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The adsorption of poliovirus antigen to glass surfaces
DISCUSSIOK The
Adsorption
of
Poliovirus
AND
Antigen
to
PRELIMINARY
St’udies in this laboratory led to the development of a poliomyelitis vaccine containing purified poliovirus antigen as the immunizing agent (1-3). An early lot of vaccine (Vaccine “1 A”j, in which the purified polio antigen was suspended in 0.85% NaCl solution, showed diminished potency test ratios after storage at 2-5” (3). Since this type of instability had not been encountered with crude poliovirus vaccine or with relatively concentrated suspensions of purified poliovirus or poliovirus antigen, it was suspected that adsorption of the diluted purified antigen to the surface of the glass vial might be responsible for this efTABLE OF ADSORPTION
Time
Adsorption-preventing substance or solution None
(saline
0.1%
BSA
0.1%
Sodium
Gelatin,
Sodium
Medium
Tween
a All
20 5 1.25 0.31
OF PURIFIED
control)
alginate
pg/ml r&l rg/ml fig/ml
nucleate,
20 pg/ml 2 crdml
100, undiluted 1:lO 1:40 1:lGO 1:640 80, 20 5 1.25 0.31 0.08 antigen
pg/mI dml pg/ml p&/ml fig/ml solutions
contained
50 pg/ml
495
feet The experiments described in t,his note confirm this hypothesis by the demonst,ration that significant quantities of poliovirus antigen can be eluted from the emptied containers (No. 283, USP Type 1 borosilicate glass vials, 5-ml size) in which the vaccine had been st,ored, using 1.25 ml of 0.1% bovine serum albumin (BSA) per 5-ml vial and rot,ating the container in a drum for 2 hours at 36°C. In addition, some results are presented on the prevention of this adsorption process by addition of nontoxic adsorpt,ion-preventing agents. Antigen concentrations were determined by complement fixation (CF) assays run in parallel with known purified poliovirus standards so that the assay results could bc stated in micrograms per milliliter. The
Glass
Surfaces
PREVENTIOX
REPORTS
1 POLIOVIRUS
of adsorption ihr)
(SAUKETT)
ANTIGEN
TO GLASS
Antigen cont. (pg/ml) before adsorption
Antigen cow. (pg/ml) after adsorption
18 FG 66 138
2.0 1.1 1.3 2.0
1.6 0.5 0.1 0.2
18 66 138
1.7 1.3 1.6
1.7 1.1 1.2
<0.04 <0.04 <0.04
66 138
1.5 1.6
3.0 3.2
<0.04 <0.04
66 86 66 66
0.56 1.3 1.1 0.6G
0.79 1.12 0.79 0 .66
<0.04 0.04 0.39 0. Mj
66 66
0.56 1.31
0.94 0.94
<0.0-l 0.10
6G 66 66 66 66
1.3 0.9 1.6 1.9 1.1
0.79 0.94 0.94 0.94 0.39
0.04 <0.04 0.10 0.33 0.66
66 66 66 66 66
0.94 1.3 1.3 0.94 0.66
1.6 0.66 1.3 1.1 0.56
<0.04 <0.04 <0.0-l 0.04 0.24
neomycin
and
25 rg/ml
Phemerol
*Antigen (pg/ml) in O.l%:, BS.1 eluate 1.3 O.GB 0.94 1.0
as preservatives.
496
DISCUSSION
AND
PRELIMINARY TABLE
CF
ASSAY Type
Formulation’
Value x2 antigen/ 0.5 ml
RESULTS 1
REPORTS
2 ON STORED
VIALS
Type
% of nominal
Value rg antigen/ 0.5 ml
2
Type
% of nominal
Value Kg antigen/ 0.5 ml
3 % of nominal
Vaccine
“1A”
Nominal* Found Desorbed
3.0 >2.7 0.27
(100) >90 9
0.6 0.08 0.06
(100) 14 10
1.0 0.08 0.40
(100) 8 40
Vaccine
“1B”
Nominal Found Desorbed
1.5 1.0 0.27
(1~) 67 18
0.3 <0.05 0.03
(100) <17 10
0.5 <0.05 0.24
(100)
Vaccine
“1C”
Nominal Found Desorbed
0.75 <0.13 0.10
(100) <17 14
0.15 <0.05 0.01
(l@J) <33 7
0.25 <0.05 0.07
(100) <20 28
a These vaccines were designed to contain sufficient antigen to permit the administration of 0.5 ml as an immunizing dose. Vaccine “1B” and “1C” were 1:2 and 1:4 dilutions, respectively, of vaccine “1A.” * The ‘Lnominal” dose is the quantity of antigen in each dose (0.5 ml) as filled. This value is based upon a physical measurement (ultraviolet absorption density) of the concentration of the virus before inactivation and subsequent dilution. A measurement of the complement-fixing activity of the formulation prior to filling was not made. c The quantitv of antigen desorbed is expressed in terms of micrograms of CF antigen calculated back to the original dose-
standards gave CF assay results ranging from 1: 3 to 1:9 per microgram, depending on the type of poliovirus and daily variation in assay sensitivity. These values coupled with the eightfold (on a dose basis) concentration factor obtained in the elution procedure permitted detection of poliovirus antigen at concentrations as low as 0.01 pg/O.5 ml. The results of these experiments are shown in Table 1. It was found that where a given type of poliovirus antigen was present at a concentration below 1 pg/O.5 ml, almost all the antigen had disappeared from suspension and that a significant fraction of the antigen lost (up to 48% for type 3, Saukett) could be recovered in the eluate. Further studies of such adsorption were made using formaldehyde-inactivated purified type 3 (Saukett) poliovirus antigen. Adsorption took place in No. 283 glass vials placed in a reciprocating shaker at low speed during various time intervals at 5”. After incubation, the vials were emptied, eluted with 0.1% BSA as described above,
and the eluates were assayed by CF simultaneously with a stored portion of the original solution to which BSA had been added to give a final concentration of 0.1%. It was found that adsorption from control (saline) suspensions progressed with time. After 18 hours, roughly one-half the antigen was still in suspension; this value decreased to about 10% at 66 and 138 hours. Approximately one-half of the original antigen could be recovered by elution with 0.1% BSA. In contrast, when 0.1% BSA was present during the incubation period, no diminution of antigen concentration occurred and no antigen could be demonstrated in the eluates (Table 2). The effectiveness of BSA in preventing adsorption led us to test other macromolecular substances of interest as nontoxic adsorption-preventing agents. Sodium alginate, sodium nucleate, and gelatin were shown to be effective (Table 2). Confirmation of the adsorption of poliovirus antigen to glass led to the consideration of the occurrence of this phenomenon
DISCUSSION
AND
PRELIMINARY
in Salk poliovaccine, in which the antigen exists in concentrations (0.1-1.0 pg/ml) at which adsorption might be expected to result in severe or complete elimination of potency. The known potency of the vaccine suggested that the suspending medium (medium 199) contained an adsorption preventative. Medium 199, slightly modified by omission of the pH indicator and a number of components of doubtful stability, was tested and proved fully effective. Further tests of the components of the medium at appropriate concentration revealed that the sole active component was Tween 80,l which is present in medium 199 at a concentration of 20 pg/ml. The complete medium prevented the adsorption of poliovirus at a dilution of 1: 10 and permitted the adsorption of a trace of elutable antigen at 1: 40. In comparison, the end point of effectiveness of Tween 80, alone, was approximately 0.3 pg/ml (Table 2). In the test system used, these end points may be regarded as identical. These results add poliovirus antigen to the substantial number of biologically active substances that have been shown to be subject to changes in potency due to adsorption to container surfaces. The phenomenon has been report,ed in connection with insulin (4)) acid prostatic phosphatase (5)) tuberculin (G-L?), and fowl plague and vaccinia viruses (9). Such adsorption may be especially troublesome in virus research. It appears that only the customary use of medium 199, cont,aining Tween 80, as a diluent for the preparation of samples, has permitted meaningful poliovirus assays and safety tests; assays performed in this laborat,ory using saline or phosphate buffers as diluents were shown to yield misleading results due to adsorption losses. It is possible that in many studies of viruses and virus antigens, losses due to adsorption to container surfaces may account for otherwise inexplicable assay, stability, filtration, and purification difficult,ies. REFERENCES 1. CHARKEY,
J., FISHER,
W. P., and
MACHLO~ITZ,
* Polyoxyethylene sorbitan mono-oleate, Powder Co., Wilmington, Delaware.
Atlas
497
REPORTS
R. d., Proc. Sot. Exptl. Biol. Med. 96, 601605 ( 1957 ) 2. HILLEMAN, M. R., CHARNEY, J., TTTELL, A. A., WIEHL, C., CORNFELD, D., ICHTER, J. T., RILEY, H. D., JR., and HUATYG, N., Acad. Med. New Jersey Spec. Bull. 6, 1-31 (1960). 3. CHARSEY, J., TYTELL, ,4. A., MACHLOWITZ, R. A., and HILLEMAN, M. R., J. Am. Med. Assoc. 177,591-595 (1961). 4. FERREBEE, J. W., JOHNSON, B. B., MITHOEFER, J. C., and GARDELLA, J. W., Endocrinology 48, 277-283 ( 1951). 5. LONDON, M., and HUDSOK, P. B., Arch. Biochem. Biophys. 46, 141-153 (1953). 6. MAGNUS, K., GULD, J., WAALER, H., and MAGNUSSON, M., Am. Rev. Tuber. Pulmonary Diseases 74, 297-303 (1956). Y. WAALER, H., GCLD, J., MAGIYUS, K., and MAGNUSSON, M., Bull. M’o& Health Organization 19, 783-798 (1958). 8. MAGKUSSOX, M., GULD, J., MAGNUS, K., and WAALER, H., Bull. World Health Organization 19,799-828 (1958). 9. VALENTINE, R. C., and ALLISON, A. C., Biochim. et Biophys. Acta 34, 10-23 (1959). JESSE CHARNEY’ ROY A. MACHLOWITZ DANIEL S. SPICER Virus and Tissue Culture Research Division Merck Institute for Therapeutic Research West Point, Pennsylvania Receiced July 5, 1962 * Present address: Research Foundation,
Preparation Al4
of
The South JerseJr Medical Camden, New Jersey.
Fluorescein-Labeled
Antibodies
from
Immune
Coxsackie Mouse
Ascitic
Fluid’
Diagnostic techniques concerned with group A Coxsackie viruses are complicated by a limited host range. The possible application of fluorescent antibody (FA) techniques toward this end would have distinct advantages over procedures involving suckling mice, but these require adequate quantities of antibody of high titer, which preferably should be induced in the homologous host in order to avoid nonspecific reactions. Herrmann and Englc (1) immunized mice 1 Aided tion.
bq. a grant
from
The
National
Founda-
Adsorption
of
Poliovirus
AND
Antigen
to
PRELIMINARY
St’udies in this laboratory led to the development of a poliomyelitis vaccine containing purified poliovirus antigen as the immunizing agent (1-3). An early lot of vaccine (Vaccine “1 A”j, in which the purified polio antigen was suspended in 0.85% NaCl solution, showed diminished potency test ratios after storage at 2-5” (3). Since this type of instability had not been encountered with crude poliovirus vaccine or with relatively concentrated suspensions of purified poliovirus or poliovirus antigen, it was suspected that adsorption of the diluted purified antigen to the surface of the glass vial might be responsible for this efTABLE OF ADSORPTION
Time
Adsorption-preventing substance or solution None
(saline
0.1%
BSA
0.1%
Sodium
Gelatin,
Sodium
Medium
Tween
a All
20 5 1.25 0.31
OF PURIFIED
control)
alginate
pg/ml r&l rg/ml fig/ml
nucleate,
20 pg/ml 2 crdml
100, undiluted 1:lO 1:40 1:lGO 1:640 80, 20 5 1.25 0.31 0.08 antigen
pg/mI dml pg/ml p&/ml fig/ml solutions
contained
50 pg/ml
495
feet The experiments described in t,his note confirm this hypothesis by the demonst,ration that significant quantities of poliovirus antigen can be eluted from the emptied containers (No. 283, USP Type 1 borosilicate glass vials, 5-ml size) in which the vaccine had been st,ored, using 1.25 ml of 0.1% bovine serum albumin (BSA) per 5-ml vial and rot,ating the container in a drum for 2 hours at 36°C. In addition, some results are presented on the prevention of this adsorption process by addition of nontoxic adsorpt,ion-preventing agents. Antigen concentrations were determined by complement fixation (CF) assays run in parallel with known purified poliovirus standards so that the assay results could bc stated in micrograms per milliliter. The
Glass
Surfaces
PREVENTIOX
REPORTS
1 POLIOVIRUS
of adsorption ihr)
(SAUKETT)
ANTIGEN
TO GLASS
Antigen cont. (pg/ml) before adsorption
Antigen cow. (pg/ml) after adsorption
18 FG 66 138
2.0 1.1 1.3 2.0
1.6 0.5 0.1 0.2
18 66 138
1.7 1.3 1.6
1.7 1.1 1.2
<0.04 <0.04 <0.04
66 138
1.5 1.6
3.0 3.2
<0.04 <0.04
66 86 66 66
0.56 1.3 1.1 0.6G
0.79 1.12 0.79 0 .66
<0.04 0.04 0.39 0. Mj
66 66
0.56 1.31
0.94 0.94
<0.0-l 0.10
6G 66 66 66 66
1.3 0.9 1.6 1.9 1.1
0.79 0.94 0.94 0.94 0.39
0.04 <0.04 0.10 0.33 0.66
66 66 66 66 66
0.94 1.3 1.3 0.94 0.66
1.6 0.66 1.3 1.1 0.56
<0.04 <0.04 <0.0-l 0.04 0.24
neomycin
and
25 rg/ml
Phemerol
*Antigen (pg/ml) in O.l%:, BS.1 eluate 1.3 O.GB 0.94 1.0
as preservatives.
496
DISCUSSION
AND
PRELIMINARY TABLE
CF
ASSAY Type
Formulation’
Value x2 antigen/ 0.5 ml
RESULTS 1
REPORTS
2 ON STORED
VIALS
Type
% of nominal
Value rg antigen/ 0.5 ml
2
Type
% of nominal
Value Kg antigen/ 0.5 ml
3 % of nominal
Vaccine
“1A”
Nominal* Found Desorbed
3.0 >2.7 0.27
(100) >90 9
0.6 0.08 0.06
(100) 14 10
1.0 0.08 0.40
(100) 8 40
Vaccine
“1B”
Nominal Found Desorbed
1.5 1.0 0.27
(1~) 67 18
0.3 <0.05 0.03
(100) <17 10
0.5 <0.05 0.24
(100)
Vaccine
“1C”
Nominal Found Desorbed
0.75 <0.13 0.10
(100) <17 14
0.15 <0.05 0.01
(l@J) <33 7
0.25 <0.05 0.07
(100) <20 28
a These vaccines were designed to contain sufficient antigen to permit the administration of 0.5 ml as an immunizing dose. Vaccine “1B” and “1C” were 1:2 and 1:4 dilutions, respectively, of vaccine “1A.” * The ‘Lnominal” dose is the quantity of antigen in each dose (0.5 ml) as filled. This value is based upon a physical measurement (ultraviolet absorption density) of the concentration of the virus before inactivation and subsequent dilution. A measurement of the complement-fixing activity of the formulation prior to filling was not made. c The quantitv of antigen desorbed is expressed in terms of micrograms of CF antigen calculated back to the original dose-
standards gave CF assay results ranging from 1: 3 to 1:9 per microgram, depending on the type of poliovirus and daily variation in assay sensitivity. These values coupled with the eightfold (on a dose basis) concentration factor obtained in the elution procedure permitted detection of poliovirus antigen at concentrations as low as 0.01 pg/O.5 ml. The results of these experiments are shown in Table 1. It was found that where a given type of poliovirus antigen was present at a concentration below 1 pg/O.5 ml, almost all the antigen had disappeared from suspension and that a significant fraction of the antigen lost (up to 48% for type 3, Saukett) could be recovered in the eluate. Further studies of such adsorption were made using formaldehyde-inactivated purified type 3 (Saukett) poliovirus antigen. Adsorption took place in No. 283 glass vials placed in a reciprocating shaker at low speed during various time intervals at 5”. After incubation, the vials were emptied, eluted with 0.1% BSA as described above,
and the eluates were assayed by CF simultaneously with a stored portion of the original solution to which BSA had been added to give a final concentration of 0.1%. It was found that adsorption from control (saline) suspensions progressed with time. After 18 hours, roughly one-half the antigen was still in suspension; this value decreased to about 10% at 66 and 138 hours. Approximately one-half of the original antigen could be recovered by elution with 0.1% BSA. In contrast, when 0.1% BSA was present during the incubation period, no diminution of antigen concentration occurred and no antigen could be demonstrated in the eluates (Table 2). The effectiveness of BSA in preventing adsorption led us to test other macromolecular substances of interest as nontoxic adsorption-preventing agents. Sodium alginate, sodium nucleate, and gelatin were shown to be effective (Table 2). Confirmation of the adsorption of poliovirus antigen to glass led to the consideration of the occurrence of this phenomenon
DISCUSSION
AND
PRELIMINARY
in Salk poliovaccine, in which the antigen exists in concentrations (0.1-1.0 pg/ml) at which adsorption might be expected to result in severe or complete elimination of potency. The known potency of the vaccine suggested that the suspending medium (medium 199) contained an adsorption preventative. Medium 199, slightly modified by omission of the pH indicator and a number of components of doubtful stability, was tested and proved fully effective. Further tests of the components of the medium at appropriate concentration revealed that the sole active component was Tween 80,l which is present in medium 199 at a concentration of 20 pg/ml. The complete medium prevented the adsorption of poliovirus at a dilution of 1: 10 and permitted the adsorption of a trace of elutable antigen at 1: 40. In comparison, the end point of effectiveness of Tween 80, alone, was approximately 0.3 pg/ml (Table 2). In the test system used, these end points may be regarded as identical. These results add poliovirus antigen to the substantial number of biologically active substances that have been shown to be subject to changes in potency due to adsorption to container surfaces. The phenomenon has been report,ed in connection with insulin (4)) acid prostatic phosphatase (5)) tuberculin (G-L?), and fowl plague and vaccinia viruses (9). Such adsorption may be especially troublesome in virus research. It appears that only the customary use of medium 199, cont,aining Tween 80, as a diluent for the preparation of samples, has permitted meaningful poliovirus assays and safety tests; assays performed in this laborat,ory using saline or phosphate buffers as diluents were shown to yield misleading results due to adsorption losses. It is possible that in many studies of viruses and virus antigens, losses due to adsorption to container surfaces may account for otherwise inexplicable assay, stability, filtration, and purification difficult,ies. REFERENCES 1. CHARKEY,
J., FISHER,
W. P., and
MACHLO~ITZ,
* Polyoxyethylene sorbitan mono-oleate, Powder Co., Wilmington, Delaware.
Atlas
497
REPORTS
R. d., Proc. Sot. Exptl. Biol. Med. 96, 601605 ( 1957 ) 2. HILLEMAN, M. R., CHARNEY, J., TTTELL, A. A., WIEHL, C., CORNFELD, D., ICHTER, J. T., RILEY, H. D., JR., and HUATYG, N., Acad. Med. New Jersey Spec. Bull. 6, 1-31 (1960). 3. CHARSEY, J., TYTELL, ,4. A., MACHLOWITZ, R. A., and HILLEMAN, M. R., J. Am. Med. Assoc. 177,591-595 (1961). 4. FERREBEE, J. W., JOHNSON, B. B., MITHOEFER, J. C., and GARDELLA, J. W., Endocrinology 48, 277-283 ( 1951). 5. LONDON, M., and HUDSOK, P. B., Arch. Biochem. Biophys. 46, 141-153 (1953). 6. MAGNUS, K., GULD, J., WAALER, H., and MAGNUSSON, M., Am. Rev. Tuber. Pulmonary Diseases 74, 297-303 (1956). Y. WAALER, H., GCLD, J., MAGIYUS, K., and MAGNUSSON, M., Bull. M’o& Health Organization 19, 783-798 (1958). 8. MAGKUSSOX, M., GULD, J., MAGNUS, K., and WAALER, H., Bull. World Health Organization 19,799-828 (1958). 9. VALENTINE, R. C., and ALLISON, A. C., Biochim. et Biophys. Acta 34, 10-23 (1959). JESSE CHARNEY’ ROY A. MACHLOWITZ DANIEL S. SPICER Virus and Tissue Culture Research Division Merck Institute for Therapeutic Research West Point, Pennsylvania Receiced July 5, 1962 * Present address: Research Foundation,
Preparation Al4
of
The South JerseJr Medical Camden, New Jersey.
Fluorescein-Labeled
Antibodies
from
Immune
Coxsackie Mouse
Ascitic
Fluid’
Diagnostic techniques concerned with group A Coxsackie viruses are complicated by a limited host range. The possible application of fluorescent antibody (FA) techniques toward this end would have distinct advantages over procedures involving suckling mice, but these require adequate quantities of antibody of high titer, which preferably should be induced in the homologous host in order to avoid nonspecific reactions. Herrmann and Englc (1) immunized mice 1 Aided tion.
bq. a grant
from
The
National
Founda-