Demonstration of type-specific influenza antibody in mammalian and avian sera by immunodiffusion.
ABSTRACT The detection of antibody against the ribonucleoprotein antigen of influenza virus is useful because its type-specificity allows the use of serological surveys to detect evidence of recent infections. Antigenic differences between strains limit the usefulness of the techniques, such as the haemagglutination-inhibition test, that detect antibody against surface antigens.This paper describes an agar-gel precipitation (AGP) test that will detect type-specific antibody in avian or mammalian sera. Convalescent levels of antibody against either type A or B influenza virus were demonstrated in human sera. Positive but inconsistent results were obtained with swine sera. The antigens used in the AGP test are non-infectious and stable. The test is easy and economical to perform. Its sensitivity compares favourably with that of the complement-fixation test using human and equine sera.While not a replacement for any of the serological tests at present in current use, the AGP test should prove useful in a variety of diagnostic and research situations.
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ABSTRACT: Techniques for studying the diffusion of antigens and antibodies in an agar medium have been applied to several strains of influenza virus. When either rabbit hyper-immune or convalescent ferret sera were mixed with agar, introduced into small tubes and the virus preparations layered upon the solid mixture, bands of precipitate formed in the appropriate tubes. This phenomenon has been shown to be strain-specific and therefore was interpreted as being due to the union of virus antigens and corresponding antibodies. The strain-specific lines of precipitate were also obtained when the virus antigens and antibodies were allowed to diffuse toward each other from depressions in agar contained by Petri dishes.The Journal of Immunology 04/1953; 70(3):321-5. · 5.52 Impact Factor
- Acta virologica 08/1966; 10(4):281-90. · 0.76 Impact Factor
- Bulletin of the World Health Organisation 02/1964; 31:129-32. · 5.25 Impact Factor
Bull.Org.mond.Sant) 1970, 42, 779-785
Bull. Wid Hlth Org.
Demonstration of Type-specific Influenza Antibody
in Mammalian and Avian Sera by Immunodiffusion
C. W. BEARD 1
The detection of antibody against the ribonucleoprotein antigen of influenza virus is
useful because its type-specificity allows the use ofserologicalsurveys to detect evidence of
recent infections. Antigenic differences between strains limit the usefulness ofthe techniques,
such as the haemagglutination-inhibition test, that detect antibody against surface antigens.
This paper describes an agar-gelprecipitation (AGP) test that will detect type-specific
antibody in avian or mammalian sera. Convalescent levels ofantibody against either type A
or B influenza virus were demonstrated in human sera. Positive but inconsistent results
were obtained with swine sera. The antigens used in the AGP test are non-infectious and
The test is easy and economical to perform. Its sensitivity compares favourably
with that ofthe complement-fixation test using human and equine sera.
While not a replacement for any of the serological tests at present in current use, the
AGP test shouldprove useful in a variety ofdiagnostic and research situations.
Influenza infections result in the production of
antibody against the type-specific ribonucleoprotein
(RNP) or soluble (S) antigen and the strain-specific
surface or envelope antigens of the virus. The detec-
tion of the antibody against the RNP antigen is
useful because its type-specificity allows the use of
serological surveys to detect evidence ofrecent infec-
tions. Antigenic differences between strains limit the
usefulness of the techniques that detect antibody
against surface antigens such as the haemagglutina-
tion-inhibition (HI), virus neutralization and strain-
specific complement-fixation (CF) tests.
Examination of sera for evidence of avian influ-
enza antibody by the HI test requires numerous
strains for antigenic coverage broad enough to give
meaning to negative results. Although all non-human
influenza viruses have been of type A, the possibility
that the antibodies are due to a new strain within
that type cannot be excluded by negative HI results,
regardless of the number of antigens used (Pereira
et al., 1966). The use of a type-specific serological
test would reduce the number of antigens needed to
no more than three (type A, B, and C) with human
serum and to one (type A) for lower animals.
Laboratory, Agricultural Research Service, US Department
of Agriculture, Athens, Georgia 30601, USA.
To utilize the benefits of type-specific antibody
identification, workers have generally relied on the
complement-fixation test and only with mammalian
sera. This report describes the use of an agar-gel
precipitation (AGP) test on a glass slide for the
demonstration of antibody against the RNP antigen
of types A and B influenza viruses in mammalian
and avian sera.
Jensen & Francis (1953) were the first to apply
the immunodiffusion technique to demonstrate virus-
serum antibody reactions. They used influenza virus
to show strain specificity with antigens prepared
from infected allantoic fluids. Immunodiffusion was
also used (Styk & Hana, 1966; Hana & Hoyle, 1966)
with disrupted influenza A and B viruses to demon-
strate numerous antigens. Schild & Pereira (1969)
recently reported the use of disrupted influenza A
viruses with rabbit antiserum to demonstrate that
is composed of a single antigenic
MATERIALS AND METHODS
The influenza viruses originally obtained from
Dr B. C. Easterday, University of Wisconsin, USA,
and Dr Gerhard Lang, University of Guelph,
Ontario, Canada, were prepared in 10-day embry-
The strains used were as follows:
C. W. BEARD
Japan/305/57, A/Swine/S15/31, A/Equi-1/Prague/56,
Duck/Czechoslovakia/56, and Duck/England/62.
Ten-day embryonated chicken eggs were inocu-
lated with 0.1 ml of 10-2 dilutions of stock virus and
incubated at 37°C for 24 hours. The eggs were
removed from the incubator, the small end of the
egg shell was cut off with scissors and the embryo
and its attachments were discarded. The chorio-
allantoic membrane (CAM) was left adhering to the
shell wall. The CAM was then removed with for-
ceps. Sterile phosphate-buffered saline (PBS) (0.8%
NaCl, pH 7.2) was used to wash the membranes,
which were re-collected by pouring into a gauze
funnel. The membranes were then homogenized in
15 000 rev/min for 1-2 minutes. After homogeniza-
tion, the suspension was put through 3 freeze-thaw
cycles before being centrifuged at approximately
700 g. The liquid phase was carefully withdrawn
and placed in screw-capped vials. Approximately
0.5 ml of antigen resulted from each membrane used.
The antigen was then treated with 0.1 % formalin
at 37°C for 36 hours to destroy any infectivity.
If the antigens formed a gel during the inactivation,
they were shaken vigorously and a small amount of
PBS was added. The antigen was centrifuged again
at 700 g after the formalin treatment to remove
aggregates of cellular debris and stored at -20°C.
The sera used in these studies were obtained from
different laboratories. They will be described at the
same time as the test results.
The medium consisted of 0.7% Ionagar No. 2
(Colabs), 8.0% NaCl, and 1: 10 000 thiomersal in
distilled water buffered to pH 7.2 by the addition
of 10 ml per 100 ml of 0.1 M phosphate buffer.
The medium was melted in the autoclave at 121°C
for 5 minutes, stored at room temperature, and
melted again as needed.
Ordinary microscope slides, frosted on one end,
were cleaned with methanol. They were positioned
on a level surface with the frosted side upwards
and 1 ml of the melted agar was placed on the clear
section with a pipette.
Wells were cut in the agar after it was cool, using
a cutter made from 7 reamed copper or brass tubes
soldered to a brass plate.
positioned around a centre well. The wells were
4 mm apart and 4 mm in diameter. After cutting,
the agar plugs were removed with a Pasteur pipette
attached with rubber tubing to a vacuum flask.
When cutting the wells, care was required to
prevent disturbing the union between the agar and
the surface of the slide. The agar came off during
the washing and staining process and reagents leaked
under the agar when this attachment was broken.
Slides were held in a humid container until the
wells were cut and the reagents added. The slides
were then returned to a level position in the con-
tainer and kept at room temperature (23°C) for
20-48 hours before examination over indirect light.
The reagents were added using disposable Pasteur
pipettes approximately 10 cm long fitted with a
Slight pressure on the bulb
would cause reagent to accumulate on the tip of
touched to the bottom. Reagent identification was
made on the frosted portion of the slide.
Six wells were evenly
It would fill the well when carefully
After 20-48 hours at room temperature, slides
were examined and discarded or placed in PBS for,
24 hours at room temperature to wash out unpre-
cipitated antigen and serum protein prior to staining.
The frequence of changing the PBS depended on
the volume of PBS and the number of slides. The
slides were placed in an " on-edge " position in a
glass slide-rack. On removal from the PBS the slides
were placed in a solution of 0.1 % thiazine red in
1% acetic acid for 15-20 minutes (Crowle, 1961).
They were then removed and decolorized in 1 % acetic
acid for several hours until the lines could be readily
distinguished from the background agar. Slides were
then left in the slide-racks to dry at room temperature
under a dust cover. Cover-slips were mounted on
the slides with Permount (Fisher), providing a
permanent record of the reaction.
Influenza occurred in a stable of horses at the
University of Wisconsin in December 1968 after
they had been assembled over a period of 30 days
from several areas of the USA." Both A/Equi-1 and
1B. Tumova, to be published.
IMMUNODIFFUSION TEST FOR TYPE-SPECIFIC INFLUENZA ANTIBODY
RESULTS OF AGAR-GEL PRECIPITATION (AGP) AND COMPLEMENT-FIXATION (CF)
TESTS ON SERA FROM HORSES DURING AND AFTER A NATURALLY OCCURRING INFECTION
WITH EQUINE INFLUENZA VIRUSES
Date of bleeding
20 Dec. 1968
20 March 1969
11 June 1969
aAcute phase bleeding.
b - -absence of precipitin line; +=presence of precipitin line; NT= not tested.
cCF titres represent the reciprocals of serum dilution at the 50 % end-point according to the method of Pereira et al. (1964);
NT = not tested. Sera and CF titres provided by Dr B. Tumova.
A/Equi-2 viruses were isolated. Sera were obtained
at intervals after the illness, tested by CF and
provided by Dr Tumova. Results of the AGP test
with type A NP antigen prepared with Turkey/
Wisconsin/66 virus are shown in Table 1 together
with the CF titres. The presence of antibody against
the A influenza NP antigen could be demon-
strated as long as 6 months after the illness with
the AGP test (Fig. 1). The intensity of the precipita-
tion lines in the last samples was considerably less
than that of those observed with earlier sera. The
lines also formed closer to the serum wells with the
later samples. The results indicated that 3 or 4 of
the 8 horses had experienced an influenza infection
within the previous 6 months as evidenced by the
results of the AGP and CF tests on the acute-phase
Paired human sera together with their CF titres
were furnished by Dr Marion Coleman, WHO
International Influenza Center for the Americas,
Atlanta, Ga., and by Mrs Julia Eubanks, Virology
Section, Georgia Department of Public Health. The
sera were obtained from patients during the acute
phase of their illnesses and 12-21 days later. AGP
tests were performed with type A antigen prepared
with Turkey/Wisconsin/66 virus and with type B
antigen prepared with B/Johannesburg/59
Results indicate good correlation between the AGP
and CF tests with both type A and type B antigens
(Table 2) when precipitin lines are graded as to
intensity and location. Some patients who experi-
enced a rise in type B antibody titres had constant
levels of type A antibody activity (Fig. 2).
Chicken and turkey antisera furnished by Dr Ger-
hard Lang and Dr B. C. Easterday were tested by
the AGP test with antigens prepared from several
different avian influenza isolates. Some of the sera,
together with a fowl-plague antiserum from the US
Department of Agriculture, Plum Island Animal
Disease Laboratory, were placed in the outer wells
with a type A NP antigen in the centre well. Lines
of precipitation formed between the antigen well
and sera representing 5 of the avian subtypes. The
lines were continuous, with no spurs between sera,
indicating antigenic identity (Fig. 3). No line formed
where normal chicken serum was used.
NP antigens prepared with 5 viruses that repre-
sented 4 avian influenza subtypes were placed in the
outer wells and tested against convalescent serum,
in the centre well, prepared
in chickens, with
C. W. BEARD
RESULTS OF AGAR-GEL PRECIPITATION (AGP) AND COMPLEMENT-FIXATION (CF)
TESTS ON ACUTE AND CONVALESCENT HUMAN SERA WITH TYPE A AND TYPE B
Type A antigen
Type B antigen
Type A antigen
Type B antigen
aAGP results + to ++++ based on intensity of precipitin lines and their proximity to serum
or antigen wells; -= absence of precipitin line.
bCF values presented as reciprocals of titre with the microprocedure; NT=not tested.
Turkey/Wisconsin/66 virus. Well-defined precipitin
lines of identity indicate specific type A antigen-
antibody reactions (Fig. 4). No line formed between
normal CAM suspension (control antigen) and the
Antigens and immune sera prepared with avian
influenza isolates were tested against antigens or
immune sera prepared with human, equine or por-
cine influenza strains. Lines of precipitation formed
that were continuous between either antigens or
sera when they were in the peripheral wells, regard-
less of the source of type A influenza virus used
to prepare them. A NP antigen prepared from
B/Johannesburg/59 influenza virus and a type B
immune serum prepared in rabbits was included in
another test as a type-specificity control (Fig. 5).
Sera obtained from pigs at intervals after aerosol
exposure to A/Swine/S15/31 influenza virus were
furnished, together with the HI titres, by Dr B. C.
Generally, very poor results were obtained with
swine sera when the AGP test was used. There were
some exceptions, as shown in Fig. 6, where a pig
was exposed to S15 virus and blood samples were
taken frequently. These sera had high HI titres and
produced clear precipitation by 7 days after exposure.
Sera with high HI titres usually gave negative results
with the AGP test, regardless of the source oftypeA
antigen or the percentage of agar or NaCl in the
Formalin-treated antigens prepared from 6 influ-
enza strains were placed at 230C and tested at
intervals for reactivity with a known positive serum.
There was no apparent loss in activity after 300 days
at this temperature. Antigens have been stored at
-20°C and repeatedly frozen and thawed with no
adverse effects on their activity.
The formalin treatment of the antigen was suf-
ficient to inactivate the virus as no infectivity was
demonstrated in embryonating eggs.
Sera from the different species that gave positive
results with the AGP test were stored at +40C.
The sera remained positive by the AGP test after
the maximum test-period of 28 days.
avian and mammalian sera were repeatedly frozen
and thawed with no apparent reduction in AGP
IMMUNODIFFUSION TEST FOR TYPE-SPECIFIC INFLUENZA ANTIBODY
While it isnot a replacement for any ofthe present-
ly used serological tests, the AGP test should prove
in a variety of diagnostic and research
Sera from naturally occurring field outbreaks of
influenza in turkeys have been examined with the
AGP TEST WITH EQUINE SERA
(HORSE " GYP ") TAKEN
AT INDICATED DAYS AFTER A
NATURAL ILLNESS OF EQUINE
AGP technique. Results will be reported in detail
elsewhere (Beard, 1970). No difficulty was expe-
rienced in demonstrating clear precipitin lines with
sera from any of the flocks that had been infected,
while uninfected flocks remained negative.
The non-infectious formalin-treated antigen prepa-
rations.have adequate stability without refrigeration
to permit shipment and use in many locations.
ST WITH ACUTE (a) AND CONVALESCENT (c) HUMAN
FTER AN ILLNESS WITH THE HONG KONG STRAIN (SI)
AND A 1969 TYPE B ILLNESS (Sm) a
aType A antigen in centre well is Turkey/Wisconsin/66; type B antigen is
at Type A antigen in centre well
sent day of blood sampling.
AGP TEST WITH AVIAN SERA
REPRESENTING DIFFERENT AVIAN
aFP= fowl plague, subtype 1;
Q It=Quail/ltaly/544/66, subtype 2;
4134=Duck/Ontario/4134/67, subtype 4;
W66=Turkey/Wisconsin/66, subtype 6;
NOR=serum from non-infected chick-
AGP TEST WITH ANTIGENS
PREPARED FROM AVIAN
REPRESENTING 4 DIFFERENT
Cz56 = Duck/Czechoslovakia/56, sub-
E62 = Duck/England/62, subtype 4;
W68= Turkey/Wisconsin/68, subtype 5;
W66= Turkey/Wisconsin/66, subtype 6;
CAM = antigen prepared from non-
Duck/Canadal53, subtype 2;