a second patient with monocytic leuke- mia whose serum lysozyme had the same mobility as the gamma globulin fraction, which in this case was polyclonal. (3).
described
as we
in Clin.
23, 754
Chem.
6-phosphate
EC 1.1.1.49
dehydrogenase,
(Reagent
B).
(1977). The latter procedure enables one to determine the true protein content. In all cases, the absorbance was read after
calibrators,
30 mm and protein content was calculated from the absorbance of standard
trols are plasma with amounts of theophylline:
sera treated
mg/liter.
similarly.
following results: (our a), and 69.3 the mean values
both
reagents,
We obtained
The
the
directly
used,
gave
practically the same but falsely high results. However, when the absorbance was read within 1-5 mm after mixing the respective reagent with lipemic serum, a result nearly corresponding to the true
protein
content
of two components: product and the velocity of the
is apparently
greater
the colored turbidity. The biuret reaction
then
the increase
detailed
results
Filter
Faculty
Hospital,
Brno
J. Fischer Research
Institute
Lachema, N.C., Czechoslovakia
of Pure
Select
paper
provided
The
Measurement of theophyllmne in plasma is clinically useful in evaluating patient dose-response and toxicity (1).
was
determined ultraviolet
by
a
method
specimens The
and
the
scaling
The
reagents
514
CLINICAL
(Reagent coupled
CHEMISTRY,
theophylline A), and to glucose-
Vol.
24,
was
4.47%
Satisfactory
0.5
was 15
ml
better
other
by re3 to 1
reagents
results
of sample, with
the
were
but 1-mi
patients’ samples (3.015.0 mg/liter) the r was 0.976. We conclude that the EMIT system
measuring
are EMIT
down
with
rapid,
antibody/substrate EMIT theophylline
the
(20 mg/liter). method (2) was
for pediatric samples sample volume from
adapted factory,
to
of
by assaying amounts of to 10 and 20
run)
ultraviolet
reagents are reconstituted kit instructions except
below.
2.5 the ab-
and 4.67%
accordingly.
obtained
modifi-
and
of at
graph
precision
(within-day
(10 mg/liter)
For
noted
a A
log-log
determined weighed-in
sample.
cations
yields
the kit.
and
was with
sensitivity
according
series
the absorbance
on the
with
accuracy
(2). Theophylline may be determined with the EMIT reagents after a single dilutipn of samples and standards. All for the
K. M., and Ogilvie, R. I., Dosage in bronchial asthma. N. Engi. 292,1218(1975).
J. Med.
2. Jatlow, P., Ultraviolet spectrophotometry of theophylline in plasma in the presence of barbiturates. Clin. Chem. 21, 1518 (1975).
Veronica Henry Jack Deutsch Gifford Lum Clinical
Chemistry
Department
Laboratory
of Pathology
State University Hospital Downstate Medical Center Brooklyn, N. Y. 11203
Binding on the Lysozyme
The electrophoretic mobility of urinary lysozyme has been thoroughly characterized, particularly in patients with monocytic and myelomonocytic leukemia (1), but references to the mobility of lysozyme (EC 3.2.1.17) in serum are scarce. In 1973, Finkle et al. (2) found complexes of a monoclonal IgG with lysozyme in the serum of a patient with acute myelomonocytic leukemia, and they presented evidence that the enzyme and the monoclonal protein had the same mobility. In 1975 we described
a second patient with monocytic leukemia whose serum lysozyme had the same mobility as the gamma globulin fraction, which in this case was polyclonal (3). At that time it appeared that serum lysozyme had a strong tendency to form
nocytic
ml,
of an
1. Piafsky,
of theophylline
sample and results obtained respectively for A - Ao for 15 specimens was 0.988 and 0.977, respectively. The CV for 15
bide,
modification
time
complexes with the gamma globulins. In three subsequent cases, however, one of monocytic leukemia, one of myelomo-
modified ducing
comparison,
a larger
theophylline corresponding mg/liter. The coefficient of correlation (r) between the 10 and 20 mg/liter
We report here a rapid, micro procedure for measuring theophylline in plasma or serum with the EMIT reagent system (Syva, Palo Alto, Calif. 94304) and a centrifugal analyzer (CentrifiChem, Models 300 and 400; Union CarFor
(A0) from
a curve
method plasma
N. Y. 10580).
that
(300
-
Method
theophylline
value
construct
To the Editor:
Rye,
= ABS Operate
=
value of standard, sample, and calibrator. The final value (A A0) is used to
Enzyme Immunoassay of Theophylline with a Centrifugal Analyzer, and Comparison with an
requires
more technician extraction steps.
Effect of Antibody Mobility of Serum
of 65 to 135 units between the 2.5 40.0 mg/liter calibrator, and a .A least 20 units between the 0 and mg/liter calibrator. Before plotting data, subtract the zero-standard
Chemicals
621 33 Brno,
method
To the Editor:
the print-out
sorbance
Ultraviolet
Terminal
=
Auto
=
Calib/operate only) T= 15s
Biochemistry
65691
as follows:
Print = 9, all Temp. = 30#{176}C Absorbance/Conc
V. Chrom J. Vozn#{233}ek
ultraviolet
sample volume, (1-2 h), and two
nm
Auto/Store T0 = 30s
will be published
of Clinical
340
=
Terminal/Rate
elsewhere.
Department
and
References
Set the analyzer
in turbidity. The clearing effect attributed to the modified biuret reagent is mostly caused by the time factor only.
Our
theophylline the conweighed-in 10, 20, 30
mg/liter,
Ml for sample, 50 Ml for sample and diluent, and 350 Ml for reagent. Start the pipettor. The last sample plug is used. (Note: When using CentrifiChem 400, leave position 1 empty.) Manually pipet 20 Ml of Reagent A carefully into the sample section of the transfer disc.
was found.
The absorbance of the reaction mixture containing lipemic serum is the resultant biuret reaction
are EMIT
0-40
The procedure is as follows: Pipet 100 Ml of sample, calibrator, or control into a 12 X 75 mm test tube, and add 200 Ml of EMIT buffer [tris(hydroxymethyl)methylamine, 55 mmol/liter, pH 7.9]. Prepare the substrate by pipetting 0.6 ml of Reagent B into 10 ml of EMIT buffer. For the pipettor, dial in 10
94.0 (Yatzidis), 92.2 (our b), all expressed as in g/liter. Evidently
when
standards
method is excellent, and no extraction procedure is necessary. In contrast, the
to the
CentrifiChem
micro-scale
is a satis-
procedure
for
theophylline in plasma. It requires only 100 Ml of sample and about 20 mm of technician time. The correlation with an established ultraviolet
No. 3, 1978
leukemia, and one of myeloma, no evidence of IgG-lysozyme complexes, and in these cases the mobility of serum lysozyme was identical to we found
that
of free urinary lysozyme. In an attempt to determine whether soluble complexes of lysozyme and anti-lysozyme
antibody
could
account
for the shift of serum lysozyme to the gamma globulin region, as seen by Finkle et al. (2) and in our earlier case (3),
we added sozyme a sheep
500
g
of purified
human
ly-
to 1 ml of serum obtained from hyperimmunized with human lysozyme. Details of the preparation of this antiserum were reported previously in this journal (4). An identical mixture was prepared with the serum from a sheep immunized with human IgG. Both mixtures were incubated for 1 hat 37#{176}C and then separated by cellulose acetate
