vvEPA
United States
Environmental Protection
Agency
Environmental Monitoring and Support EPA-600/4-80-034
Laboratory June 1980
Cincinnati OH 45268
Research and Development
Identification and
Detection of
Water-Borne Viruses
by Immunoenzymatic
Methods
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate; further development and application of en-
vironmental technology. Eliminatioh of traditional grouping was consciously
planned to foster technology transfef and a maximum interface in related fields.
The nine series are: :
1, Environmental Health Effects Research
2. Environmental Protection [Technology
3. Ecological Research :
4, Environmental Monitoring;
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7, Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL MONITORING series.
This series describes research conducted to develop new or improved methods
and instrumentation for the identification and quantification of environmental
pollutants at the lowest conceivably significant concentrations. It also includes
studies to determine the ambient cortcentrations of pollutants in the environment
and/or the variance of pollutants as la function of time or meteorological factors.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/4-80-034
June 1980
IDENTIFICATION AND DETECTION OF WATER-BORNE VIRUSES BY
IMMUNOENZYMATIC METHODS
by
John E. Herrmann
Harvard School of Public Health
Boston, Massachusetts 02115
Grant No. R-803360
Project Officer
Robert S. Safferman
Biological Methods. Branch.
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.si Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
11
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati, conducts research to:
Develop and evaluate techniques to measure the presence and
concentration of physical, chemical, and radiological pollutants in
water, wastewater, bottom sediments, and solid waste.
Investigate methods for the concentration, recovery, and
identification of viruses, bacteria and other microbiological
organisms in water; and to determine the responses of aquatic
organisms to water quality.
Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for
monitoring water and wastewater.
Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
This research was conducted in the interest of applying immunoenzymatic
techniques to the identification of enteroviruses isolated from varying
qualities of waters including sewage, river, sea and tap water. Such
methodology may bring about significant improvement over techniques currently
in use in the detection and identification of waterborne viruses.
Dwight G. Ballinger
Director
Environmental Monitoring and Support
Laboratory - Cincinnati
±±±
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ABSTRACT
A quantitative enzyme-linked immunosorbent assay (ELISA) was used for
identification of viruses selected las representative waters-borne viruses:
poliovirus 1, echovirus 6, coxsackievirus A9, and coxsackie B viruses.
Partially purified viral antigens or virus-specific antibodies were adsorbed
to polystyrene spectrophotometer cuvettes, which permitted the assays to be
reported and compared in terms of enzyme units specifically reacting. Both
the adsorbed antigen and adsorbed antibody methods were approximately
equal in terms of sensitivity and specificity of reaction. By use of C-
leucine-labelled enteroviruses, the amount of virus that binds to the
plastics used was dependent on the [purity of the virus preparation used,
but was higher than the amount that! was bound by plastics coated with viral
antibody. It was also shown that the inhibitors in diluents used to prevent
non-specific adsorption of immunoreagents caused desorption of virus or
antibody during an immunoassay; the1 amount of virus desorption varied with
the type of preparation used, and antibody desorption was dependent on the
concentration of antibody initially adsorbed. For specific identification
of a given enterovirus type by this ELISA method,, approximately 105 plaque-^
forming units of virus per assay tube were required, To alleviate the
problem of antibody and virus desorption,, and to increase the amount of
each that could be bound to plastiqs, antibodies and virus were immobilized
by covalent linkage on nylon balls |for use in solid-phase enzyme-linked
immunoassays. Covalent linkage of [antibody or virus to nylon was accomplished
by treatment of partially hydrolized nylon with glutaraldehyde or carbodii-r
mides. Up to 0.74 yg of immunoglobulin G per mm2 nylon could be immobilized,
whereas only 0.02 yg per mm2 could [be adsorbed to polystyrene, and the
binding to nylon was stable. This eliminated the problem of antibody
desorption and gave more reproducibjle results. Also, antibodies coupled to
nylon balls remained bound under conditions that dissociate antibody-antigen
complexes, which permitted reuse of the immobilized antibodies for immuno-
assays. A higher percentage of virus could be immobilized by this method
than was possible by adsorption to polystyrene, and enzyme-linked immuno-
assay on nylon was sufficiently specific to differentiate the three polio-
virus types.
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CONTENTS
Foreword.
Abstract.
Figures..
Tables
.111
. iv
. vi
.vii
1,
•?,.
3.
4.
5.
6.
Introduction ,
Materials and Methods ,
Results ,
Discussion
1
4
5
30
References.
.33
v
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[FIGURES
Number j
i
1 Detection limits of coxsackievirus- type A9 (CA-9) by enzyme
Page
linked immunoassay J 19
2 Titration of antibody to coxsackievirus type A-9 (CA-9) by enzyme-
linked immunoassay 1 ' 20
i
3 Enzyme-linked immunoassay of rabbit IgG on nylon balls coupled with .
goat anti-rabbit IgG , 22
VI
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TABLES
Number,
Page
1 Virus Antigen Purification Procedure: Removal of Cellular Protein.,. ..10
2 Inhibition of Non-Specific Adsorption of Immunoreagents 11
3 Adsorption of Peroxidase-Labeled Antibody to Polystyrene Tubes 12
14
4 Adsorption of C-Leucine-Labeled Enteroviruses
to Polystyrene Tubes 13
14
5 Binding of C-Leucine-Labeled Coxsackieviirus Type B3(CB-3) By
Antibody Coated Tubes 15
6 Precision of Enzyme-Linked Immunoassay for Coxsackievirus Type B-2 16
7 Reaction of Enterovirus Types with Type-Specific and Heterotypic
Antisera by Enzyme-Linked Immunoassay 17
8 Comparison of Adsorbed Antigen and Adsorbed Antibody Methods for
Enterovirus Identification by Enzyme-Linked Immunoassay 17
9 Enzyme-Linked Immunoassay of Rabbit IgG. Use of Spacers for
Coupling Goat Anti-Rabbit IgG to Nylon Balls 23
125
10 Efficiency of Attachment of Ir-Labeled IgA to Nylon Balls 24
11 Comparison of Methods for Immobilizing IgG on Plastics 24
12 Re-use of Nylon Balls Coupled with Goat Anti-Rabbit IgG for Enzyme-
Linked Immunoassay 26
13 Enzyme-Linked Immunoassay of Rabbit IgG Immobilized on Nylon Powder 27
14 Coupling of Enteroviruses to Nylon Balls 27
15 Enzyme Immunoassay of Enteroviruses on Activated Nylon 28
vii
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SECTION 1
INTRODUCTION
There are approximately 100 enteric viruses that infect humans, including
those in the enterovirus group, adenoviruses, reoviruses, hepatitis agents,
and gastroenteritis agents. Many of them are highly virulent, and 'cause a
wide variety of clinical diseases. Despite concern for these viruses, there
is as yet no rapid practical assay for their- identification and detection,
which is limited to.clinical diagnosis, epidemiological studies on their
occurrence and impact, and their control. The purpose of the present study
was to develop immunochemical methods which would be applicable to rapid
viral diagnosis. The methods we concentr.aitedi'.on were'solid-phase enzyme-linked
immunoassays, and we selected enteroviruses as model water-borne viruses to
be assayed.
Solid phase enzyme immunoassays, usually called enzyme-linked immunosor-
bent assays (ELISA, ref. 1), and also enzyme-linked immunospecific assays
(2), have been applied to the detection of antibodies to a variety of micro-
bial and parasitic infections, as well as to toxin detection, hormone assay,
and a number of other chemical substances. The application of the method
to virus identification .is more limited, but has been used to identify
plant viruses (3), hepatitis B antigen (4), hepatitis A antigen (5),
herpesviruses (6), and human reovirus-like agent (7).
For enterovirus identification, the most common method used is virus
neutralization which, because of the large number of enterovirus types can
be both time-consuming and costly. Immunofluorescent (8,9,10) and immuno-
peroxidase techniques (11) for enterovirus identification have been described,
but require subjective judgements which are always a factor in histochemical
tests. For this reason, the suitability of enzyme-linked immunoassay for use
in enterovirus identification was investigated. The present report describes
the identification of selected enterovirus types by this technique, the
factors involved in the assay, and the development of an enzyme-linked
immunoassay based on coupling of antibodies and viral antigens to nylon.
The latter aspect was undertaken because the ELISA method, which is based
on simple adsorption of antigens or antibodies to plastics, is limited by
the amounts of antigen or antibody that can be adsorbed (2,3) to a given
plastic, and the problem of elution of what is adsorbed during an immuno-
assay procedure (2). Lack of reproducibility is also a problem, and antisera
adsorbed cannot be re-used.
To alleviate this problem, more efficient solid-phase supports were
sought,1 utilizing some of the technology developed for preparing immobilized
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enzymes. Among the supports used for enzyme immobilization have been various
types of nylon, which chemically referable polypeptides in structure.
Activation of nylon tubing by chemical treatments permits it to be coupled
covalently to enzymes (14,15), and activated nylon fibers have been used
as immunosorbents for cell fraction'ation (16) . Treatment of nylon with HC1
for limited periods of time causes [partial hydrolysis, which results in free
amino and carboxyl groups. Thus, proteins can be covalently coupled through
either group by use of the appropriate bifunctional reagents. We adapted
this to our purposes, namely the co'upling of antibodies and virus antigens to
solid-phase carriers for immunoassa.y, by use of chemically activated nylon
balls. These methods were compared with methods based on adsorption for both
immobilization of virus antigens and.antibodies, and for usefulness in enzyme^.
linked immunoassay of enteroviruses.
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SECTION 2
CONCLUSIONS
The methods developed were designed for identification of enteroviruses
but should be applicable to other water-borne viruses as well. In terms of
identification, enzyme-linked immunoassays based on either adsorption to
polystyrene or covalent linking to nylon were satisfactory for identifying
selected enterovirus types. The limitations of the method appear to be
related to the specificity of the viral antisera available. Both individual
sera and serum pools are satisfactory for typing enteroviruses by virus
neutralization, but all are not satisfactory for use in immunochemical
studies. This appears to be due in part to antibody against cell antigens,
but "also appears to be due to spurious cross-^reactions of undetermined origin.
In terms of sensitivity, the amount of antigen needed to give a positive
reaction by enzyme-linked immunoassays developed to date is approximately
Ing. For the enterovirus we tested, 105 plague^forming units of virus were
needed per assay. Although this amount is readily available from infected
tissue cultures, it is well above the amount that would be found in water.
Use of radioactive enzyme substrates have been reported to increase the
sensitivity of enzyme-r-linked immunoassays by up to 1000 times (17) , which
would thus give a sensitivity of 100 plaque-forming units per assay>
utilizing antisera currently available. Preparation of antisera that is
immunospecific and .of _htgh titer could possibly increase the sensitivity another
order of magnitude, which would allow for direct detection of virus in at
least some water- samples which have been processed and concentrated for virus
detection.
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SECTION 3
RECOMMENDATIONS
Enzyme-linked immunoassays have been found useful for identification of
a number of viral antigens, including those in the enterovirus group reported
here. The limitations on the methojd for type-specific identification of all
enterovirus types appears to be the! specificity, for immunochemical work, of
the antisera that is readily available to all investigators. Because of this,
it is recommended that sera be prepared from purified enterovirus virions,
and subsequently tested for type-sp;ecificity by immunochemical methods. For
use as a detection method for viruses in water, the maximum sensitivity that
would appear to be obtainable by current technology is 100 infectious units
of virus per assay. This is close to the amount found in some concentrated
water samples and the sensitivity cpuld undoubtedly be improved by use of
high titer, immunospecific viral antisera. Although preparation of such
antisera for all the virus types considered to be water-borne would be an
extensive project, preparation of antisera for a few selected types most
frequently isolated would not be difficult. This might permit a pilot study
to be made for direct testing of these virus types in concentrated water
samples and in waste water.
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SECTION 4
MATERIALS AND METHODS
VIRUSES AND TISSUE CULTURES
Poliovirus types l-r3, echovirus type 6, coxsackievirus B types 1, 4-6,
and coxsackievirus A type 9 were obtained from the NIH Research Resources
Branch, Bethesda, Maryland. Coxsackievirus types B2 and B3 were obtained
from D.O. Cliver, University of Wisconsin, Madison. Viruses were propagated
in vero cells (obtained from D.O. Cliver) or in BGM cells (obtained from
R.S. Safferman, Environmental Protection Agency, Cincinnati, Ohio).
VIRUS ANTIGENS
f\
Virus was innoculated onto cell monolayers in 75 cm flasks and incubated
with serum-free MEM medium, 5 ml per flask. After the cells showed 3 to 4 +
cytopathic effects, they were frozen-thawed twice. Cell debris was removed
by centrifugation at 40,000 xg (Sorvall RC-2B) for 30 min. Five grams of
anion exchanger (Bio Rad AG2-X8) was washed in distilled water and added to
30 ml virus solution. The mixture was stirred with an overhead stirrer at
300 rev/rain for 1 h and filtered through a Millipore fritted-glass filter.
By plaque titration, no virus losses were noted by use of the anion-exchange
resin or the filtration step. Uninfected cells were processed in the same
way for use as cell antigen controls.
Virus antigens were also prepared by extraction with diethyl ether.
Virus suspensions clarified by centrifugation as above were mixed with an
equal volume of cold (4°C) ether, and held on ice for 2h. The aqueous
layer was collected, and the residual ether removed under vacuum.
RADIOACTIVE VIRUS
14.
For preparation of C-leucine-labelled poliovirus 2 and coxsackievirus
B3, BGM or vero cultures were starved of leucine for 18h by incubation
with Earle's balanced salt solution (EBSS), The cultures were inoculated
with virus suspended in EBSS at a concentration of ca. 10 plaque forming
units (PFU) per cell, adsorbed 30 min at room temperature, and rinsed twice
with EBSS. Uniformly labeled 14C-leucine (specific activity 270 mCi/mM),
(New England Nuclear, Boston, Mass.) in EBSS (20 yCi/ml) was added with or
without 1 yg/ml Actinomycin D, and the cultures incubated for 24 h at 37°C
The cultures were freeze-thawed three times, and the harvested virus
centrifuged at 2500 x g to remove cell debris. The supernatant- fluids were
collected and the labelled virus purified (18) by passage through a DEAE-
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Sephadex A-50 column (1.5 x 30 cm),jusing 0.06M phosphate buffer, pH7.5,
as eluent. The effluent column fractions in which peak counts/min (Searle
Model 6880 Liquid Scintillation Counter) and peak virus PPU coincided were
the fractions used as labelled virus.
IMMUNQGLOBULJNS: AND ANTISERA. ;
Rabbit IgG was obtained from Miles Laboratories, Elkhart, IN. Goat
anti-rabbit IgG sera was obtained from Antibodies, Inc., Davis, CA. The
IgG fractions of all sera were prepared by (NH ) SO precipitation at one-
third saturation, followed by dialylsis against 0.015M phosphate-buffered
saline, pH7.2 (PBS).
!
Viral antisera used were horse1 antiviral sera obtained from the NIH
Research Resource Branch,. Bethesda, Maryland, or rabbit antiviral sera
obtained from Microbiological Associates (Bethesda, Maryland). For use in
the assays, viral antisera were absorbed with cell debris prior to use. This
was done by freeze-thawing cell cultures, centrifuging the cell debris at
2500 x g, and resuspending the pellet (0.1 ml) in ml of antisera diluted
1:10 PBS plus 1% bovine serum albumin (BSA). The cell-sera mixture was
incubated 24 h at 4°C, and the cell! debris removed by centrifugation at
2500 x g. Sera were tested for absence of antibody to cell antigens by
enzyme immunoassay prior to use. [
IODINATED IgA
IgA, (mouse myeloma protein),[obtained from G. Kelsoe, Harvard
University, was labelled with 125I[by use of the method described by Bolton
and Hunter (19). The preparation used had approximated 3 x 10^ counts/mm/
ng protein. I
ENZYME-LABELLED ANTIBODIES -
i
Peroxidase from horseradish (E.G. 1.11.1.7), Sigma type VI, Sigma
Chemical Co., St. Louis, MO, sp. act. 274 units/mg, was coupled to the
IgG fraction of goat anti-rabbit IgG or rabbit,anti-horse IgG by use of .
periodate (20). Peroxidase-labelled globulin was separated from unlabelled
material by gel filtration on 2.5 x 80 cm columns of Sepharose 6B.
Fractions that gave maximum absorption in a spectrophotometer at both
403 nm (enzyme) and 280 nm (protein) were pooled and precipitated by
addition of (NH ) SO to 40% saturation. The precipitate was suspended
in a volume of PBs to give a protein concentration of approximately 2 mg/ml,
and dialysed against PBS for 3 days at 4°C. The preparations were tested
for immunologic reactivity in gel-diffusion plates against the appropriate
globulin, and stored at -20°C until used.
ENZYME IMMUNOASSAY '
i
The procedure for the conventional solid-phase enzyme-linked immuno-
assay was based on the ELISA of Engvall and Perlman (1), as modified by
Ruitenberg, et al. (21). Virus antigens diluted 1:4 in PBS were added to
wells of polystyrene Microtiter plktes CCooke Engineeringf Alexandria,
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Virginia), 0,1 ml/well, QJ? to polystyrene spectrophotometer cuvettes
(Variable Volumetrics, Inc., Woburn, Mass.), 0.2 ml/cuvette. Both plates
and cuvettes were pre-treated with 25 yg/ml poly-L-Lysine in PBS (22) to
enhance antigen binding. The antigens were adsorbed 1 h at 37°C, plus
overnight at 4°C.
The plates or cuvettes were washed three times with distilled water or
PBS plus 0.05% (v/v) Tween 20. Dilutions of test and control sera were added
(0.05 ml/well, 0.1 ml/cuvette) and incubated 30 min at 37°C. The samples
were washed as above and peroxidase-conjugated antiglobulin added (0.05 ml/
well, 0.1 ml/cuvette) at a 1:100 dilution. The diluent for the sera and
the conjugates was PBS with 2% w/v BSA and 0.15% (v/v) Tween 20 added.
Optimal dilutions of viral antigens and immunoreagents used were determined
by "checkerboard" titrations. For the Microtiter plate assay the plates
were incubated 30 min at 37°C, washed as above, and 0.2 ml/well substrate
(0.05% 5-amino salicylic acid in distilled water adjusted to pH 6.0 with
IN NaOH, plus 0.005% H^) added plus one drop of 1% w/v gelatin to help
prevent precipitation of the reaction product. After 30 to 60 min at room
temperature, the reaction was stopped with one drop of 1.5M sodium azide.
A red-brown reaction product is formed; the end points were read visually
by comparison with controls (antigen plus normal serum and/or heterotypic
viral antisera, and ant jgen plus PBS).
For spectrophotometric assays, the cuvettes were incubated at 37°C
for 30 min, washed as above, 0.1 ml of phosphate buffer, 0.01M, pH 6 added
and 2.9 ml of enzyme substrate (3 x 10~"% O-dianisidine dihydrochloride,
0.001M HO) in the same buffer added. The reaction was monitored (A)
with a Zeiss PM6-KS recording spectpophotometer and enzyme units calculated.
One unit of peroxidase is the amount of enzyme decomposing 1 y mol of HO
per min at 25°C under the conditions above.
For comparison to the above method, an adsorbed antibody method was
also used. Virus antisera (horse) at a 1:50 dilution were adsorbed to
cuvettes as above. After adsorption and washing the samples, ether-
extracted virus preparations diluted 1:4 in PBS plus 2% BSA and 0.15%
Tween 20 were added (0.1 ml/cuvette), and incubated 1.5 h at room
temperature. The samples were washed with PBS-Tween, 0.1 ml diluted rabbit
antiviral sera (1:100) added, and incubated 30 min at 37°C. Subsequent
treatment and addition of conjugates and enzyme substrates were the same
as that for the adsorbed antigen method above. In addition to the use of
poly-L-lysine to enhance antibody binding to plastics, glutaraldehyde was
also tested, as described (23).
ACTIVATED NYLON
Nylon 6/6 (poly-hexamethylene-adipamide) balls (Precision Plastic
Ball Co., Chicago, IL), 3.2mm diameter, were incubated 30 min in 3,5M HC1.
The balls were washed in distilled water, treated with concentrated acetic
anhydride 1 min, washed in distilled water, washed 1 min in 0.1M carbonate
buffer (pH9.5), washed'dn phosphate-buffered saline and reacted with either
l-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide metho-p-toluene sulfonate
(CMC), 4% v/v in distilled water for 10 min, or with glutaraldehyde (acetic
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anhydride treatment omitted), 8% v/v in distilled water, for 2 h. All
procedures were done at room temperature.
I
Activated nylon powder was also prepared for use in antibody immobiliz-
ation. This was done by dissolving nylon 6/6 in cone. HC1 (approx. Ig
nylon per 5ml HC1), The dissolved[nylon was precipitated as powder
(particles of approx. 1 ym diameter) by dropwise addition to distilled
water at room temperature. The precipitate was washed by centrifugation
at 3500 X g in 0.1M sodium carbonate buffer (pH9.5), washed in distilled
water, and activated for coupling by incubation with glutaraldehyde, as
above. The effectiveness of the HC1 hydrolysis procedure in exposing free
amino groups on nylon balls and powder was monitored by a color reaction
with 2,4,6-trinitrobenzenesulfoniciacid, used as described for determination
of amines (24).
COUPLING OF ANTIBODY AND VIRUS TO 1JIYLON
The activated nylon balls or powder were washed in distilled water and
incubated with IgG fractions of antisera, normal, sera or virus solutions
for 2 h at room temperature plus 18 h at 4°C for CMC-activated nylon and
4 h at room temperature for glutaraldehyde-activated nylon. IgG solutions
used for coupling were diluted,in PBS at concentrations of approx. 100 yg/ml.
Virus was diluted as for the conventional ELISA tests above. The nylon was
washed in PBS, and a 2% w/v solution of BSA in PBS added for 1 h to bind
unreacted CMC or aldehyde groups.
In some experiments, chemical'spacers were added to the modified nylon
to lessen steric hindrance of immobilized antibody. For this purpose,
nylon balls were treated with 3.5M|HC1 as above and rinsed with phosphate-
buffered saline several times, until the pH of the mixture was 7.0. Poly-
L-Lysine, 1 mg/ml, in phosphate-buffered saline, was added followed by a
4% v/v solution of CMC in the same buffer. The nylon was rinsed in phos-
phate-buffered saline, 4% v/v glutaraldehyde was added, and incubated 30
min at room temperature. The'balls[were rinsed again, and solutions of
antibody added, as above. \
ENZYME-LINKED IMMUNOASSAY ON NYLON?
The method used for enzyme-linked immunoassay of antibody on nylon was
similar to that as described for tlie conventional ELISA tests above. Four
to 6 activated nylon balls were placed in 12 x 75mm glass tubes and IgG
fractions of antiglobulin coupled to them as described above. Globulin,
diluted in PBS with 2% w/v BSA and!0.05% v/v Tween 20, was added (0.5 ml/
tube) and incubated 1 h at 37 C. The balls were washed, and 0.25ml of peroxi-
dase-labeled antiglobulin, dilutedi1:100 in the same diluent as that used
for globulin, added. After 30 minjat 37°C, the balls were washed with PBS
with 0.05% v/v Tween 20 added, and[removed to cuvettes. Enzyme substrate
(1.5 ml/cuvette of5-amino-salicylic acid plus HO, ref. 21) was added.
After 15 min incubation at room temperature, the reaction was stopped with
0.1 ml of 1.5M NaN , and the absorbance at 460mm determined. For comparison
to the above, a conventional ELISA;test was done utilizing antibody adsorbed
to 3.2mm polystyrene balls. All assays were done in duplicate, and recorded
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as mean values,
Two methods were used for enzyme-linked immunoassay on nylon for entero-
viruses, the coupled antigen and coupled antibody methods. The general
procedure for the coupled antigen method was to 1) react viral antigens with
activated nylon balls, 2) add antiviral sera, 3) add enzyme-labelled antir
globulin, 4) add enzyme substrate and read reactions. Viral antigens were
either unpurified virus (fluid from infected cell cultures) or partially
purified virus. Virus antigens (purified or unpurified) were diluted 1:4
in PBS and immobilized on activated nylon balls as described. Four to six
balls were placed in 12 x 75mm tubes, and dilutions of virus antiserum in PBS
with 2% w/v BSAQand 0,15% Tween 20 added CO,5 ml/tube). The'tubes were
incubated at 37 C, 30 minutes, washed as above, and 0.25 ml of peroxidase-
conjugated antiglobulin added. The conjugate was diluted 1:100 in PBS with
2% BSA and 0.15% Tween 20 added. After 30 min at 37°C, the balls were
washed 3 times with PBS plus 0.05% Tween 20, and removed to cuvettes.
Enzyme substrate was added and the reactions monitored as above.
The genera.1 procedure for the coupled antibody method was to 1) react
Viral antibody with activated nylon balls, 2\ add viral antigen, 3) add anti-
viral sera, 4) add enzyme-conjugated antiglobulin. When used as an indirect
test, it was necessary to use antiviral sera prepared in two different .
species of animals, igG fractions of viral antisera (horse) diluted in
PBS were coupled to nylon balls as described. Viral antigen, diluted 1:4
in PBS wa.s added (0,5 ml per 4 balls, in 12 x 75 mm tubes) and incubated for
30 min, at room temperature plus overnight at 4°C, The balls were washed
3 times with PBS plus 0,05% Tween 20, and incubated with viral antisera
rabbit, diluted in PBS, The procedure from this point on followed that
described just above. The enzyme conjugate used was directed against the
rabbit antisera,,
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SECTION 5
RESULTS
VIRUS PURIFICATION PROCEDURES
Because there is a limited amount of protein that can be adsorbed to
plastics (12,B), viral antigens were p|artially purified. The efficiency of the
virus purification procedures for removing cellular protein was tested with
uninfected BGM cell cultures, treated in the same manner as virus-infected
ones. The amount of protein removed after each treatment with Bio Rad AG 2X8
anion exchange resin, or by ether extraction was measured by absorption at
280 nra and the method of Lowry, et al. (25). The protein standard used for
comparison was bovine serum albumin , (BSA). It can be seen from the results
shown in Table 1 that one extraction with ether removed as much protein as
one ion-exchange treatment. Subsequent ether: extractions (not shown) did
not give additional protein reduction, but further ion-exchange treatments
did. The same extraction procedures used for virus preparations did not
cause any loss of virus as measured [by the plaque method. For use in enzyme
immunoassay tests below based on adsorption to plastics, one ether extraction
or two anion-exchange treatments proved to be sufficient.
TABLE 1. VIRUS ANTIGEN PURIFICATION PROCEDURE:
REMOVAL OF CELLULAR PROTEIN
Procedure Protein concentration Protein reduction
(mg/ml) (%)
Centrifugation* 0.61 (0)
Ion-ei:change,
extraction 1
2
3
Ether extraction
0.42
0.26
0.15
0.38
31
57
75
38
Cells removed from suspension by centrifugation at 40,000 x g for 30 min.
10
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NON-SPECIFIC ADSORPTION
To test for non-specific adsorption of immunoreagents to plastics, '
peroxidase-labeled anti-rabbit globulin: was diluted in PBS with varying'
amounts of BSA and Tween 20 added and adsorbed to spectrophotometer cuvettes
(1 h at 37°C). The substrate addition and reading of results were as de--
scribed for enzyme immunoassay tests above.
The results in Table 2 show that at lower concentrations of BSA and
Tween 20 that some peroxidase-labeled antiglobulin adsorbs to polystyrene
spectrophotometer cuvettes non-specifically. Concentrations of Tween 20
at 0.15%^ (v/v) plus BSA at 2% (w/v) gave about as low a background as any
combination tried. Also, these concentrations did not interfere with
antigen-antibody reactions, as measured by precipitin tests and enzyme
immunoassay tests. Thus, we selected this combination as a standard
diluent (PBS-BSAT diluent) for both viral antibody and peroxidase-labeled
antiglobulin.
TABLE 2. INHIBITION OF'NON-SPECIFIC ADSORPTION OF IMMUNOREAGENTS
Inhibitor*
Bovine serum albumin Tween 20
(% w/v) (% v/v)
0 0
1
2
3
4
0 0
0.05
' 0.10
0.15
0.20
2 0
0.05
0.10
Enzyme conjugate
adsorbed
(Enzyme units)
0.50
0.16
0.08
0.11
0.06
0.58
0.28
0.04
0.02
0.02
0.08
0.03
0.02
(continued)
11
-------
TABLE 2, (.continued)
Inhibitor*
Enzyme conjugate
Bovine serum albumin
(% w/v)
Tween 20
|(% v/v)
O.;15
0.20
(Enzyme units)
0.01
0.01
Inhibitors added to PBS in the amounts indicated.
ANTIBODY ADSORPTION
To determine the effect of antiibody concentration on adsorption to poly-
styrene, and to find if the above diluent would cause elution of antibody
adsorbed to plastics, rabbit anti-horse IgG labeled with peroxidase was
added to normal rabbit IgG to give ifinal IgG concentrations of 2, 10, and 100
Ug/ml. These were adsorbed to polystyrene tubes in the same manner as that
which is used for an immunoassay (1 h at 37°C plus overnight at 4°C). The
tubes were incubated for 1 h at 37°C with diluents (PBS with BSA and/or Tween
20), washed, and enzyme substrate added. The product was determined spectro-
photometrically, and the number of enzyme units bound to the tubes calculated.
The results are shown in Table 3. It can be seen from the table that the
diluent used did not cause elution of adsorbed antibody at 10 yg/ml. This
could cause some loss of sensitivity if the serum antibody to be adsorbed
needed to be used at a low dilution (<1:100), assuming an IgG level of
10 mg/ml in serum. Prior treatment of the tubes with BSA and glutaraldehyde
(23) did not increase the number of enzyme units that could be adsorbed.
TABLE 3. ADSORPTION OF PEROXIDASE-LABELED ANTIBODY
TO POLYSTYRENE TUBES
Enzyme units bound (%) *
PBS diluent added
BSA (%)
Tween-20 (%)
Antibody protein concentration (yg/ml)
! 2 10 100
0
1
2
0
0.15
0
0.15
0
42.6
38.5
38.5
38.1
30.9
44.4
42.8
50.1
47.6
49.0
47,2
27.1
37.3
30.1
33.1
(continued)
12
-------
TABLE 3. (continued)
PBS diluent added
BSA (%)
Tween-20 (%)
Enzyme units bound (%)*
Antibody protein concentration
10
100
0.15
31.7
41.5
29.0
*Based on the number of units bound/number of units in adsorbing solutions,
after treating tubes coated with peroxidase-labeled antibody for 1 h
at 37°C with the diluent indicated.
ADSORPTION OF ENTEROVIRUSES
The sensitivity of a solid-phase immunoassay depends in part on the
amount of antigen adsorbed to the solid-phase surface, or to an antibody-
coated surface. The amount of enterovirus that adsorbs was determined by
use of 14C-leucine labeled coxsackievirus type B3(CB-3) and/or 14C-leucine
labeled poliovirus type 2 (PO-2). For direct antigen adsorption, labeled
virus at a 1:4 dilution in PBS was adsorbed to polystyrene tubes, as described
in Materials and Methods for enzyme immunoassay, and either tested for virus
adsorbed directly, or incubated with PBS-BSAT diluent prior to assay. The
bottoms of the tubes were _cut off, dissolved in scintillation fluid, and
counted. It can be seen from the results shown in Table 4 that both types
of labeled virus, in which >95% of the radioactivity is virus associated (26)
when purified by column chromatography, adsorbed better when used alone, than
when paritally purified (batch method ion-exchange resin) unlabeled virus
was added. This might be due to the relatively high amounts (ca. 250 yg/mlbf
cellular protein present in unlabeled preparations. For both types of
viruses and preparations used, there was considerable elution of adsorbed
virus by incubation with PBS-BSAT diluent. Based on the initial infectivity
titer (PFU/ml) of input virus used, the maximum amount remaining adsorbed in
unlabeled preparations would be 1.0 x 106 PFU/tube for PO-2, and 8.1 x 105
PFU/tube for CB-3. Virus also adsorbed to the glass tubes used as controls.
There was less initial adsorption for most samples but what did adsorb was
less readily eluted.
TABLE 4. ADSORPTION OF 14C-LEUCINE-LABELED ENTEROVIRUSES
TO POLYSTYRENE TUBES
Virus
preparation
Virus adsorbed (%)*
treated +
polystyrene polystyrene1
Initial
(D*
Eluted
(E) § I E
I E
14
C-labeled PO-2
65.5 30.8 64.5
(continued)
13
40.85
29.1
28.9
-------
TABLE 4.! (continued)
Virus
preparation
Virus adsorbed (%)*
polystyrene
treated
polystyrene'
Initial
(I)*
Eluted
(B)§
E
E
14C-labeled PO-2
+ P0-2e
14C-labeled CB-3
!4C-labeled CB-3
•f CB-3*
11.8
5.5
65.9 34.2
20.0 6.3
11.6
6.1
18.3 14.3
68.6 38.0 32.7 23.9
24.1 8.1 17.0 15.4
*Counts per rain (cpm)/cpm of input virus, x 100.
"•"Tubes treated with poly-L-lysine, as described in Methods.
I
^Percent of input cpm adsorbed to Itubes after three washes with PBS plus
0.05% Tween 20.
^Percent of input cpm adsorbed to tubes after incubation for 1 hr at 37 C
with PBS plus 0.15% Tween 20 and 2% BSA, and washing as above.
$^4C-leucine-labeled enteroviruses plus unlabeled enterovirus at the
concentration used for immunoassays.
For comparison to the direct adsorption of virus to plastics above,
the uptake of virus by antibody coated tubes was measured. Labeled CB-3
and labeled plus unlabeled CB-3 were diluted as above in PBS-BSAT diluent,
incubated for 1.5 h at room temperature, washed, and the tubes assayed for
bound cpm. The results presented in Table 5 show that the maximum amount'of
virus uptake was 5.2% of input virus for labeled virus alone, and 4.1% of
labeled plus unlabeled virus. Thus J the amount of virus available for -
immunoassay is approximately the same as that obtained by direct adsorption
above for unlabeled plus labeled virus, but is less for the labeled virus
alone. The data also show that because there was only a slight increase in
uptake of the labeled virus alone, purity of virus, i.e., absence of cell
protein, is not as important a factor in this type of assay, which is based
on an immune reaction for virus binding.
14
-------
TABLE 5, BINDING OF 14Cr-LEUCINE LABELED COXSACJCIEVIRUS
TYPE B3(CB-3) BY ANTIBODY COATED TUBES
Virus
preparation
14
C-labeled
CB-3
Anti-CB-3
dilution
adsorbed
1:10
1:50
1:100
none
Cpm bound (%) *
1.4
5.2
1.5
0.2
14
C-labeled
CB-3 +CB-3t
1:10
1:50
1:100
none
1.2
4.1
0.9
0.5
*Counts per min (.cpm) bound to tubes/cpm of input virus, xlQQ,
tl4C-leucine labeled CB-3 plus unlabeled CB-3 at the concentration
used for immunoassay.
Use of glutaraldehyde (data not shown), used either directly or in con-
junction with adsorbed BSA, did not increase the uptake of labeled virus by
either of the two methods used.
VIRUS IDENTIFICATION
The Microtiter plate method was used as a preliminary test to insure
that viral antisera did not visibly react with cellular antigens, to demon-
strate reactivity of viral antisera with specific virus types, and to deter-
mine the optimal dilutions of viral antigens and immunoreagents. The
spectrophotometric assay, which yields quantitative data/ was used for the
virus identifications reported here. Enzyme units bound were calculated for
both positive (type-specific) sera and for control sera.
To determine the positive/negative (P/N) ratio required for these
assays to be considered positive, an enzyme-linked immunoassay for cox-
sackievirus type B2 (CB-2) was used. CB-2 antigen was adsorbed to poly-
styrene cuvettes in replicate samples,- sera used were rabbit anti-CB2 and
15
-------
normal rabbit serum both diluted 1:100 in PBSA-T. The indicator of the
reaction was peroxidase-labeled goat anti-rabbit IgG. The data obtained
are shown in Table 6. From these data, it was calculated by the Mann-
Whitney test that the two groups of values represent distinct populations
at the 95% confidence level, and tluit the difference between a test and
control sera would need to be 0.07 4nzyme units for a positive identification.
Thus, from the value shown for normal rabbit serum the P/N ratio for a test
sera would need to be 2.0 or higher!to be considered positive.
TABLE 6. PRECISION OF ENZYME-LINKED IMMUNOASSAY FOR COXSACKIEVIRUS TYPE B-2
Serum
Replicate
Enzyme
units
bound
Mean
units
bound
Standard
deviation
Normal
rabbit serum
Anti-
coxsackie
virus B-2
1
2
3
1
2
3
0.073
0.065
0.076
6.625
6.552
0.575
0.071
•0.006
0.584
0.037
For identification of selected! enterovirus types (poliovirus type 1,
PO-1, coxsackievirus type Bl, CB-l,tCB-2, and coxsackievirus type A-9, (CA-9)
the adsorbed antigen method was utilized. Homotypic and heterotypic rabbit
antiviral sera were used; the indicator of the reaction was peroxidase-
labeled goat anti-rabbit IgG as above. The results given in Table 7 show
that all of the viruses tested are positively identified when compared with
normal sera, giving P/N ratios of 2.5 or higher, and all gave higher P/N
ratios with homotypic than with heterotypic sera. Some of the heterotypic
sera did give stronger reactions than normal sera, but with the exception
of the two CB viruses, all had P/N ratios of less than 1.7. The higher
values obtained with some of the heterotypic sera could indicate a degree
of antigenic relatedness, but more extensive.studies utilizing viral antigens
of greater purity would be required before any definitive .conclusions are
reached. For the coxsackie B viruses, there was significant cross reaction
between types B-l and B-2. Subsequent testing of all the viruses in the B
group (data not shown) indicated that these viruses could be accurately
identified as to group only. Reaction of B-group viral antigens with anti-
sera to virus types in other groups1 was minimal, as measured by either
Microtiter plate assays (not shown)! or by spectrophotometric tests (Table 7).
16
-------
TABLE 7. REACTION OF ENTEROVIRUS TYPES WITH TYPE-SPECIFIC
.AND HETEROTYPIC ANTISERA BY ENZYME-LINKED IMMUNOASSAY
Viral
antisera
used
PO-1
CB-1
CB-2
CA-9
Normal
rabbit
serum
Enzyme units bound
PO-1
0.32
0.09"
0.17
0.14
0.11
*
P/N
2.9
0.8
1.5
1.3
•
(1.0)
Virus
CB-1
' 0.17
0.30
0.25
0.10
0.12
antigen
P/N
1.4
2.5
2.0
0.8
(1.0)
adsorbed
CB-2
0.23
0.21
0.47
0.19
0.14
P/N
1.6
1.5
3.4
1.4
(1.0)
CA-9
0.09
0:08
0.10
0.24
0.07
P/N
1.3
1.1
1.5
.3.4
(1.0)
Enzyme units bound with viral antisera (positive)/units bound with
normal rabbit serum (negative).
To compare the adsorbed antigen with the adsorbed antibody method,
enzyme-linked immunoassays were tested on several virus types. The results
shown in Table 8,indicate that both methods gave approximately the same
results for identification of echovirus type 6 (EC-6), PO-1, CB-1 and CA-9,
with P/N ratios >2.0. Cell antigen preparations were tested against virus-
specific antisera for all types, and gave P/N ratios varying from <1.0 to
the 1.3 value shown for anti-CA-9 in Table 8.
TABLE 8. COMPARISON OF ADSORBED ANTIGEN AND ADSORBED ANTIBODY
METHODS FOR ENTEROVIRUS IDENTIFICATION BY ENZYME LINKED IMMUNOASSAY
Adsorbed antigen method
Antigen
tested
EC-6
PO-1
CB-1
CA-9
' Type-specific
antiserum
0.28
0.34
0,31
0.50
NRS
0,08
0,11
0.13
0,12
P/N
3,5
3,1
2.4
4.2
Adsorbed antibody method
Type-specific
antiserum
0.27
0.24
0.19
0.43
NRS
0,12
0.11
0.09
0.08
P/N
2.3
2.2
2.1
5.3
(continued)
17
-------
Antigen
tested
cell
extract"''
Adsorbed antigen met?
Type-specific
antiserum NRS
0.15 0.11
lod
P/N*
1.3
Adsorbed antibody method
Type-specific
antiserum NRS
0.10 0.08
P/N
1.3
*Enzyme units bound with viral antisera (positive)/units bound with
normal rabbit sera (NRS) (negative).
"""Serum used for cell extract was anti GA-9.
SENSITIVITY OF ASSAYS
The sensitivity of the assays for enteroviruses, in terms of the number
of plaque-forming units (PFU) required to give a positive test, was deter-
mined for CA-9. Virus was adsorbed!to polystyrene tubes at dilutions of 1:5
to 1:500 from an initial concentration of 2.0 x 10? PFU/ml for CA-9, and
5.6 x 10^ PFU/ml for EC-6 which was jused as a control for possible cell anti-
body presence in viral antisera. Rabbit anti-CA-9 sera was added at a
1:100 dilution, followed by goat anti-rabbit IgG labeled with peroxidase.
The substrate used to indicate presence of bound enzyme was 0.08% w/v
5-aminosalicylic acid (5-AS) in distilled water at pH6.0 plus 0.005% H^.
The absorbance at 460 nm was measured after 15 min reaction. The results
in Fig. 1 show that the highest dilution of CA-9 giving A4go values 2.0 or
more times that of either the EC-6 or cell antigen controls was between
1:200 and 1:100, which is equivalent to 1.0 to 2.0 x 105 PFU/assay'-.tube.
A similar assay was done to determine the titer of antisera used for
CA-9 identification. Virus and cell antigen was adsorbed to polystyrene
tubes at a 1:10 dilution, and rabbit anti-CA-9 sera was added at dilutions
from 1:50 to 1:1600. The titer of the antisera used, after absorption with
cell debris, was 1:1600 as determined by plaque reduction. Serum controls
tested were rabbit anti-CB-1 and normal rabbit serum. Peroxidase-labeled
antiglobulin and 5-AS substrate were added as above. The results in Fig. 2
show that up to a 1:800 dilution ofjanti-CA-9 sera was clearly positive
(P/N=2.1 from data), when compared with the control most strongly reactive.
18
-------
0.8
0.7
0.6
0.5
.
0.3
0.2
0.1
// I
10 50 100 200
1/ Antigen Dilut ion
500
Fig. 1. Detection limits of coxsackievirus type A9 (CA-9) by enzyme-linked
immunoassay. Virus concentration per sample tube at a 1:5
dilution was 4 x 106 PFU. Symbols (O), CA-9 antigen reacted with
anti-CA-9 sera; (®) , CA-9 antigen reacted with anti-echovirus type
6 sera; (D), cell antigen reacted with anti-CA-9 sera.
19
-------
< 0.4
SO
100 200 400
1/Serum Dilution
goo
1*01}
Fig. 2.
Titration of antibody to coxsackievi^us type A-9 CCA-r9) by enzyme-
linked immunoassay. CA-9'was used at a 1:10 dilution, as was cell
antigen. Symbols (O), CA-9 antigen reacted with anti-CA-9 sera;
(9) , cell antigen reacted|with anti-CA-9 sera; (D) , CA-9 antigen
reacted with anti-coxsackievirus type B2 sera; (•) , CA-9 antigen
reacted with normal sera.
20.
-------
IMMOBILIZED ANTIBODY FOR IMMUNOASSAY
Because of the ^problem of elution of virus and immunoreagents non-
specifically adsorbed to plastics, we tested the effectiveness of covalently
linking antibody to nylon, utilizing rabbit IgG as a, model antigen. To do
this, an enzyme-linked immunoassay or nylon for rabbit IgG was done. Goat
anti-rabbit IgG was coupled to nylon balls through amino groups with glutaral-
dehyde or through carboxyl groups with CMC as described. HCl-treated nylon,
which we found in preliminary experiments to adsorb antibody more readily
than untreated nylon, was used as a nonr-coupled control. Dilutions of rabbit
IgG were added; peroxidase^labelled goat anti-rabbit IgG was the indicator
of the reaction.
The results in Fig. 3 show the relative sensitivity of*the assay
procedures. All showed a linear response over a, 50T-5000 ng range, with
the glutaraldehyde coupled immunosorbent showing the greatest capacity for
binding IgG. Antibody adsorbed to HCl-treated nylon showed a far less
binding capacity than either of the covalently-linked immunosorbents.
Although the purpose of the experiment was to compare relative, not
absolute sensitivity, the coupled nylon gave about the same sensitivity for
IgG as that reported for methods utilizing insoluble antibody or acrylamide
bead immunosorbents (27), The antisera used in the above experiment and the
ones to follow were not immunospecific, i.e. not purified by affinity
chromatography,
A problem that has been noted for immobilized enzymes is that enzymes
bound close to a polymer surface may cause a decrease in enzyme activity
due to steric hindrance. To overcome this, straight-chain spacers, such
as polyamino acids, have been used (.15) . Because there may be loss of
apparent avidity of antibody covalently bound to solids, also due to steric
hindrance (28,29), an immunoassay utilizing spacers was done. To test if
spacers would enhance the binding capacity of goat anti^rabbit IgG immobilized
on nylon, poly-L-lysine was bound to nylon balls with CMC prior to coupling
antibody with glutaraldehyde. An enzyme-linked immunoassay for rabbit IgG
was done in the same manner as that described above (Fig. 3). The results
presented in Table 9 show that the use of poly-L-lysine as a spacer increased
the total binding capacity of the immunosorbent for IgG at high concentrations.
The sensitivity was about the same as that obtained by immunosorbents
prepared with glutaraldehyde alone, but was more sensitive than that obtained
by use of CMC alone (Fig. 3) .
The relative efficiency of immunosorbents prepared by covalent coupling
were higher for binding IgG, as measured by enzyme immunoassay, than non-
coupled ones (Fig. 3, Table .9). To determine if the efficiency of immuno-
assay was correlated with the efficiency of antibody attachment, IgA
(mouse myeloma protein) labelled with 125I was adjusted to 1 mg/ml with
unlabelled immunoglobulin and reacted with nylon balls. The nylon balls
were activated with glutaraldehyde or CMC, or were HCl-treated or untreated.
After immunoglobulin attachment, the balls were washed in a solution of PBS
plus 2% w/v BSA and 0.15% v/v Tween 20,. and incubated further in the same
solution for 3 h at room temperature, or 3 h at room temperature plus 21 h
at 4°C. This solution is the one most often used for preventing non-specific
21
-------
1.2
1.0
0.8 r
0.6
0.4
0.2
0
5000
500 50 5
IgG, ng/assay
0.5
Pig. 3. Enzyme-linked imrounoassay of rabbit IgG on nylon balls coupled
with goat anti-rabbit IgG, (O). antiglobulin glutar-aldehyde
coupled; (•) antiglobulin CMC coupled; (A), antiglobulin
adsorbed (HC1-treated nylon).
22
-------
TABLE 9. ENZYME-LINKED IMMUNOASSAY OF BABBIT IgG. USE OF
SPACERS FOR COUPLING GOAT ANTI-RABBIT IgG TO NYLON BALLS
Rabbit IgG
assayed (ng/ml)
Gliitaraldehyde
A460
5000
500
50
5
, of
1.
0.
0.
0.
0.
144
540
081
042
015
P/N*
76.2
36.0
5.4
2.8
1.0
Poly-L-lysine+CMC
A460
1.
0.
0.
0.
0.
333
581
086
032
016
P/N
83.
36.
5.
2.
1.
3
3
4
0
0
HC1 -treated
A460
0.333
0.200
0.040
0.014
0.020
P/N
16.9
10.0
2.0
0.6
1.0
Untreated
A460
0.222
0.125
0.040
0.014
0.021
P/N
10.6
5.9
1.9
0.7
1.0
P/N, ratio of A460 IgG samples/A of bovine serum albumin control samples.
A P/N £ 2.0 shows differences between test and control samples at a 95%
confidence level (Mann-Whitney test) .
'''Bovine serum albumin, 1 mg/ml, was used as control.
adsorption in enzyme immunoassays, and, as we showed previously
(Tables 3 and 4) , will desorb proteins attached by simple adsorption to
plastics. After incubation, the balls were washed in PBS and counted in
an automatic gamma counter.
The results given in Table 10 show that little if any desorption
occurred on nylon balls treated with glutaraldehyde , which was also about
15 times as effective in attaching immunoglobulin as untreated nylon. CMC-
coupled and HC1 -treated nylon balls gaVe intermediate values, all of which
correlated well with the results shown in Fig. 3. Because desorption of
antibody from the surface of a solid-phase carrier can bind free antigen
and thus lower the sensitivity of an immunoassay, the advantage of covalent
coupling is apparent.
23
-------
TABLE 10. EFFICIENCY OF ATTACHMENT OF
125
I-LABELED IgA TO NYLON BALLS
Desorption
time*
(h)
0
3
24
1 ' t
IgA bound (%)
Glutaraldehyde
20.9
21.0
18,9
Nylon coupling
| CMC
15.1
10.3
9.3
agent or nylon
HC1
11.5
9.3
7.3
treatment
None
3.1
1.5
1.3
Time treated with phosphate buffered saline with 2% w/v bovine serum
albumin and 0.15% v/v Tween-20 added.
t!25
125
I-labelled IgA remaining/total I-labelled IgA input, X 100.
EFFICIENCY OF IMMOBILIZATION
The relative amount of immunoglobulin (Ig) that can be immobilized on
various plastics was also determined. The amount of IgG in yg/mm2 that
could be immobilized on nylon (Table 11) was 37 times the amount which
could be immobilized on polystyrene, the plastic most often used for ELISA
tests, or on poly-methyl-methacrylate (PMMA), a plastic used in some types
of Microtiter plates.
TABLE 11 COMPARISON OF METHODS FOR IMMOBILIZING IgG ON PLASTICS
Desorption time*
Plastic Treatment
Nylon Poly-L-lysine+CMC
Glutaraldehyde
CMC
HC1
None
None
Percent
bound lag
31.7
20.9
15.1
11.3
2.5
(continued)
24
2
IgG/mm
0.98
0.65
0.47
0.35
0.08
24
Percent
bound
23.9
18.9
10.3
7.3
1.3
hours
2
yg IgG/mm
0.78 .
0.59
0.32
0.23
0.04
-------
TABLE 11 (continued)
Desorption time*
Plastic
Treatment
None
24 hours
Percent
bound yg IgG/mm
Percent
bound yg IgG/mm
PMMA None
Polystyrene None
1.1
0.7
0.03
0.02
0.8
0.7
0.02
0.02
*Time treated with phosphate-buffered;saline with 2% w/v bovine serum
t!25i_iabelled Ig remaining/total I-labelled Ig input, X 100.
RE-USE OP IMMOBILIZED ANTIBODY
Another advantage of covalent coupling of antibodies to insoluble
carriers is the potential for antibody re-use. This was tested for goat
anti-rabbit IgG coupled (glutaraldehyde method) to nylon balls. An enzyme-
linked immunoassay was done for rabbit IgG with peroxidase-labelled goat
anti-rabbit IgG as the indicator of the reaction, as above. After the
results were recorded forthe initial reaction (Table 12), the balls were
regenerated by treatment with low pH buffers, as indicated in the table.
Between assays, enzyme substrate was added to the regenerated balls to insure
that no residual enzyme activity remained. Once this was established, the
immunosorbents were tested for IgG binding as before (data not shown),
regerated and re-assayed. The results given in Table 12 show that both
the A460 and P/N values obtained were approximately the same at all IgG
concentrations tested, indicating that cpv'alently coupled antibody
remains bound to the nylon used, and the bound antibody is suitable for
re-use at least two times.
25
-------
TABLE 12. RE-USE OF NYLON BALLS COUPLED WITH GOAT ANT^r-RABBET EgG FOR
ENZYME LINKED IMMUNOASSAY
Enzyme-linked immunoassay of rabbit IgG
Rabbit IgG Initial
assayed (ng/ml) assay
5,000
500
50
5
0§
A460
1.269
0.599
0.111
0.045
0.015
P/Nt
84.6 i
39.9
7.4
3.0
1.0
Treatment prior to re-assay
HCl-NaCl*
A460
1.161
0.537
0.103
0.057
0.014
P/N
82.9
38.4
7,4
4.1
1.0
Glycine-HClt
A460
1.210
0.557
0.098
0.026
0.015
P/N
80.7
37.1
6.5
1.7
1.0
*Nylon treated for 0.5 h with 0.003M HC1 in 1 M NaCl, and washed in
phosphate-buffered saline.
'''Nylon treated for 0.5 h with 0.1M glycine-HCl (pH 2.2), rinsed in 1 M
NaCl, and washed in phosphate-buffered saline.
P/N, ratio of £450 of I9G samples/A460 of bovine serum albumin control
samples.
^Bovine serum albumin, 1 mg/ml, was used as control,
IMMUNOASSAY ON NYLON POWDER
The use of nylon ba.lls provides a convenient solid-phase for- performing
enzyme-linked immunoassays, and the washing procedures,require no centrifug-
ation. The use of nylon powder, though, should provide more surface area per
volume or weight of nylon, and thus be a mo-re efficient immunosorbent.
To test the usefulness of nyIon, powder for antibody immobilization, an
enzyme-linked immunoassay of rabbitj IgG was done. Approximately 0.5ml of
activated, packed nylon powder was washed in PBS and suspended in 0.5ml of
an IgG fraction of goat anti-rabbit; IgG. After 2 h at room temperature, 0.5ml
of PBS was added and the mixture inbubated for 18-24 h at 4 C. For assay, the
anbibody-coupled powder was washed 3 times in PBS by centrifugation at 3500 x
g, and suspended 1:5 in PBS plus 2% w/v BSA and 0.05% Tween 20. A 0.02ml
amount of this suspension was added to 0.5ml of rabbit IgG, diluted in PBS
containing 10% v/v normal goat serum, and incubated 1 h at 37 C. The procedures
from this point on were the same as that described for immunoassay on nylon
balls, except that washing procedures were done by centrifugation. Peroxi-
dase-labelled goat anti-rabbit IgG was the indicator used. The results given
26
-------
in Table 13 show that the sensitivity of enzyme-linked immunoassay on nylon
powder was approximately that obtained by use of nylon balls, with low back-
ground values for control samples (normal goat sera).
TABLE 13. ENZYME-LINKED IMMUNOASSAY OF RABBIT IgG IMMOBILIZED ON NYLON POWDER
Rabbit IgG
assayed (ng/ml)
5000
500
50
5
Normal goat
serum
Nylon
Glutaraldehyde
A460
0.735
0.433
0.045
0.014
0.008
P/N*
91.9
54.1
5.6
1.8
1
powder treatment
None
A460
0.478
0.181
0.061
0.035
0.051
P/N
9.3
3.5
1.2
0.7
1
*P/N, ratio of A IgG samples/A of normal goat serum control samples.
BINDING OF ENTEROVIRUSES TO ACTIVATED NYLON,
Direct coupling of virus to activated nylon wa,s tested with Oleucine
labelled poliovirus 2 (PO-2) and coxsackievirus B3 (CB-3). Dilutions of
each, 1:10 in PBS, were added to untreated nylon or glutaraldehyde-activated
nylon balls (0.5 ml/6 balls), incubated and treated with BSA as for the IgG
experiments'above, and washed. The amount of virus remaining was determined
by counting x C disintegration in a liquid scintillation spectrometer. The
results in Table 14 show that the coupling procedure was about two to three
times as effective in attaching virus than that obtained by simple non-
specific adsorption (untreated nylon or HCl-treated nylon).
TABLE 14. COUPLING OF ENTEROVIRUSES TO NYLON BALLS
Virus
added
14C-labelled PO-2
14C-labelled CB-3
Active ted
nylon
44.0
45.0
Virus bound (%)
HCl-treated
nylon
18.0
22.0
Untreated
nylon
10.0
7.6
27
-------
There was also more virus bound than was obtained by simple adsorption to
polystyrene (Table 4), although the results aren't directly comparable.
I
ENZYME-LINKED IMMUNOASSAY OF ENTEROVIRUSES ON NYLON
Assay of enteroviruses by use bf activated nylon was done by a di-
rect and indirect method, specific |enterovirus antisera (rabbit) or control
sera was added, followed by goat anti-rabbit IgG labelled with peroxidase.
Enzyme substrate was added, and thej product read at 460 mm after 10 min.
For the indirect test, specific horse antiviral sera was coupled to nylon
balls as described for antibody rabbit IgG above. These were incubated with
virus, and rabbit antiviral sera added for specific identification as in the
direct test. The results are shown1 in Table 15. By the direct method,
poliovirus 2 (PO-2) and poliovirus [3 (PO-3) could be identified, and the
test could distinguish them from the other poliovirus types. By the indirect
test, PO-2 gave a strong reaction cpmpared with coxsackievirus type B2 (CB-2),
and PO-3 was distinguished from pol|iovirus 1 (PO-1) . In both cases, the use
of chemical coupling methods gave better results than the adsorption methods
used in the conventional ELISA tests, especially for the coupled antibody
method. However, it was not possible to obtain as low background (normal
sera) values as those obtained for the experiments utilizing rabbit IgG -as
a model antigen.
TABLE 15. ENZYME .I^MUNQASSAY OF ENTEROVIRUSES ON ACTIVATED NYLON
Method
Virus
Type
Antisera
Enzyme
Units
Bound
P/N*
Direct
PO-2
PO-2
PO-1
NRS
0.24 3.0
0.075 0.9
0.08 1.0
PO-3
PO-3
PO-2
NRS
0.26
0.16
0.10
2.6
1.6
1.0
Indirect?
PO-2
PO-2
CBT2
NRS
0.49
0.11
0.08
6.1
1.4
1.0
Legend (continued)
28
-------
P/N, ratio of enzyme units bound with viral antisera/units bound with
normal rabbit sera (NRS),
"^Virus coupled to nylon,
+Viral antisera coupled to nylon.
29
-------
SECTION 6
DISCUSSION
The enzyme-linked immunoassay i technique used was found suitable for type
specific identification of the enterovirus types selected, and for group
identification of coxsackie B viruses. The reason for the lack of specific-
ity within the B group is not clear, but may be related to the viral antigens
that adsorb most strongly to the plastic, and/or to the antisera used. Group
antigens have been demonstrated in this group (3), which could adsorb more
strongly, and for diagnosis of coxsackie B viruses by immunofluorescence,
neither horse nor rabbit antisera were found entirely satisfactory for type-
specific identification, whereas hamster antisera and mouse immune ascitic
fluid were (8).
Because of the low amount of yiral antigen present in enterovirus prep-
arations (ca. 1 ng/108 virions) and because some of the types are antigen-
ically related, immunoassay of these viruses requires optimal conditions.
It was found that for direct adsorption of viral antigen to polystyrene tubes
or plates, some type of partial purification was necessary. Extraction of
the virus samples with ether or a batch-method treatment with an anion-
exchange resin removed sufficient cellular protein to permit adsorption,
although maximum adsorption was obtained with virus purified by passage
through a column of DEAE-Sephadex A-50. For routine diagnostic work the
simpler methods are sufficient, and "no gain in accuracy was obtained by use
of purified virus (data not given).
The precision of the assay was high, when a given set of reagents were
tested under the same conditions. Over a period of time, when different
reagents are used, the precision would undoubtedly be less, although our
background values for normal sera were quiJpe similar over periods of several
months. Most reports consider a positive/negative ratio of 1.8:1 or more to
indicate a positive result in an immunoassay. The ratio selected, though,
depends on the precision of an individual set of assays, as there is not
as yet a standard method. Increasing the precision can be done by decreas-
ing non-specific adsorption; we found that use of PBS plus 2% BSA and 0.15%
Tween 20 for diluting immunoreagents gave the lowest background values.
Others have found that PBS plus 0.05% Tween 20 is sufficient for inhibiting
non-specific reactions (2). In our study, use of this diluent permitted 0.28
units of enzyme conjugate to adsorb to polystyrene cuvettes, which is as
high a number as some of our positive tests. However, the disadvantage of
using diluents with inhibitors is ^hat desorptlon of antigen and antibody
during an immunoassay is increased}. We found that for adsorbed viral antigen,
30
-------
a high percentage of virus elates when incubated with the diluents us.ed f,or-
immune-assay, Desorption of adsorbed antibody- was minimal at concentrations
of 2 and 10 yg/ml. Engvall, ejt aJU (31), however, found that desorption
of antibody was about 40% for antibody adsorbed at 2 yg/ml and incubated
with PBS plus 0.05%
-------
selected enterovirus types, for developmental purposes. At the present
stage of development, the method would be useful for presumptive identifica-
tion of group B coxsackieviruses in situations where clinical or other
evidence suggests involvement of these viruses, as an alternate means of
confirming a type-specific identification of an enterovirus isolate, or as
a means to verify the identity of laboratory strains, but not for direct
identification of virus in water. ; If the method is to be useful for
identification of field isolates, which could be any of a large number of
enterovirus types, the use of pooled sera in an intersecting serum scheme
(34) would be needed for rapid typing by any form of enzyme-linked immuno-
assay. Preliminary data not reported here indicate that some virus types
can be identified by use of pooled sera whereas others cannot, due to cross-
reaction, which is a current limitation of the method. The reasons for
these cross-reactions and possible means to eliminate them need to be
investigated.
32
-------
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Riggs, J.L., and G.C. Brown. Application of Direct and Indirect
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Taber, L.H., R.R. Mirkovic, V. Adam,. S.S. Ellis, M.D. Yow, and -J.L.
Melnick. Rapid Diagnosis of Enterovirus Meningitis by Immunofluorescent
staining of CSF Leukocytes. Intervirol., 1:127-134, 1973.
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11. Herrmann, J.E., S.A, Morse and M,R, Collins-. Comparison of Techniques*
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12. Herrmann, J.E., and M.R. Collins. Quantitation of Immunoglobulin
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15. Sundaram, P.V. Potentials of Enzymes Attached to Nylon Tubes in
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35
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO
EPA-600/4-80-034
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Identification and Detection of Water-B0rne Viruses by
Immunoenzymatic Methods
5. REPORT DATE
JUNE 1980 ISSUING DATE.
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
John E. Herrmann, Ph.D.
8. PERFORMING ORGANIZATION REPORT NO.
3. PERFORMING ORGANIZATION NAME AND ADDRESS
Harvard School of Public Health
Boston, MA 02115
10. PROGRAM ELEMENT NO.
A38B1D
11. CONTRACT/GRANT NO.
R-803360
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
Environmental Monitoring and Support Lab.-Cincinnati
Office of Research and Development
U. S. Environmental Protection Agency
Cincinnati, OH 45268
Final 4/75-3/80
14. SPONSORING AGENCY CODE
EPA/600/06
15. SUPPLEMENTARY NOTES
Project Officer: Dr. Robert S. Safferman (513) 684-7334
16. ABSTRACT
A quantitative enzyme-linked immunosorbejrt assay (ELISA) was used for identification
of viruses selected as representative wa;ter-borne viruses: poliovirus 1, echovirus 6,
coxsackievirus A9, and coxsackie B viruses. Partially purified viral antigens or
virus-specific antibodies were adsorbed ;to polystyrene spectrophotometer cuvettes,
which permitted the assays to be reported and compared in terms of enzyme units
specifically reacting. Inhibitors in diluents used to prevent non-specific adsorption
of immunoreagents caused desorption of virus or antibody during an immunoassay; the
amount of virus desorption varied with the type of preparation used, and antibody
desorption was dependent on the concentration of antibody initially adsorbed. For
higher percentage of virus could be immobilized by this method than was possible by
adsorption to polystyrene, and enzyme-linked immunoassay on nylon was sufficiently
specific to differentiate the three poliovirus types.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Viruses,* water, wastewater, immunoassay
Detection, identification.
monitoring
06M
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport/
Unclassified
21. NO. OF PAGES
44
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
36
U.S. GOVERNMENT PRINTING OFFICE: 1980--657-165/0003
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