I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and Its antibody, In serum.
CLIN. CHEM. 24/12, 2139-2144 (1978)
Assayof an AntitumorProtein,Neocarzinostatin,and ItsAntibody by FluorescencePolarization Hlroshi Maeda
I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and Its antibody, In serum. The antigen (neocarzinostatin), labeled with fluorescein isothiocyanate, was allowed to interact with its antibody in a cuvet, in the instrument, yielding an increase in the fluorescence polarization value. Antibody content was determined in the presence of a definite amount of the labeled antigen, fluorescence polarization values increasing in parallel with each addition of antibody. Antigen content was determined with a known amount of antibody, which reacted at first with an unknown amount of antigen in samples, followed by addition of a definite amount of the labeled antigen (competition). I used the method to determine a pharmacokinetic parameter, the apparent volume of distribution for neocarzinostatin in rabbits, using drug-injected rabbit sera. I evaluated precision, accuracy, and reproducibility, using various samples or possible interfering substances such as bilirubin and hemoglobin, and also compared resuIts for antigen with those by single radial immunodiffusion
assay. The present assay is fast (50
30
-
2.6
Labeled neocarzlnostatin with its antibody(30X dii.antiserum,50 1ziin 2 ml) Effect of normal serum, 50 ol in2 ml Changes after 10 times freezing-thawing in PBS, pH 7.0,for 2 months (-20 #{176}C/room temp)a Effectof hemoglobin (80 tI of dilutedhemolytic serum in 2 ml buffer)
204.0
245.5
202.8
258.9
41.5
-
56.1 ± 6.73
200 202.8
203.5
0
Effectof lipidemicserum (50 jslof patient sera in 2 ml buffer) with 50 l antibody(see above second row)
204.5
332.2 ±
127.5 ±
Effect of icteric serum (20 I of patient sera with liver cirrhosis or hepatitis In 2 mi buffer) with 50 ol antibody (see above)
204.5
0.78
>80
-
2.37
27
-
0.65
10
0.93
10
40.0
plus 50 ol antibody 27.3 375 ±
18.1
36.7
13.6
12
37.3
6.6
6
27 169 ±
18.5
Measurements were carrIed out In 0.1 mol/Ilter borate or 0.1 mol/llter TrIs.HCI buffer, pH 8.0, at 20 #{176}C unless otherwise Indicated. P value Is an arbItrary unit. Indicates an Increased P value due to serum viscosity.’ Indicates an IncreasedP value due to antigen-antibodyreaction (LPag., = Pag.atPwurn). Effects of repeated freezing and thawing or storage at 4 #{176}C for a long period varies from proteIn to protein. Neocarzlnostatln and ribonuclease are proteins less prone to such effect; lgG and anglotensin are susceptible.
between results by the two methods (r > 0.90). The modified single radial immunodiffusion was found also to be very sensitive and reproducible, and in fact the two methods were almost equivalent. The minimum and maximum amount o use
of the error in the P value (Table 1), but this error is usually
in both methods
blocked, it must be an effect of sample viscosity (see Table 1). Yellow bilirubin or red hemoglobin did not interfere with the measurement, owing to an efficient cut-off filter; however, unusually high serum viscosities perhaps caused by increased fibrinolytic activity in patient sera, were observed frequently. Precision,accuracy, and reproducibility of the instrument was
were similar
sion assay, however, required less sensitive when gradual degeneration (see Discussion).
(about
0.3 g total).
The diffu-
more than 20 h, and it became
antigen was present in serum, of the antigen with proteolytic
owing to enzymes
DIscussion The results
show that antibody
or antigen
tolerable
when
diluted
sera are used.
Subtraction
of back-
ground viscosity may be useful, or use of unlabeled antigen to block pseudo-antibody for false-positive cases-if not
can be readily
determined by the fluorescence polarization method based on the changes in the apparent relative molecular mass of the
complex formed. An advantage of the present method is its extreme rapidity. For instance, the single radial immunodiffusion assay takes at least 18 h, while the present method requires
only 82 s. Furthermore,
it requires
no separation
pro-
cedures such as are used in radioimmunoassay for determination of a single component in a mixture. In addition, the accuracy of the measurement can be seen in Figures 1 through 4 and Table 1, even in the picomole range in 1 ml. The result depicted in Figure 1 (neocarzinostatin-antineocarzinostatin system) showed a linear and large increase in the P value, up to about 50%, when antibody IgG concentrationwas less than 15 mg/liter. This indicatesthat the increase in relative molecular mass is due to formation of antigen-antibody (IgG) complex. Although undescribed in this report, FITC-neocarzinostatin and its antibody complex, on use of 50 l of antiserum, resulted in a P value equivalent about 2 X 10 daltons (as high as 530) (19, 29).
The results depicted
labeledantigen
in Figures 2 and 3 show that any unbe so assayed when an antibody and
on the viscosity of the solution being tested is the major cause CLINICALCHEMISTRY,Vol. 24,No. 12,1978
I
0.4
!
0.2
‘I ‘I’
to
could antigen are available. This application has been previously reported to be quite useful, not only by Dandliker et al. (10, 11, 14), but also by other groups for insulin (13), the gentamicin (21), and phenytoin (23). The influence of serum itself
2142
0.6
0 0
0.2
0.4
0.6
Fluorescence polarizMion (
Fig. 4. Comparison of antigen determinationby fluorescen#{243}e
polarization and by single radial immunodiffusion assay The antIgen (neocarzlnostatln) was dissolved In PBS, and Is shown Inmlcrogams (total amount) used
satisfactory. Variations within run and between runs are very small, and reproducibility after one year is satisfactory. Day-to-day variation is negligibly small. However, the temperature must be kept constant. The polarizer accumulated considerable dust on its surface during a year of operation, but still there were no serious errors in the measurements. The dust eventually will interfere with polarizability and the sensitivity will decline. Colored substances in serum such as hemoglobin, bilirubin, and lipid particles appear to have very little effect
when Pserum
Pharmacokinetic
is subtracted
application
ocarzinostatin-injected application is indeed
(Table
1).
with use of plasma from ne-
rabbits showed that this practical feasible with this method (Figure 3).
Results by the present method were comparable to those
for antigen
determination
in sensitivity
by single radial
immuno-
diffusion (Figure 4). However, the diffusion assay requires a longer time. When neocarzinostatin in normal human sera was subjected to the diffusion assay, sensitivity declined and more than fivefold as much sample had to be used. This is explained by proteolytic degradation of neocarzinostatin, accompanied by the loss of antigenicity, during incubation (6, 21, 22).
Therefore, fluorescence polarization has an advantage of rapidity of determination, especially when the substance being measured is inactivated by the serum. The advantage of using fluorescein as the fluorochrome is that the labeled compounds (or antigens) retain their original antigenicity (13, 19, 20, 23-26), perhaps because the fluo-
rescein is primarily conjugated at N-terminal amino groups (17) which may be remote from the antigenic determinant (antibody-combining site). Furthermore, fluorescein has a very high quantum efficiency and its spectral properties differ from those of proteins or substances contained in the clinical samples, thus minimizing noise levels. In the present instrument, no grating monochrometer was used, which is known to cause depolarization (27,28), thus no correction factor is necessary for I in equation 1. Instead, a high-quality filter (three cavities) system was used, together
with a high-performance photomultiplier and a microcomputer. These improvements made the total operation very simple ic.
and the calculation
Although differences
of P value
completely
data are not shown, different in the relative
molecular
automat-
P values reflect
mass (or brownian
motion
of the labeled molecules) (29); therefore the fluorescence polarization method can be applied not only to antigen-antibody reactions, but also to many other systems, such as proteolysis (29), protein/nucleic acid, enzyme/inhibitor (13), enzyme! cofactor, enzyme/substrate, hormone-binding protein, and hormone receptors (30). Some assays now utilizing radioisotopes could be replaced by the present method. In addition, this method
is quite
of hydrolytic
powerful
processes
above advantages
for
for the kinetic macromolecules
(velocity)
study
because
of the
(29).
7. Maeda, H., Aikawa, S., and Yamashita, A., Subcellular fate of protein antibiotic neocarzinostatin in culture of a lymphoid cell line from Burkitt’s lymphoma. Cancer Res. 35, 545 (1975). 8. Perrin, M. F., Polarization de Ia lumiere de fluorescence. Vie myenne des molecules dans l’etat excite. J. Phys. Radium 7, 390 (1926).
9. Weber, G., Rotational brownian motion and polarization of the fluorescence of solution. Adu. Protein Chem. 8,415(1953). 10. Dandliker, W. B., and Feigen, G. A., Quantification of the antigen antibody reaction by the polarization of fluorescence. Biochem. Biophys. Res. Commun. 5,299 (1961). 11. Dandliker, W. B., Schapiro, H. C., Meduski, J. W., et al., Application of fluorescence polarization to the antigen-antibody Theory and experimental method. Immunochemistry (1964).
12. Haber, E., and Bennett, measure of antigen-antibody 48, 1935 (1962).
J. C., Polarization
interaction.
reaction. 1, 165
of fluorescence
as a
Proc. Nat!. Acad. Sci. USA
13. Spencer, R. D., Toledo, F. B., Williams, B. T., and Yoss, N. L., Design, construction, and two applications for an automated flow-cell polarization fluorometer with digital readout: Enzyme-inhibitor
(antitrypsin) assay and antigen-antibody (insulin-insulin antiserum) assay. Clin. Chem. 19, 838 (1973). 14. Dandliker, W. B., Investigation of immunochemical reactions by fluorescence polarization. In immunochemistry of Proteins, 1, M. Z. Atassi, Ed., Plenum Press, New York, N.Y., 1977, pp 231-261. 15. Mancini, G., Carbonara, A. 0., and Heremans, J. F., Immunochemical quantitation of antigens by single radial immunodiffusion. immunochemistry
16. Peterson, I. Cellulose (1956). 17. Maeda,
2,235
(1965).
E. A., and Sober, H. A., Chromatography ion-exchange adsorbents. J. Am. Chem. H., Ishida,
of fluorescein
N., Kawauchi,
isothiocyanate
valent and non-covalent fluorescein to proteins.
H.,
and Tsuzimura, K., Reaction
with proteins
binding
and amino acids. 1. Co-
of fluorescein
J. Biochem.
of proteins. Soc. 78, 751
(Tokyo)
isothiocyanate
and
65, 777 (1969).
18. Fingl, E., and Woodburg, D. M., General principles. I. Pharmacokinetics. In The Pharmacological Basis of Therapeutics, L. S. Goodman and A. Gilman, Eds., Macmillan, New York, N.Y., 1975, pp 1-45. 19. Maeda, H., Application of fluorescence polarization technique in biology and medicine: Determination of antigen-antibody reaction and assay of proteolytic enzymes. Igaku no Ayomi 106, 773 (1978).
In Japanese.
I thank Messrs. Adachi and Mizuta, of Japan Immunoresearch Co. Ltd., Takasaki, Japan, for the supply of the antibody used and loan of a polarization spectrofluorophotometer. I am also grateful to Professor Yorio Hinuma, of this University, for his helpful suggestions and encouragement, and to Dr. C. B. Glaser (Institute for Medical Sciences, San Francisco, Calif.) for help with the manuscript.
References 1. Ishida, N., Miyazaki, K., Kumagai, K., and Rikimaru, M., Neocarzinostatin, an antitumor antibiotic of high molecular weight. Isolation, physicochemical properties, and biological activity. J. Ant ibiot. 18A, 68 (1965). 2. Maeda, H., Glaser, C. B., Kuromizu, K., and Meienhofer, J., Structure of the antitumor protein quence. Arch. Biochern. Biophys.
3. Sakamoto, S., Ogata, J., Ikegami, K., and Maeda, H., Effect of systemic administration of neocarzinostatin, a new protein antibiotic, on human bladder cancer. Cancer Treat. Rep. 62, 453 (1978). 4. Maeda, H., Yamamoto, N., and Yamashita, A., Fate and distribution of 1’4Clsuccinyl neocarzinostatin in rats. Eur. J. Cancer 12, 869 (1976). 5. Maeda, H., Sakamoto, S., and Ogata, J., Mechanism of accumulation of the antitumor protein antibiotic neocarzinostatin in bladder tissue: Intravenous administration, urinary excretion, and absorption into the bladder tissue. Antimicrob. Agents Chemother. 11, 941 (1977). 6. Maeda, H., and Takeshita, J., Degradation of neocarzinostatin by blood sera in vitro and its inhibition by diisopropyl fluorophosphate and N-ethylmaleimide. Gann 66, 523 (1975).
neocarzinostatin. 164,379(1974).
Amino
acid se-
20. Maeda, quantitation anese.
H., Application of antigen
of fluorescence
or antibody.
Biomed.
polarization
for the
J. 2, 11(1978).
In Jap-
21. Maeda, H., and Takeshita, J., Inhibitor of proteolytic enzymes prevents the inactivation by blood of neocarzinostatin and its succinyl derivative. J. Antibiot. 29,111(1976).
22. Maeda, H., Yamamoto, N., and Yamashita, A., Fate and distribution of [‘4C)succinyl neocarzinostatin in rats. Eur. J. Cancer 12, 865 (1976). 23. Watson, R. A. A., Landon, larization fluoroimmunoassay 51(1965).
J., Shaw, E. J., and Smith, D. S., Poof gentamicin. Clin. Chim. Acta 72,
24. McGregor, A. R., Crookall-Greening, D. S., Polarization
fluoroimmunoassay
J. 0., Landon, J., and Smith, Clin. Chim. Acta
of phenytoin.
83, 161 (1978). 25. Bromer, W. W., Sheehan, S. K., Berns, A. W., and Arguilla, E. R.,
CLINICALCHEMISTRY,Vol. 24, No. 12, 1978 2143
Preparation
and
properties
of fluorescein-thiocarbamyl
insulin.
Biochemistry 6, 2378 (1967). 26. Kierszenbaum, P., Dandliker, J., and Dandliker, W. B., Investigation of the antigen-antibody reaction by fluorescence polarization. Measurement of the effect of the fluorescent label upon the bovine serum albumin (BSA) anti-BSA equilibrium. Immunochemistry 6, 125 (1969).
27. Stewart, J. E., and Gallaway, W. S., Diffraction anomalities in grating spectrophotometers. App!. Optics 1,421(1962).
2144
CLINICAL CHEMISTRY, Vol.24,No. 12,1978
28. Chen,
R. F., and Bowman, R. L., Fluorescence polarization: ultraviolet-polarizing filters in a spectrophoto-
Measurement with
fluorometer. Science 147, 729 (1965). 29. Maeda, H., Assay of proteolytic enzymes by fluorescence ization. Anal. Biochem., in press (1979). 30. Levinson, S. A., Dandliker,
W. B., Grawn, R. J., and Vanderlan,
W. P., Fluorescence polarization measurement binding site interaction.
polar-
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of the hormone
99, 1129 (1976).
