with a high affinity but they did not discriminate the different phospho-
rylated forms of AZT. In contrast, AS2 clearly discriminated AZTTP from AZT,
AZTMP and AZTDP. The cross-reactivity of antibodies with dTTP and d4TP must
be assessed.
Table 1. Study of Specificity of Anti-AZTTP Antibodies
AZTTP
0 %
AZT
AZTMP
50%
AZTDP
50%
5
50%
inhibition inhibition % cross inhibition % cross inhibition % cross
Antis e´ rum
nM.
3.7
40
2
nM.
reaction
nM.
reaction
nM.
6.7
913
1.3
reaction
AS1
AS2
AS3
94
2397
356
3.9
1.6
0.5
16.3
663
1.9
22.7
6
105
55
4.4
154

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46
AKEB ET AL.
Figure 1. Calibration curve of immunoassay of AZTTP.
In conclusion, this study shows that antibodies specially directed against
AZTTP can be raised in rabbits in spite of the poor chemical and in vivo stability
of the pyrophosphate linkage. An AZTTP-AChE conjugate can also be prepared
and allowed the development of a specific ELISA that does not require the use of
labelled AZTTP. These tools will be used to assay intracellular AZTTP levels in
peripheral blood monocytes of AIDS patients.
EXPERIMENTAL SECTION
AZT was donated by Glaxo Wellcome (France), phosphorous oxychloride,
tributylamine, 3-chloroperbenzoic acid, EDC (1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide) and tributylammonium pyrophosphate were purchased from
Sigma-Aldrich (Saint-Quentin, France). All other reagents were of analytical
grade.
Anion-exchangeresinusedforseparationofAZTTPwasSEPHADEX-DEAE
A-25 ion-exchange resin (40–120 micron) purchased from Aldrich. The HPLC sys-
tem for purification or control of purity of AZTTP consists on WATERS apparatus

ORDER
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ENZYME IMMUNOASSAY OF AZTTP
247
including 600 pump, 996 photodiode array detector and Millennium chromato-
graphic manager. HPLC purification of AZTTP was achieved on analytical
/semi-preparative TSK-GEL DEAE-5PW column with a linear gradient of triethy-
lammonium buffer (TEAB) [0.1–1 M].
Keyhole Limpet Hemocyanin (KLH) was purchased from Pierce. Acetyl-
cholinesterase (AChE) was purified from electric eels (Electrophorus electricus)
by affinity chromatography as previously reported (29). Ellman’s reagent was a so-
−
4
−4
ꢀ
lution of 7.5 10 M acetylthiocholine iodide (enzyme substrate) and 5 10 M 5,5 -
dithiobis (2-nitrobenzoic acid) (chromogen) in 0.1 M phosphate buffer (pH = 7.4).
EIA buffer (pH = 7.4) : [K HPO ] = 0.08 M; [KH PO ] = 0.02 M; [NaCl] =
2
4
2
4
0
.15 M; [BSA] = 145 µM; [NaN ] = 1.5 mM.
3
ꢀ
Synthesis of AZT-TP (5 -O-Triphosphate-AZT)
To a solution of AZT (100 mg; 0.37 mmol), in 2 ml of triethylphosphate cooled
◦
to −10 C was added freshly distilled phosphorous oxychloride (125 µl; 3.6 eq).
◦
The mixture was stirred for 2 h at −10 C under a nitrogen atmosphere. Then trib-
utylamine (1.1 ml; 4.9 mmol) and a anhydrous dimethylformamide solution of
anhydrous tributylammonium pyrophosphate (0.5 M; 2.5 ml) were added. The
reaction was quenched after 5 min by addition of aqueous solution of NaOH
◦
(
0.1 M) under vigorous stirring for 20 min at 0 C. The mixture was evaporated
◦
under vacuum at room temperature (bath temperature should be kept below 30 C)
and lyophilised. The residue was kept in methanol/ethyl ether (1:10, v/v) and cooled
at 4 C. The white precipitate was evaporated at room temperature, remained in 5 ml
◦
of distilled water and loaded onto DEAE-SEPHADEX A-25 column that was equi-
librated with 0.01 M TEAB. The column was eluted with a linear gradient of TEAB
(
pH = 7.6), [0.1 M–0.3 M; 250 ml each followed by 0.3–0.8 M; 250 ml each]. The
ꢀ
appropriate fractions were collected and lyophilised. The 5 -O-triphosphate-AZT
(AZT-TP) was purified on analytical/semi-preparative ion-exchange HPLC with a
linear gradient of TEAB described in the following table.
Time (min) %H O %TEAB (0.3 M) %TEAB (1 M)
2
0
5
5
0
100
50
0
0
50
100
0
0
0
0
1
2
4
0
100
ꢀ
The 5 -O-triphosphate-AZT was converted into its sodium salt using sodium
ꢀ
1
iodide in acetone. The yield of 5 -O-triphosphate-AZT was around 30%. H NMR
D O, 200 MHz) δ 7.8 (s, 1 H, H ), 6.1 (t, 1 H, H
), 4.3 (m,1H, H4
P NMR (D O, 81 MHz) δ −8.6 (d, 1P, P ,J
3
(
ꢀ
, J
ꢀ
ꢀ
= 6.8 Hz), 4.4 (m, 1 H,
2
6
1
H1 -H2
H
ꢀ
ꢀ
), 4.1–4.2 (d, 2H, H5 ), 2.6 (m, 2H, H ), 1.9 (s, 3H, CH );
ꢀ
ꢀ
3
2 3
3
1
2
= 20.1 Hz), −11.0 (d, 1 P, Pα,
2
γ
Pγ -Pβ

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2
48
AKEB ET AL.
J2
(
= 19.1 Hz), −21.8 (dd, 1P, P , J
2
= 20.1 Hz, J
2
= 19.1 Hz); MS
Pβ-Pα
β
Pβ-Pγ
Pβ-Pα
-
+
ESI ) (m/z) 506 [M-H], 527.9 [M-2H + Na ]; t (HPLC) : 26 min.
R
ꢀ
KLH-5 -O-Triphosphate-AZT
To an aqueous solution (0.5 ml) of 1 mg (1.6 µmol) of triethylammonium salt
of 5 -O-triphosphate-AZT and 325 µg (1.1 eq) of EDC, 1 mg of KLH are added at
C. The pH is adjusted at 7.5 by dropwise addition of Tris-HCl buffer (1M). After
ꢀ
◦
0
an overnight incubation at room temperature in the dark, the mixture was dialyzed
◦
ꢀ
four times against Tris-HCl buffer (0.01 M) at 4 C. KHL-5 -O-triphosphate-AZT
◦
was lyophilised and kept frozen at −20 C until use.
Rabbits were immunized by intradermal injection. Antisera were supple-
◦
mented with 0.01% sodium azide and stored at +4 C.
ꢀ
AChE-5 -O-Triphosphate-AZT
ꢀ
One milligramme (1.6 µmol) of triethylammonium salt of 5 -O-triphosphate-
AZT, 325 µg (1.1 eq) of EDC and 50 µg (156 pmol) of acetylcholinesterase were
dissolved in 0.5 ml distilled water. 250 µl of Tris-HCl buffer (1M; pH = 7.4) were
added. After an overnight incubation at room temperature in the dark, the enzymatic
tracer was purified by size exclusion chromatography on a Biogel A1.5M and stored
◦
at −80 C until use. The acetylcholinest e´ rase activity of the tracer was determined
after addition of Ellman’s reagent.
Competitive Immunoassay
These assays were performed in 96-well microtiter plates coated with mouse
monoclonal anti-rabbit IgG antibodies as specific secondary antibodies. The total
reaction volume was 150 µl. Each component (tracer, AZTTP standard or sample
and rabbit antiserum) being added in a 50 µl volume. AZTTP-AChE conjugate was
used at a 2U Ell/ml.
All components of the immunoreaction were dissolved in EIA buffer. All
concentrations refer to the concentrations of the reagent in the initial volume before
mixing with the other reagents. The sensitivity of the assay was characterized by
the dose of AZTTP inducing a 50% lowering of the binding observed in the absence
of competitor (B/Bo = 50%).
◦
After an 18 h incubation at +4 C, the plates were washed with 10 mM phos-
phate buffer pH 7.4 containing 0.05% Tween 20. The enzyme activity was measured
by addition of 200 µl of Ellman’s reagent.

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ENZYME IMMUNOASSAY OF AZTTP
249
ACKNOWLEDGMENTS
This work was supported by the Agence Nationale de Recherches sur le SIDA
ANRS) and by Ensemble contre le Sida.
(
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Received June 27, 2000
Accepted November 17, 2000

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