Clinical Chemistry 43: 2345-2352, 1997;
(Clinical Chemistry. 1997;43:2345-2352.)
© 1997 American Association for Clinical Chemistry, Inc.
7
-Biotinylated testosterone derivatives as tracers for a competitive chemiluminescence immunoassay of testosterone in serum
Peter Luppa1,a,
Christine Brückner1,
Ingrid Schwab1,
Sabine Hauck1,
Stefan Schmidmayr1,
Christian Birkmayer2,
Birgit Paulus2 and
Hagen Hauptmann2
1
Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technical University Munich, Ismaninger Str. 22, D-81675 Munich, Germany.
2
Institute of Organic Chemistry, University of
Regensburg, Universitätsstr. 31, D-93053 Regensburg,
Germany.
a Author for correspondence. Fax 0049 89 4140 4875; e-mail peter.luppa{at}edv1.klinchem.med.tu-muenchen.de
 |
Abstract
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Ring core-biotinylated testosterone tracers were synthesized with
bridges of three different lengths connecting the biotin moiety to the
steroid core (7
-Cn-Bio-T, n = 3, 6, or 11).
Together with a position 7-specific polyclonal anti-testosterone
antibody, we used the 7-C11-Bio-T tracer to develop a
novel, labeled-hapten competitive immunoassay for total testosterone in
serum. (The C3 and C6 tracers proved to be not
suitable for analogous immunoassays.) Enhanced chemiluminescence signal
was generated by use of a second immobilized antibody and a
streptavidin–horseradish peroxidase conjugate. The measuring range of
the assay is 0.2–20.0 nmol/L, linearity of serial dilutions can be
demonstrated, the lower detection limit is 0.125 nmol/L, and the
interassay imprecisions are 13–16%. Accuracy determinations in mass
spectrometry-controlled reference specimens showed a mean recovery of
95%. In addition, the assay shows low cross-reactivities,
demonstrating the favorable specificity of the combination of a
"nearly native" tracer with a position analog antibody. The
optimized steric structure and the long spacer arm of the biotinylated
testosterone tracer make this chemiluminescence assay well-suited for
measuring total testosterone concentration in serum.
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Introduction
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Nonisotopic immunoassays for the determination of
steroids in serum are at present widely used in clinical laboratories
(1)(2). Besides limitations attributable to
the competitive technique, another disadvantage must be considered: Use
of enzyme-labeled steroid tracers may diminish the sensitivity and
specificity of the assay when the structures of the tracers are altered
by the derivatization method.
To address this problem, we developed novel biotinylated steroid
tracers (3)(4). Our concept, in accordance
with Landsteiner's principle (5) that antibody
specificity is directed primarily at that portion of the hapten
furthest from the functional group linking it to the carrier protein,
leads to an effective competition between the immunogen and the steroid
tracer when the tracer is a ring core-biotinylated steroid. The biotin
residue is attached via a defined spacer group to the steroid at the
same position as the immunogen that was used to produce the specific
anti-steroid antibody used in the assay. This synthetic approach leads
one to expect high specificity because of the greater conformational
similarity of the hapten to the native steroid.
The performance of these tracers is directly related to the chemical
nature and length of the spacer arm. We previously demonstrated that
not only the specificity but also the sensitivity (detection limit) of
such an assay format is advantageous over original RIA techniques
(4) by establishing a chemiluminescence immunoassay for
estrone. This improvement was due to the high affinity of biotin to the
streptavidin
(sAv)1
reporter enzyme conjugate, which can be used for an
appropriate chemiluminescence substrate. However, the measuring range
for estrone in serum could not be improved. We therefore believe that
this concept is an attractive alternative to other non-RIA developments
for steroid measurements.
To further confirm this concept, we have synthesized different
sterically optimized ring core-biotinylated testosterone
tracers—17ß-hydroxyandrost-4-en-3-one-7-(biotinyl)-6-N-propylamide
(7-C3-Bio-T), -6-N-hexylamide
(7-C6-Bio-T), and -6-N-undecylamide
(7-C11-Bio-T) (6)—and subsequently
developed a competitive testosterone (T) chemiluminescence immunoassay.
Here we report the characteristics of the assay and its comparison with
two commercially available RIAs.
|
Materials and Methods
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reagents
Chemicals.
sAv-conjugated horseradish peroxidase (HRP)
was obtained from Vector. [1,2,6,7-1
H]T (specific
activity: 3.7 TBq/mmol) was from Amersham-Buchler. Androstenedione,
19-nortestosterone, and 5-dihydrotestosterone were from Fluka;
19-hydroxyandrostenedione and progesterone from Sigma;
5-androstanediol, 5ß-androstanediol, 11-keto-testosterone,
3ß,17ß-dihydroxy-5-androstane, 17-methyltestosterone,
epitestosterone, and 11ß-hydroxy-testosterone from Research Plus,
Bayonne, NJ. All other laboratory chemicals were from Merck.
Reagent solutions.
The assay buffer consisted of 1.50
mmol/L NaH2PO4, 8.15 mmol/L
Na2HPO4, 149 mmol/L NaCl, and 0.30 mmol/L (20
g/L) bovine serum albumin (BSA), pH 7.4. Borate washing buffer (pH 8.4)
and luminol signal reagent were both used in the original format of the
Amerlite System (Johnson & Johnson).
Antibodies and antibody-coated microtiter plates.
The
polyclonal rabbit anti-T antibody 7A [raised against
T-7-(O-carboxymethyl)-thioether-BSA] was obtained from
Accurate Chemical, Westbury, NY (lot no. F9046, antibody titer
1:10 000). For capture antibody we used a goat anti-rabbit antibody
from Biogenesis, Poole, UK (batch no. 960124D/961129D, concentration
2.7 g/L), coated to Microlite 2F wells (Dynatech). The coating
procedure consisted of incubating 250 µL of 50-fold-diluted capture
antibody solution (54 mg/L) in assay buffer without BSA in the
Microlite wells for 18 h at room temperature. After the wells are
washed with borate washing buffer, they are incubated with 200 µL of
assay buffer plus 20 g/L BSA for 30 min at 37 °C in a
shaker-incubator. After additional washing, the wells are ready to use
or can be stored in a dry place for up to 4 weeks.
Tracers.
As described in detail for
7-C6-Bio-T (6), the synthesis for the
biotinylated tracers started from 6-dehydrotestosterone 17ß-acetate,
which was alkylated at the 7-position in a 1,6-Michael addition
reaction by applying 6-(tert-butyldimethylsilyloxyalkyl)
bromide Grignard reagents. After cleavage of the respective silyl
ethers, the HPLC-isolated 7-isomers of the alcohols were transformed
to primary amines and subsequently biotinylated with
biotinyl-N-hydroxysuccinimide ester.
Calibrators.
T calibration specimens were prepared by
dissolving T (from Merck, lot no. K 02983215, purity >98%) in 960
mL/L ethanol (storable at -70 °C) and diluting to the following
final concentrations in assay buffer: 0.2, 0.5, 1.0, 2.0, 5.0, 10, and
20 nmol/L (equivalent to 10, 25, 50, 100, 250, 500, and 1000
fmol/well). The lyophilized control samples (I–III) for T were based
on a human serum matrix (to be reconstituted with 3 mL of distilled
water) and checked for T content by a definitive mass spectrometry (MS)
method (7).
serum specimens
Blood was taken from 96 patients (both sexes) and from 74
apparently healthy men and pre- and perimenopausal women. All subjects
gave their informed consent to the investigation according to the
standards of the Ethics Committee of the Technical University Munich.
Health was assumed on the basis of a medical and clinical chemistry
examination. Premenopausal women were regularly menstruating. A
perimenopausal status was presumed in case of the following serum
constellation: luteinizing hormone <20 IU/L,
follicle-stimulating hormone <30 IU/L, estradiol >120 pmol/L.
All sera were stored at -70 °C after centrifugation. For
measurements, 250-µL serum samples were extracted with 1.5 mL of
diethyl ether. The aqueous phase was frozen in a
CO2/ethanol mixture, and the extract was removed and
evaporated in a stream of N2. The residue was subsequently
reconstituted in 250 µL of assay buffer. Recovery of T was
reproducible at 89–92%, as checked by analyzing 3 different serum
pools that had been supplemented with various amounts of tritiated T
(up to 50 nmol/L). The measured counts of 3 x 7 samples after
extraction were referred to those in the respective unextracted samples
measured on 3 separate days. The final T values found with the assay
were therefore corrected by the above extraction factor (multiplied x
10/9).
procedures
Assay protocol.
The following final concentrations (in
assay buffer) of the key components were used: 7A antibody, 1:600;
sAv-conjugated HRP, dilution 1:20 000 (50 µg/L);
7-C11-Bio-T tracer, 3.0 nmol/L (600 fmol/well). The
assay procedure was performed as described previously (4),
with 50 µL of specimen/calibrator and 150 µL of tracer stock
solution. Total assay time is ~5 h, with the incubation steps taking
a total of 2.5 h.
Displacement experiments.
The technique for controlling
the degree of T displacement by the 7-Bio-T tracers was equivalent
to that described previously (4). In brief, serial
dilutions of antibody solution (1:102 to 1:105)
were incubated with 7-Bio-T (C3-, C6-, or
C11-tracer; 0 to 35 nmol/L each) and tritiated T (3.5
nmol/L). The percent binding (%B) of tritiated T after separation of
bound from free label was plotted vs log of antibody dilution for
different tracer concentrations. At a given antibody dilution, the
difference in %B values in the presence or absence of the respective
tracer is represented by d, with the highest d
values indicating optimal antibody titers. At various 7-Bio-T
concentrations and at the optimum antibody dilution, the ratios of %B
(7-Bio-T concn >0) to %B (7-Bio-T concn = 0) are then
calculated and plotted against the tracer concentration. The 7-Bio-T
concentration at which 50% of the bound tritiated T is displaced
(%Brel = 50%) reflects the molar ratio of the
respective biotinylated tracer and tritiated T for competition.
Comparison assays.
The 125I-labeled direct T
RIA kits with coated tubes were obtained from ICN Biomedicals and from
Diagnostic Products Corp. (DPC).
analytical performance of the chemiluminescence immunoassay
Accuracy.
Accuracy testing was performed by adding known
amounts of T to 3 pooled serum samples with low endogenous T content.
The concentrations were measured before and after addition of T and
recoveries were calculated. Additionally, 3 control materials with
different T concentrations were tested in measurements on separate days
and compared with the results of a definitive MS method. These samples
were extracted before use in the same way as the serum specimens.
Precision.
Different serum pools were used to determine
the intraassay and the interassay CVs.
Lower detection limit.
The threshold for detection of T
was calculated as the concentration corresponding to the mean + 3 SD of
the light intensity for zero T, measured in 20 serial (intraassay)
determinations.
Functional sensitivity.
The functional sensitivity was
assessed (8)(9) by repeated interassay
measurements of specimens with T at 0.2, 0.5, 1.0, 1.5, 2.0, and 3.0
nmol/L (n = 12). Interpolation of the minimum analyte
concentration at a CV of 20% from the precision–dose profile was used
to define the minimum detection limit.
Linearity.
Dilution linearities were tested by
consecutively diluting 8 sera with high endogenous T concentrations;
the measured T concentrations, pmol/well, were plotted against the
effective sample volumes in microliters.
Cross-reactivity.
The calculation of percent
cross-reactivities for the different antibodies was made according to
Abraham (10) and expressed as the ratio of the apparent T
concentrations to the added concentration of cross-reacting steroid at
50% binding of an almost T-free serum sample.
|
Results
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preparation of the biotinylated t tracers and displacement
experiments
The 7-Bio-T immunochemical tracers (Fig. 1
) were prepared by attaching biotin via C3-,
C6-, and C11-alkyl spacer arms at the C-7
position of T as described above.
The degree of competition between the three different T tracers and the
endogenous steroid at the binding site of the polyclonal rabbit anti-T
antibody was investigated in displacement experiments. Examining
the correlation between the difference values d and the log
of antibody concentrations, we determined that the optimal 7A dilution,
at which 50% of the [1
H]T is bound, was 1:200,
independent of the tracer concentrations. All three tracers displaced
the tritiated T (3.5 nmol/L) from the binding site of the antibody in a
competitive manner at different molar ratios: 3.0 nmol/L for
7-C3-Bio-T, 8.5 nmol/L for 7-C6-Bio-T,
and 17.0 nmol/L for 7-C11-Bio-T.
signal generation
For optimal signal generation in the chemiluminescence immunoassay
system with the 7-C11-Bio-T tracer, the best 7A
antibody dilution for the coated wells was found to be 1:600. For a
dynamic assay range of 0.2–20.0 nmol/L T, the C11
long-chain tracer was best in a 3.00 nmol/L (600 fmol/well) final
concentration.
The 7-C3-Bio-T tracer was not suitable for
establishing a chemiluminescence assay because of lack of binding to
the sAv-linked HRP. The impaired signal generation was such that no
decline of the %B values below 80% in the dose–response curve could
be recorded. The 7-C6-Bio-T tracer showed similar
characteristics. A direct comparison of the maximum light intensities
(T = 0 nmol/L) was performed for the C6- and
C11-tracers (1.5 and 3.0 nmol/L, respectively) with the
conditions for the 7-C11-Bio-T assay. Whereas 8000–8500
relative light units were measured for 7-C11-Bio-T, only
180–220 units were counted for 7-C6-Bio-T;
nevertheless, a complete dose–response curve from 100% to 25% B
could be calculated when measuring all T calibrators. By using
affinity-purified 7A antibody (6) for a
7-C6-Bio-T assay, binding to the coated microtiter plate
wells was improved, which thus improved the assay signal. The
performance data of that assay, however, were less satisfying than for
the 7-C11-Bio-T assay because of unfavorable
signal-to-noise characteristics (data not shown).
assay performance data
Figure 2
shows the calibration curve of the assay for known amounts of T
in assay buffer. Mean values ± 2 SD for 10 interassay
determinations of the calibrators are also given. The lowest T
concentration that was significantly different from zero was 6.5
fmol/well (equivalent to a serum concentration of 0.125
nmol/L).
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Figure 2. Dose–response curve of the T chemiluminescence
immunoassay.
Plotted are means ± 2 SD from measurements on separate days
(n = 10) of the calibrators.
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The measured T concentrations for control samples (I-III) are presented
in Table 1
. The chemiluminescence immunoassay measured T at 95% of the
values found by the MS method. The analytical recoveries of T added to
sera with low T content were 95–104% (Table 1
).
Intra- and interassay imprecisions, assessed from the SD of
multiplicates of various serum samples with low, medium, and high T
content (4 pool sera for intraassay and interassay imprecisions), are
summarized in Table 2
.
Figure 3
shows the precision–dose profile of various T values
determined in measurements on separate days in the lower range to
assess the minimum detection limit. At a CV of 20% the T value was 0.8
nmol/L. The functional sensitivity is ~6 times the lower detection
limit.
For 8 sera with different endogenous T contents, the linearities of
dilutions were determined (Fig. 4
). The data for 50% inhibition points and respective
cross-reactivities of a series of putative cross-reacting steroids are
presented in Table 3
for the 7A antibody and both of the RIA antibodies. Because of
the extraction step, no interferences were observed in hemolytic sera,
sera containing increased lipoproteins, or sera from patients with
either hyperbilirubinemia or monoclonal hypergammaglobulinemia (data
not shown).
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Figure 4. Linearity of the 7-C11-Bio-T assay,
determined by serial dilutions of 8 sera with high endogenous T
content.
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measurements of t serum concentrations in adults
The mean values, SD, and 95% ranges of T concentrations in 74
sera from healthy men and pre-/perimenopausal women are given in Table 4
.
intermethod comparisons
We compared the results for T concentrations by the
chemiluminescence assay in 170 sera with those by two
125I-RIA kits (from DPC and ICN). All linear
regression parameters, determined according to Passing and Bablok
(11), and Spearman correlation coefficients are given in
Table 5
for the whole concentration range up to 29 nmol/L, and for the
ranges 0.4–3.0 and 8.0–29 nmol/L. Fig. 5
(top) shows the paired values for DPC vs the
7-C11-Bio-T assay. In Fig. 5
(bottom) a Bland–Altman
plot (12) depicts the difference between both methods vs
the mean values.
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Figure 5. Intermethod comparison of the chemiluminescence
immunoassay with DPC RIA: (top) scatter diagram
(insert: an enlargement of the lower range of
concentrations); (bottom) Bland–Altman plot,
showing the mean ± 1.96 SD.
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Discussion
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The determination of T as the key androgen steroid in human serum
is of great importance in the endocrinological laboratory, especially
in the evaluation of hyperandrogenemic states in women. T is
circulating primarily in bound form, mostly to sex-hormone-binding
globulin (SHBG), only 1–2% being unbound
(13)(14). Hitherto nonradioactive T
immunoassays reported were neither entirely specific for this steroid
alone, nor as sensitive as RIA methods (15). However,
recent developments give fair promise that new nonisotopic assays will
have superior performance data for measuring T in serum. A recently
evaluated automated direct chemiluminescence immunoassay from Chiron
ACS:180 seems to be particularly attractive as a routine assay
(16)(17). By using a conventionally
synthesized T tracer (an acridinium ester labeled to position 3) and a
position 3-complementary rabbit anti-testosterone antibody for the
competitive assay format, this immunoassay is probably subject to
cross-reactivity with an unidentified steroid compound; this is seen
when comparing the ACS results with a GC-MS method (18).
Other novel automated T immunoassays have been developed by DPC
(Immulite system) and by Boehringer Mannheim (Elecsys system). External
evaluations of these assays have still to be described. Thus, the
versatile and reliable application of nonisotopic immunoassays in
various endocrine disease states is still in debate (19).
Identification of hyperandrogenemia in women, for example, requires
highly accurate and precise measurements of total or free
(non-SHBG-bound) T concentrations in serum (14).
We have developed and used "nearly native" tracers, which possess
an optimized ability to compete with analyte molecules, thanks to the
attachment point we used. As an alternative approach to the problem
inherent in use of the steroids' functional groups to attach reporter
groups to these molecules, we inserted a prosthetic biotin label in a
steroid ring core position furthest from the critical functionalized
positions. However, the characteristics of tracers of this nature are
also related to the chemical nature and length of the spacer arm. As
Bieniarz et al. (20) have shown, an extended length of
spacers between the reporter enzyme and antibody conjugates for
different protein analytes leads to a marked increase in signal
strength when increasing the length of the linker from 9 to 30 atoms.
In light of these data, we investigated different analogs of the
7-Bio-T tracers, using various spacer lengths to improve the signal
generation. The addition of a prefunctionalized Grignard compound at
the C-7 position is the key step of the tracer syntheses and offers the
possibility of inserting CH2-groups in various lengths to
6-dehydrotestosterone, which is advantageous for modeling different
immunochemical tracers.
The signal-generation step of the antibody-bound
7-Bio-T/sAv-coupled HRP system is affected by the length of the
linker, the lowest light intensities being achieved with
7-C3-Bio-T and the greatest with
7-C11-Bio-T. Steric hindrances possibly interfere with
the bipolar binding of the tracer to the antibody and to the sAv–HRP
when the spacer arm length is insufficient. Care must be taken that the
spacer not be too long, however, to avoid potential loss in antibody
binding through the diminished hydrophilic character of the tracer.
These considerations are supported by investigations of Tiefenauer and
Andres (21), who also discussed structural requirements of
biotinyl-estradiol derivatives for optimal antibody binding.
As expected, displacement experiments depicted that the 7-Bio-T
tracers displace tritiated T from the antibody in a competitive manner.
7-C3-Bio-T had the lowest displacement
concentration at an equimolar ratio to tritiated T. For the two
long-chain T tracers, the concentrations required to displace 50% of
the [1
H]T increased markedly with the linker length. This
may reflect the diminished hydrophilicity of the long-chain tracers.
Especially remarkable are the performance data of the assay concerning
the lower detection limit (0.125 nmol/L), the excellent dilution
linearity and recovery data, and the functional sensitivity (0.8
nmol/L). Suitability of the minimum detection limit for specimens with
T values in the female reference range can thus be assumed. The
accuracy of the assay with MS-defined control specimens
(7) reveals a well-acceptable 95% recovery.
Differences were observed in the 12 potentially cross-reacting
C19 steroids with regard to the antibodies of the
different assays (7A, ICN, DPC). Overall, the data for the 7A and the
DPC antibodies were similar, whereas greater cross-reactivities were
observed with the ICN antibody (Table 3
). This finding reflects the
influence of the different bridge positions on specificity during
antibody formation. The ICN assay uses a rabbit anti-T antibody
(immunogen T-19-(O-carboxymethyl)ether-BSA) and a T molecule
as tracer that is 125I-substituted at the C-19 position.
The DPC assay uses a coated rabbit anti-T antibody; additional
information concerning the immunogen and the 125I-T tracer
is not available, but we presume a position 3- or 7-immunogen and a
respective position analog tracer. In particular, 19-nor- and
17-methyl-T have quite similar low cross-reactivities with the 7A
and DPC antibodies, in contrast to a very high cross-reaction with the
ICN antibody. The cross-reactivity of androstenedione, present in
relevant concentrations in serum, was the only one that was greater for
the 7A antibody than for the other two antibodies; nevertheless, this
cross-reaction (1.6%) is still quite acceptable.
These characteristics allow the sensitive and reproducible
determination of T over a large dynamic range with the
chemiluminescence assay. Although the assay requires a serum extraction
step before measurement, its handling is easy and the reagents have a
long shelf-life. Results are available within 5 h.
Applying the 7-C11-Bio-T assay to measure T
concentrations in 74 sera from healthy adults gave results (Table 4
) in
accordance with reference values given in literature
[24], [25]. Intermethod comparisons with two
commercially available nonextraction RIAs yielded acceptable
correlations in the ranges 0–29 (all), 0–3.0 (female), and 8–29
nmol/L (male) T. Correlations with the DPC method are given in Table 5
and Fig. 5
(top). At low concentrations the DPC results are lower than
and less well correlated (r = 0.7119) with the
7-C11-Bio-T assay results—findings confirmed by the
Bland–Altman plot (Fig. 5
, bottom). With increasing T concentrations,
however, no relevant divergences between the two methods could be
found.
The performance characteristics of the
7-C11-Bio-T assay are similar to those of the two
RIAs, the latter showing comparable precision and recovery data (Table 6
). Accuracy measurements with MS-defined control materials
showed comparable results for DPC, whereas ICN had lower recoveries
(80%). The lower detection limit of the DPC was equal to that of the
chemiluminescence assay. In the DPC kit information, an intraassay
precision–dose profile presents a functional sensitivity of
~0.7 nmol/L. This is also comparable with the minimum detection limit
of the 7-C11-Bio-T assay.
In conclusion, the determination of total T in serum with the
7-C11-Bio-T tracer in an HRP-labeled,
ligand-binding chemiluminescence assay offers good sensitivity and
specificity and is easy to use. However, the imprecision still needs to
be improved. The superior performance data of the
7-C11-Bio-T assay demonstrate the importance of an
optimized steric structure and a long spacer arm of the biotinylated T
tracers. In addition, the 7-C11-Bio-T tracer provides
higher specificity through better availability of the critical antigen
sites, in accordance with suggestions by Fránek
(24).
|
Acknowledgments
|
|---|
We are grateful to L. Siekmann, Institut für Klinische
Biochemie, Universität Bonn, for providing us with reference
material, and M. Page for reading the manuscript. We thank also
the Fond der Deutschen Chemischen Industrie for a financial grant. C.B.
is supported by the Stiftung zur Förderung
Körperbehinderter Hochbegabter, Vaduz, Principality of
Liechtenstein.
|
Footnotes
|
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1 Nonstandard abbreviations: 7-C3-Bio-T, 17ß-hydroxyandrost-4-en-3-one-7-(biotinyl-6-N-propylamide); 7-C6-Bio-T, 17ß-hydroxyandrost-4-ene-7-(biotinyl-6-N-hexylamide); 7-C11-Bio-T, 17ß-hydroxyandrost-4-ene-7-(biotinyl-6-N-undecylamide); HRP, horseradish peroxidase; sAv, streptavidin; SHBG, sex-hormone-binding globulin; T, testosterone; BSA, bovine serum albumin; DPC, Diagnostic Products Corp.; MS, mass spectrometry 
|
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Abstract
Introduction
