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Sheets obtained by radiation polymerization for enzyme immunoassay
hr. J. dppl. Radix
Isor. Vol. 35. ?;o. 6. pp. 471-471.
0020-708x:84
1981
Copyright
Printed m Great Britain. AU rights resewed
33.00 + 0.00
,s 1981 Pergamon Press Ltd
Sheets Obtained by Radiation Polymerization for Enzyme Immunoassay MINORU Takasaki Radiation
Chemistry
KUMAKURA
and ISA0 KAETSU
Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma, Japan
(Receiced 15
April
1983; in recisedform 8 August 1983)
Immobilized anti-3-fctoprotein sheets, which were attached to sticks, for enzyme immunoassay of z-fetoprotein were prepared by radiation polymerization of hydrophilic monomers. The relationship between the preparation conditions and the activity of the sheets was studied. The activity varied with monomer concentration, hydrophilicity of polymer matrix, the amount of coating solution, and antibody concentration. The sheets obtained at relatively low monomer and antibody concentrations appeared to give a high activity. It was found that the sheets are applicable for the enzyme immunoassay of r-fetoprotein with high sensitivity.
Materials and Methods
Introduction At present there are two widely accepted assays of enzyme and radioimmunoassay that employ labeled antibodies and antigens. Enzyme immunoassay employs antibodies or antigens conjugated to enzymes in such a way that the immunological and enzyme activity of each moiety is maintained. This assay gives objective results and is extremely sensitive, though it is not usually quite as precise as radioimmunoassay. The range of application of enzyme-immunoassay is potentially as wide as that of radioimmunoassay and it may also reinforce or replace other serological tests. Various kits containing immunoreagents have been studied for enzyme- and radioimmunoassay.(‘b’ AS immunoreagent, beads, plates, and tubes have been used, in which antibodies were coated or bound on their surfaces. The immunoreagents resulting from physical coating method have a weak point such as leakage of antibodies, and those resulting from the chemical covalent method require necessary longer preparation times and excess amounts of antibodies, though the leakage does not occur. To increase the surface area of immunoreagents, for example, the glass beads have been frosted and antibodies have been immobilized. These problems on the preparations of immunoreagents led us to study the preparation of immunoreagents by the radiation polymerization technique. In this work, sheets consisting of paper and polymer matrices for enzyme immunoassay were prepared by radiation polymerization and its characteristic was studied, in which anti-z-fetoprotein was used as antibody. The assay of z-fetoprotein has been noticed as a diagnostic test for cancer patients.“.J)
Materials
Anti-sera to z-fetoprotein (anti-AFP) were produced in rabbits by injecting an emulsion of human AFP with an equal volume of complete Freund’s adjuvant. Human AFP obtained from the plasma of a patient with primary hepatoma, then purified by afinity chromatography on anti-AFP Sepharose 4B column (Pharmacia Japan Co., Ltd), foliowed by gel-filtration on Sephacryl S-300 column (Pharmacia Japan Co., Ltd). To prepare peroxidase-labeled antiAFP, we purified anti-AFP serum by ammonium sulfate fractionation, followed by the ion-exchange chromatography on diethylaminoethyl then coupled the cellulose, purified IgG fraction from anti-AFP sera with peroxidase according to the method of Wilson and Nakane.“) Standard human AFP was obtained from Dinabott Radioisotope Lab., Ltd. The buffer solution was phosphate-buffered saline (PBS), 10 mmol/L, pH 7.2. Hydroxyethyl methacrylate (HEMA). hydroxyethyl acrylate (HEA), hydroxypropyl methacrylate (HPMA), and methoxytetraethyleneglycol dimethacrylate (M-4G) were used as monomer for immobilization. Thin papers (10 x 5 mm, 8 pm in thickness; Efuron # 100 No. 3) used as a base material of the sheets were obtained from Fukuko Paper Mill Co., Ltd. Preparation
of immobilized
anti-AFP
sheets
Anti-AFP serum dissolved in the PBS solution was mixed with monomer and it was coated onto the sheet (coat area; 5 x 5 mm) using a micro-syringe, in which the edge (5 mm wide) of the sheet was attached to the 471
472
MINORUKWAKURA and
Monomer
1%)
Fig. 1. Effect of HEA monomer concentration. stick (5 x 100 x 0.5 mm) made of polyvinyl chloride which had been heated for handling in the assay. These coated sheets were put into a vessel, frozen at -78°C and irradiated with 1 Mrad in N, gas atmosphere by y-rays from a @‘Co source. After irradiation, immobilized anti-AFP sheets obtained by radiation polymerjzation at low temperatures were dried at room temperature. Enzyme-immunoassay One immobilized anti-AFP sheet was placed in a test tube containing 100 PL of sample or the standard AFP solution, and incubated at room temperature for 1 h. After incubation, the solution was removed and the immobilized anti-AFP sheet was washed with the PBS solution three times. Then 5OpL of peroxidase-labeled anti-AFP and 100 ,uL of the PBS solution were added to the tube and incubated at 37°C for 1 h. Again, the immobilized anti-AFP sheet was washed with the PBS solution three times to remove the unreacted peroxidase-labeled anti-AFP. The enzyme reaction was carried out with 200 p L of a solution of H202 (0.3 g/L) and o-phenylenediamine (3 g/L) at room temperature for 30 min and terminated by adding 1 N HCl solution, 1 mol/L. After the reaction, we measured the adsorbance of the solution at 492 nm with a spectrophotometer, to detect the peroxidase activity.
ISAO KAETSU
crease of the activity at high monomer concentrations is caused by the entrapment of anti-AFP into the polymer matrix. Since the mechanism of the immobilization of anti-AFP in the present method is a physical trapping by radiation polymerization, the trapping capacity is related to the amount and surface area of the polymer matrix on the paper. As monomer concentration increases, the amount (quantity of polymer per unit area) of the polymer matrix is increased and it leads to the decrease of the surface area of the sheet, in which anti-AFP would be entrapped, and disappear into the polymer matrix. On the other hand, in the immobilization with lower monomer concentrations, anti-AFP becomes attached to the surface of cellulose fibril in the paper with the polymer matrix. The polymer matrix played the role of adhesion agent for anti-AFP and cellulose fibril, so that a relatively low monomer concentration was recommended. However, the sheets from low monomer concentrations below 5% cause anti-AFP to leak due to a weak trapping; the leakage of anti-AFP in 3% monomer concentration was about 7%. Therefore, optimum monomer concentration appeared to be 10-20x. Effect of the amount of coating solution on activity Effect of the amount of the coating solution containing HEA monomer, anti-AFP, and the buffer solution on the activity was examined. The relationship between absorbance and the amount of the coating solution is shown in Fig. 2. The activity increased with increasing the amount of the coating solution which was clearly due to the increase in the quantity of anti-AFP. From these results, it was found that the sensitivity of the sheets increased with an increase in the amount of coating solution. Thus, the improvement of the sensitivity was easily compared to other immobilization methods with a chem-
20 r
Results and Discussion Effect of monomer concentration on activity The preparation method of immobilized anti-AFP sheets was based on the radiation polymerization of the monomer solution coated on the sheets, so that it is expected that the activity of the sheets is related to the polymerization condition. Here the effect of monomer concentration on the activity of the sheets was examined. Figure 1 shows the relationship between absorbance and HEA monomer concentration. The absorbance decreased with increasing monomer concentration, indicating that the activity is markedly dependent on the monomer concentration. The de-
L 0
I
2 ArnOU”,
3
c
(&I
Fig. 2. Effect of the amount of the coating solution. monomer concentration: 20%.
HEA
473
Obtaining sheets for enzyme immunoassay
Fig. 3. Effect of the hydrophilicity of monomers. Monomer concentration: ZOO/,.
ical covalent. This was one of the characteristics of the present method. The polymer matrix of the sheets obtained by radiation polymerization of hydrophilic HEA monomer at low temperature had a porous structure, in which anti-AFP was trapped on the surface of the polymer matrix with cellulose fibrils. The formation of the porous structure in the polymer matrix resulted from the ice in the matrix system melting at room temperature, after radiation polymerization at a low temperature. Such a porous structure led to a further increase in the surface area of the sheets, though not in the sheets consisting of the network structure with cellulose fibrils. Immobilization of anti-AFP with other monomers Anti-AFP was immobilized on the sheets with other slightly hydrophobic monomers such as HEMA and HPMA in comparison with the case of HEA monomer. The resultant absorbance in the sheets from HEMA and HPMA is shown in Fig. 3 together with that in HEA monomer as a function of the water content of the polymers. The activity of the sheets appeared to be related to the water content of the polymer matrix, giving a maximum activity at 26% water content (polymer of HEMA monomer). From this result, it is suggested that the activity of the sheets decreased by a decrease or increase in the water content. Thus, a monomer such as HEMA appeared to be of optimum hydrophilicity for the immobilization of anti-AFP. The molecule of y-globulin such as anti-AFP has Fab and F, chains and as recently reported the adsorption of plasma proteins such as y-globulin on polymer might be related to the hydrophilicity of polymer.(‘“.“’ Okano et al. have reported that the F, chain of y-globulin is selectively adsorbed on relatively hydrophobic sites.“*’ In the sheets obtained with the present method, it is reasonable that the F, chain of anti-AFP is trapped on the polymer matrix and the Fab chains of anti-AFP are free because the Fab chains are reacted with antigen (AFP) to form an antigen-antibody complex by a sandwich reaction. It was proposed that anti-AFP was trapped on the polymer matrix, taking such a conformation state, because the activity resulting from the sandwich reaction was very high.
Fig. 4. Effect of the dilution of anti-AFP. HEA monomer concentration: 20%. 0-l PL coating solution, O-2 PL coating solution.
Eflect of the dilution of anti-AFP on actbity The effect of the dilution of anti-AFP serum on the activity of the sheets was studied. The various concentrations of anti-AFP serum diluted with normal rabbit serum were used for the immobilization. The relationship between absorbance and dilution ratio is shown in Fig. 4. As dilution ratio increased, the absorbance in 1 PL coating solution decreased moderately but that in 2pL coating solution decreased markedly. This result indicated that the activity of the sheets obtained with 1 PL coating solution varies slightly with anti-AFP concentration within concentrations studied in this work. In Fig. 4, the absorb ante in 1 and 2 PL coating solution at high dilution ratios (l/10) gave almost the same value, though those at nondilution gave a 2-fold difference. This suggests that anti-AFP trapped in the sheets obtained from thin coating solutions with low anti-AFP concentrations is located on its surface, taking the conformation of the F, chain trapping. As can be seen in Fig. 4, the activity did not decrease according to the
.I t f 0.
./ 100 !
200 I
300 1
Fig. 5. Standard curve. HEA monomer concentration: 20%.
474
MINORU KIJMAKURAand
dilution ratio of anti-AFP, indicating that the excess amounts of anti-AFP in the sheets do not contribute to the sandwich reaction and they might be entrapped in the polymer matrix. Thus, it was reasonable to assume that anti-AFP serum, diluted to a certain concentration, is employed in immobilization. Assay characteristics
This typical standard curve in the enzymeimmunoassay with the sheets is shown in Fig, 5. The slope of the curve was linear over the range of 0-300ng/mL. Since the concentration of AFP in normal human serum is about 20 ng/mL, it is found that the sheets obtained by the present method was applicable for the enzyme-immunoassay of AFP. The minimum detectable concentration of AFP was 2 ng/mL, and the assay precision was 44%. This sensitivity of the present method using the sheets was higher than that of other enzyme-linked immunosorbent assay using CNBr-activated cellulose (5 ng/mL),“’ and was the same range as that described bv Belaneer et a/.f’3)
ISAOKAETSL’
References 1. Wisdow G. B. Clin. Chem. 22, 1213 (1976). 2. Scharpe S. L., Cooreman W. M. and Blomme W. J.
Clin. Chem. 25, 733 (1979). 3. Woo J. and Cannon D. C. Am. J. Clin. Pathol. 66, 854 (1976). 4. Voller A., Bidwell D. E. and Bartlett A. Bull. WHO 53, 55 (1976). 5. Belanger L., Hamel D.. Dufour D. and Pouliot M. Clin. Chem. 22. 198 (1976). 6. Maiolini R.. Ferrua B. and LMasseyeff R. J. Immunol. Methods 6, 3% (1975). 7. IMaiolini R. and Masseyeff R. J. fmmunol. Merho& 8, 223 (1975). 8. Belanger L., Syivestre C. and Dufottr D. Clin. Chem. Acta 48, 15 (1973). 9. Wilson M. B. and Nakane P. K. tmmunofluorescence and Related Staining Techniques (Ed. Knapp W.) p. 215 (Elsevier/Nonh-Holland, Amsterdam. 1978). IO. Baskin A. and Lyman D. J. J. Biomed. Mater. Rex 14, 393 (1980). II. Holly F. J. J. Polym. Sci. Polym. Symp. 66,409 (1979). 12. Okano T.. Nishimura S., Schinohara I.. Akaike T. and Sakurai Y. Polymer J. 10, 223 (1978). 13. Belanger L., Sylvestre C. and Dufour D. C/in. Chim. Acra 48, 15 (1973).
Isor. Vol. 35. ?;o. 6. pp. 471-471.
0020-708x:84
1981
Copyright
Printed m Great Britain. AU rights resewed
33.00 + 0.00
,s 1981 Pergamon Press Ltd
Sheets Obtained by Radiation Polymerization for Enzyme Immunoassay MINORU Takasaki Radiation
Chemistry
KUMAKURA
and ISA0 KAETSU
Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma, Japan
(Receiced 15
April
1983; in recisedform 8 August 1983)
Immobilized anti-3-fctoprotein sheets, which were attached to sticks, for enzyme immunoassay of z-fetoprotein were prepared by radiation polymerization of hydrophilic monomers. The relationship between the preparation conditions and the activity of the sheets was studied. The activity varied with monomer concentration, hydrophilicity of polymer matrix, the amount of coating solution, and antibody concentration. The sheets obtained at relatively low monomer and antibody concentrations appeared to give a high activity. It was found that the sheets are applicable for the enzyme immunoassay of r-fetoprotein with high sensitivity.
Materials and Methods
Introduction At present there are two widely accepted assays of enzyme and radioimmunoassay that employ labeled antibodies and antigens. Enzyme immunoassay employs antibodies or antigens conjugated to enzymes in such a way that the immunological and enzyme activity of each moiety is maintained. This assay gives objective results and is extremely sensitive, though it is not usually quite as precise as radioimmunoassay. The range of application of enzyme-immunoassay is potentially as wide as that of radioimmunoassay and it may also reinforce or replace other serological tests. Various kits containing immunoreagents have been studied for enzyme- and radioimmunoassay.(‘b’ AS immunoreagent, beads, plates, and tubes have been used, in which antibodies were coated or bound on their surfaces. The immunoreagents resulting from physical coating method have a weak point such as leakage of antibodies, and those resulting from the chemical covalent method require necessary longer preparation times and excess amounts of antibodies, though the leakage does not occur. To increase the surface area of immunoreagents, for example, the glass beads have been frosted and antibodies have been immobilized. These problems on the preparations of immunoreagents led us to study the preparation of immunoreagents by the radiation polymerization technique. In this work, sheets consisting of paper and polymer matrices for enzyme immunoassay were prepared by radiation polymerization and its characteristic was studied, in which anti-z-fetoprotein was used as antibody. The assay of z-fetoprotein has been noticed as a diagnostic test for cancer patients.“.J)
Materials
Anti-sera to z-fetoprotein (anti-AFP) were produced in rabbits by injecting an emulsion of human AFP with an equal volume of complete Freund’s adjuvant. Human AFP obtained from the plasma of a patient with primary hepatoma, then purified by afinity chromatography on anti-AFP Sepharose 4B column (Pharmacia Japan Co., Ltd), foliowed by gel-filtration on Sephacryl S-300 column (Pharmacia Japan Co., Ltd). To prepare peroxidase-labeled antiAFP, we purified anti-AFP serum by ammonium sulfate fractionation, followed by the ion-exchange chromatography on diethylaminoethyl then coupled the cellulose, purified IgG fraction from anti-AFP sera with peroxidase according to the method of Wilson and Nakane.“) Standard human AFP was obtained from Dinabott Radioisotope Lab., Ltd. The buffer solution was phosphate-buffered saline (PBS), 10 mmol/L, pH 7.2. Hydroxyethyl methacrylate (HEMA). hydroxyethyl acrylate (HEA), hydroxypropyl methacrylate (HPMA), and methoxytetraethyleneglycol dimethacrylate (M-4G) were used as monomer for immobilization. Thin papers (10 x 5 mm, 8 pm in thickness; Efuron # 100 No. 3) used as a base material of the sheets were obtained from Fukuko Paper Mill Co., Ltd. Preparation
of immobilized
anti-AFP
sheets
Anti-AFP serum dissolved in the PBS solution was mixed with monomer and it was coated onto the sheet (coat area; 5 x 5 mm) using a micro-syringe, in which the edge (5 mm wide) of the sheet was attached to the 471
472
MINORUKWAKURA and
Monomer
1%)
Fig. 1. Effect of HEA monomer concentration. stick (5 x 100 x 0.5 mm) made of polyvinyl chloride which had been heated for handling in the assay. These coated sheets were put into a vessel, frozen at -78°C and irradiated with 1 Mrad in N, gas atmosphere by y-rays from a @‘Co source. After irradiation, immobilized anti-AFP sheets obtained by radiation polymerjzation at low temperatures were dried at room temperature. Enzyme-immunoassay One immobilized anti-AFP sheet was placed in a test tube containing 100 PL of sample or the standard AFP solution, and incubated at room temperature for 1 h. After incubation, the solution was removed and the immobilized anti-AFP sheet was washed with the PBS solution three times. Then 5OpL of peroxidase-labeled anti-AFP and 100 ,uL of the PBS solution were added to the tube and incubated at 37°C for 1 h. Again, the immobilized anti-AFP sheet was washed with the PBS solution three times to remove the unreacted peroxidase-labeled anti-AFP. The enzyme reaction was carried out with 200 p L of a solution of H202 (0.3 g/L) and o-phenylenediamine (3 g/L) at room temperature for 30 min and terminated by adding 1 N HCl solution, 1 mol/L. After the reaction, we measured the adsorbance of the solution at 492 nm with a spectrophotometer, to detect the peroxidase activity.
ISAO KAETSU
crease of the activity at high monomer concentrations is caused by the entrapment of anti-AFP into the polymer matrix. Since the mechanism of the immobilization of anti-AFP in the present method is a physical trapping by radiation polymerization, the trapping capacity is related to the amount and surface area of the polymer matrix on the paper. As monomer concentration increases, the amount (quantity of polymer per unit area) of the polymer matrix is increased and it leads to the decrease of the surface area of the sheet, in which anti-AFP would be entrapped, and disappear into the polymer matrix. On the other hand, in the immobilization with lower monomer concentrations, anti-AFP becomes attached to the surface of cellulose fibril in the paper with the polymer matrix. The polymer matrix played the role of adhesion agent for anti-AFP and cellulose fibril, so that a relatively low monomer concentration was recommended. However, the sheets from low monomer concentrations below 5% cause anti-AFP to leak due to a weak trapping; the leakage of anti-AFP in 3% monomer concentration was about 7%. Therefore, optimum monomer concentration appeared to be 10-20x. Effect of the amount of coating solution on activity Effect of the amount of the coating solution containing HEA monomer, anti-AFP, and the buffer solution on the activity was examined. The relationship between absorbance and the amount of the coating solution is shown in Fig. 2. The activity increased with increasing the amount of the coating solution which was clearly due to the increase in the quantity of anti-AFP. From these results, it was found that the sensitivity of the sheets increased with an increase in the amount of coating solution. Thus, the improvement of the sensitivity was easily compared to other immobilization methods with a chem-
20 r
Results and Discussion Effect of monomer concentration on activity The preparation method of immobilized anti-AFP sheets was based on the radiation polymerization of the monomer solution coated on the sheets, so that it is expected that the activity of the sheets is related to the polymerization condition. Here the effect of monomer concentration on the activity of the sheets was examined. Figure 1 shows the relationship between absorbance and HEA monomer concentration. The absorbance decreased with increasing monomer concentration, indicating that the activity is markedly dependent on the monomer concentration. The de-
L 0
I
2 ArnOU”,
3
c
(&I
Fig. 2. Effect of the amount of the coating solution. monomer concentration: 20%.
HEA
473
Obtaining sheets for enzyme immunoassay
Fig. 3. Effect of the hydrophilicity of monomers. Monomer concentration: ZOO/,.
ical covalent. This was one of the characteristics of the present method. The polymer matrix of the sheets obtained by radiation polymerization of hydrophilic HEA monomer at low temperature had a porous structure, in which anti-AFP was trapped on the surface of the polymer matrix with cellulose fibrils. The formation of the porous structure in the polymer matrix resulted from the ice in the matrix system melting at room temperature, after radiation polymerization at a low temperature. Such a porous structure led to a further increase in the surface area of the sheets, though not in the sheets consisting of the network structure with cellulose fibrils. Immobilization of anti-AFP with other monomers Anti-AFP was immobilized on the sheets with other slightly hydrophobic monomers such as HEMA and HPMA in comparison with the case of HEA monomer. The resultant absorbance in the sheets from HEMA and HPMA is shown in Fig. 3 together with that in HEA monomer as a function of the water content of the polymers. The activity of the sheets appeared to be related to the water content of the polymer matrix, giving a maximum activity at 26% water content (polymer of HEMA monomer). From this result, it is suggested that the activity of the sheets decreased by a decrease or increase in the water content. Thus, a monomer such as HEMA appeared to be of optimum hydrophilicity for the immobilization of anti-AFP. The molecule of y-globulin such as anti-AFP has Fab and F, chains and as recently reported the adsorption of plasma proteins such as y-globulin on polymer might be related to the hydrophilicity of polymer.(‘“.“’ Okano et al. have reported that the F, chain of y-globulin is selectively adsorbed on relatively hydrophobic sites.“*’ In the sheets obtained with the present method, it is reasonable that the F, chain of anti-AFP is trapped on the polymer matrix and the Fab chains of anti-AFP are free because the Fab chains are reacted with antigen (AFP) to form an antigen-antibody complex by a sandwich reaction. It was proposed that anti-AFP was trapped on the polymer matrix, taking such a conformation state, because the activity resulting from the sandwich reaction was very high.
Fig. 4. Effect of the dilution of anti-AFP. HEA monomer concentration: 20%. 0-l PL coating solution, O-2 PL coating solution.
Eflect of the dilution of anti-AFP on actbity The effect of the dilution of anti-AFP serum on the activity of the sheets was studied. The various concentrations of anti-AFP serum diluted with normal rabbit serum were used for the immobilization. The relationship between absorbance and dilution ratio is shown in Fig. 4. As dilution ratio increased, the absorbance in 1 PL coating solution decreased moderately but that in 2pL coating solution decreased markedly. This result indicated that the activity of the sheets obtained with 1 PL coating solution varies slightly with anti-AFP concentration within concentrations studied in this work. In Fig. 4, the absorb ante in 1 and 2 PL coating solution at high dilution ratios (l/10) gave almost the same value, though those at nondilution gave a 2-fold difference. This suggests that anti-AFP trapped in the sheets obtained from thin coating solutions with low anti-AFP concentrations is located on its surface, taking the conformation of the F, chain trapping. As can be seen in Fig. 4, the activity did not decrease according to the
.I t f 0.
./ 100 !
200 I
300 1
Fig. 5. Standard curve. HEA monomer concentration: 20%.
474
MINORU KIJMAKURAand
dilution ratio of anti-AFP, indicating that the excess amounts of anti-AFP in the sheets do not contribute to the sandwich reaction and they might be entrapped in the polymer matrix. Thus, it was reasonable to assume that anti-AFP serum, diluted to a certain concentration, is employed in immobilization. Assay characteristics
This typical standard curve in the enzymeimmunoassay with the sheets is shown in Fig, 5. The slope of the curve was linear over the range of 0-300ng/mL. Since the concentration of AFP in normal human serum is about 20 ng/mL, it is found that the sheets obtained by the present method was applicable for the enzyme-immunoassay of AFP. The minimum detectable concentration of AFP was 2 ng/mL, and the assay precision was 44%. This sensitivity of the present method using the sheets was higher than that of other enzyme-linked immunosorbent assay using CNBr-activated cellulose (5 ng/mL),“’ and was the same range as that described bv Belaneer et a/.f’3)
ISAOKAETSL’
References 1. Wisdow G. B. Clin. Chem. 22, 1213 (1976). 2. Scharpe S. L., Cooreman W. M. and Blomme W. J.
Clin. Chem. 25, 733 (1979). 3. Woo J. and Cannon D. C. Am. J. Clin. Pathol. 66, 854 (1976). 4. Voller A., Bidwell D. E. and Bartlett A. Bull. WHO 53, 55 (1976). 5. Belanger L., Hamel D.. Dufour D. and Pouliot M. Clin. Chem. 22. 198 (1976). 6. Maiolini R.. Ferrua B. and LMasseyeff R. J. Immunol. Methods 6, 3% (1975). 7. IMaiolini R. and Masseyeff R. J. fmmunol. Merho& 8, 223 (1975). 8. Belanger L., Syivestre C. and Dufottr D. Clin. Chem. Acta 48, 15 (1973). 9. Wilson M. B. and Nakane P. K. tmmunofluorescence and Related Staining Techniques (Ed. Knapp W.) p. 215 (Elsevier/Nonh-Holland, Amsterdam. 1978). IO. Baskin A. and Lyman D. J. J. Biomed. Mater. Rex 14, 393 (1980). II. Holly F. J. J. Polym. Sci. Polym. Symp. 66,409 (1979). 12. Okano T.. Nishimura S., Schinohara I.. Akaike T. and Sakurai Y. Polymer J. 10, 223 (1978). 13. Belanger L., Sylvestre C. and Dufour D. C/in. Chim. Acra 48, 15 (1973).