Assay of an AntitumorProtein,Neocarzinostatin,and ... - Semantic Scholar

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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-

Endocrinology

of the hormone

99, 1129 (1976).