1916 CLINICAL CHEMISTRY, Vol. 26, No. 13, 1980. ]1#{174}ID. Antiferritin Labeled with the. Bolton-Hunter Reagent. To the Editor: The Bolton. & Hunter.
]1#{174}ID 30,000
Antiferritin Labeled with the Bolton-Hunter Reagent
surement
‘a
To the Editor: The Bolton & Hunter reagent for protein iodination (1) has been used successfully to label the ligand (ferritin) in a radioimmunoassay of serum ferritin (2) but has not been tried in the labeling of antiferritin in immunoradiometric assays. We have labeled antiferritin with the Bolton & Hunter reagent (iodinated p-hydroxyphenylpropionic acid Nhydroxysuccinimide ester), and used it in the two-site immunoradiometric assay developed by Miles et al. (3). In addition, inclusion of an extra step in the assay procedure has improved the sensitivity
and
the
linearity
of
the
standard curves. We have used the assay procedure described by Miles et al. (3) with slight modifications in volume and with an extra step included: after the polystyrene tubes are coated overnight with 200 sL
of cold
antiferritin
and
washed,
they
are again incubated overnight with 400 sL of isotonic saline-phosphate buffer (0.1 mol/L, pH 7.4) containing 5 g of bovine serum albumin per liter, and washed before proceeding with the successive coating with standards (or unknowns) and labeled antiferritin. Our seven attempts to iodinate the antiferritin with the Bolton & Hunter reagent have all been successful. Iodine uptake has ranged from 25 to 35% of
added radioiodine,
and the infinite dose
response in the assay (up to 1000 zg of ferritin per liter) has been very reproducible in the seven iodinations, ranging from 46 to 56% of the labeled antiferritin added. The stability of the labeled antiferritins has been evaluated by the changes in the infinite dose response in the course of time. Figure 1 shows the findings in three of our preparations
100
Xavier
0
25 (.4g/
I
Fig. 2. Effect on the ferritin standard curve of an extra step in the method of
which show some differences in stability, i.e., T112 of about 40 days for preparation B and about 80 and 100 days for preparations A and C, respectively. The reasons for these differences are unknown to us but all three preparations gave adequate standard curves.
of an extra incubation
step with buffered bovine serum albumin (either 5, 10, or 20 g/L) usually increases the slope of the standard curves and tends to linearize the concentration/cpm curve over the tested range of 0 to 25 zg of ferritin standard per liter (correlation coefficients of 0.990 to 0.999 in 20 batches in which a straight line was calculated by the least-squares method). The latter finding contrasts with the logit-log response seen by Miles et al. (3), and with the nonlinear response seen by us when the extra incubation is
means
for
candidate. Capillary
blood
obtained
by finger-
prick from normal adults was applied as
munoassay
(which
would
then
bind
molecules.
from CONACYT
in part by grant
(Consejo
Nacional
de
de Mexico).
1. Bolton, A. E., and Hunter, W. M., The Iabelling of proteins of high specific radioactivity by conjugation to an ‘251-containing
Fig. 1. Stability of three antiferritins labeled with the Bolton & Hunter reagent, as
acylating munoassay.
evaluated by changes in infinite dose response
(1973).
1916
to be a practical
screening newborn infants for hypothyroidism (2, 3). Pang et al. (4) first applied the filter paper method for measuring a steroid, 17a-hydroxyprogesterone. The advantages in terms of sample size, handling, and transport provide a potent stimulus for broader applications of the method. The relatively high concentrations of cortisol in blood, as well as its clinical importance, make this steroid hormone a suitable
antiferritin
References
DAYS AFTER LARELING ANTIFERR’IN
shown
1-cm spots onto filter paper (Schleicher & Schuell, no. 903) identical to that used in screening for phenylketonuria and hypothyroidism. Venous blood specimens were simultaneously obtained. A paper disc, 6.4 mm in diameter, was punched out from the central region (5) of the air-dried blood spots with a hole puncher. 125J serum cortisol radioim-
Ciencia y Tecnologia
200
Heel-prick blood samples, collected on filter paper, are widely used nowadays for neonatal screening of phenylketonuria (1). Radioimmunoassay of thyroxine and thyrotropin in blood specimens on filter paper has also been
omitted (Figure 2), or when the bovine serum albumin concentration is decreased to 1 g/L. The effects observed do not appear to be the result of a decrease in nonspecific binding, because the zero dose counts are very similar with and without the extra step. We think it may be due to a “reorientation” of the cold
This work was supported
00
Radloimmunoassay of Cortisol in Blood Collected on Filter Paper To the Editor:
Miles et at. (3).
The inclusion
de Ia Nutrici#{243}n
50
FERRIrIM
O2-C
I-
Alvarez-Hern#{225}ndez Alvar Loria
Instituto Nacional Dept. of Hematology Mexico 22, D.F. 5
assay.
61, 209-224 (1974).
.‘
10,000
antiferritin
MI1 0 0,oi
by a 2-site immunoradiometric
/oRIeN:L
0
15, 102-108
3. Miles, L. E. M., Lipschitz, D. A., Bieber, C. P., and Cook, J. D., Measurement of serum Anal. Biochem.
/,,#{248}
EXTRA STEP\
cpm
by radioimmu-
ferritin
Ann. Clin. Riochem.
(1978).
ferritin
20,000
ferritin more easily) brought about by the presence of albumin molecules sticking to the tube walls in between the
z
of serum
noassay.
agent.
to the radioim-
Application
Biochem.
J.
2. Goldie, I). .J., and Thomas,
CLINICAL CHEMISTRY, Vol. 26, No. 13, 1980
133, 529-539 M. .J., Mea.
kits,
kindly
donated
by
Clinical Assays, Cambrigde, MA 02139, were used to estimate cortisol in the paper disc and in the corresponding venous serum. The procedure involves the following steps: addition of phosphate-buffered isotonic saline + 1251 labeled cortisol and serum (or standard) to antibody-coated tubes, and 45-mm incubation at 37 #{176}C, followed by decantation and counting. It was modified as
follows
to
measure
cortisol
in
the
paper discs. Buffer and tracer were added to the antibody-coated tubes containing the paper disc, including the tubes with cortisol standards (0, 100, 300, 1000, 2500, and 6000 pg) to which a blank non-spotted paper disc was
