Immunoassays for discriminating between brucellosis infections and vaccinations

A method is disclosed for discriminating between cattle vaccinated against and those infected with Brucella spp. The method involves immunoassay using a purified polysaccharide containing 4,6-dideoxy-4-acylamido-D-mannopyranosyl units obtained from B. abortus or from cross-reacting organisms, and results in improved differentiation between vaccinated and infected animals. Test kits are also disclosed for performing the assay and a process is disclosed for obtaining the O-chain polysaccharides in high purity and yield.

FIELD OF THE INVENTION 
This invention relates to a method of immunoassay for discrimination 
between animals vaccinated against and those infected with Brucella spp. 
BACKGROUND OF THE INVENTION 
Brucella is a genus of Gram-negative bacteria which are the causative 
agents of brucellosis, an important disease of animals and humans. In 
animals, for example cattle, brucellosis causes abortions and in addition 
decreased meat and milk production. In humans, it produces intermittent 
debilitation with high fever which may resist antibiotic therapy and recur 
over a period of several years. The disease can therefore be the cause of 
serious health problems and substantial economic losses. 
The species B. abortus, which is one example of this genus, remains a 
problem throughout the world. In South American countries, for example, up 
to 40% of cattle herds are affected by brucellosis, thousands of human 
cases are known, and economic losses are estimated at tens of millions of 
dollars annually. Other economically significant species include B. 
melitensis (which affects humans, sheep and goats), B. suis (which affects 
pigs, reindeer and humans), B. ovis (affecting sheep) and B. canis 
(affecting dogs and humans). 
There have been advances in the development of vaccines against 
brucellosis. For example, in the case of B. abortus, there is common use 
of an attenuated live strain (S19) of B. abortus. However, two problems 
persist. The first is that, although the vaccine is effective, the 
protection it affords is not absolute, hence allowing some infection by 
field strains of B. abortus. The second problem is that, with current 
diagnostic techniques, vaccinated animals appear serologically similar to 
those infected. In Canada, limited vaccination of cattle by B. abortus S19 
still occurs for export purposes. In the United States, 2.9 million cattle 
were vaccinated in 1985, in part because some of the southern states 
(Texas, Florida, Louisiana and Arkansas) still have high prevalences of 
brucellosis in their cattle populations. There is therefore an urgent need 
for a method of discriminating vaccinated from infected cattle. 
To date, some discrimination is possible by setting limits for the amount 
of antibody against B. abortus produced using whole 
smooth-lipopolysaccharide (sLPS) complex in enzyme immunoassay (EIA or 
ELISA). FIG. 1 depicts graphically the distribution of cattle sera with 
antibodies having affinity or specificity for this antigen in one case 
study. In the Figure, the frequency or number of cattle, is plotted 
against the lower class limit (i.e. five sera with A.sub.414nm on the 
enzyme immunoassay of 1.00, 1.01, 1.02, 1.03 and 1.04 would be represented 
by a bar at 1.00 having a frequency of 5 sera). It can be seen that, in 
general, the vaccinates have readings below an A.sub.414nm of 0.50, while 
culture positives (i.e. B. abortus was isolated from these animals) have 
sera that gave an A.sub.414nm above 0.50 on this test. However, there is 
still considerable overlap, mostly in the 0-0.50 range but also some 
degree of overlap at higher readings (note the high positive readings of 
vaccinate sera around A.sub.414nm of 1.5). 
The difficulty in differentiating vaccinates from infected cattle is again 
evident in a study that compared the sensitivities and specificities of 
five serodiagnostic tests used for the detection of bovine antibodies to 
B. abortus. The results of this study are given in the following Table 1. 
TABLE 1 
__________________________________________________________________________ 
COMATIVE ASSAY RESULTS 
Status Number 
of of Number of Positive Readings 
Cattle Cattle 
BPAT.sup.1 
STAT.sup.2 
CFT.sup.3 
HIGT.sup.4 
EIA.sup.5 
__________________________________________________________________________ 
FREE LISTED.sup.a 
1 non- 1067 12 (1.1%) 
6 (0.6%) 
0 (0%) 
0 (0%) 
1 (0.1%) 
vaccinated 
2 vaccinated 
76 6 (7.8%) 
2 (2.6%) 
0 (0%) 
8 (10.5%) 
3 (3.9%) 
REACTOR.sup.b 
1 non- 798 74 (9.3%) 
50 (6.3%) 
9 (1.1%) 
4 (0.5%) 
5 (0.6%) 
vaccinated 
2 vaccinated 
253 20 (7.9%) 
15 (5.9%) 
5 (2.0%) 
12 (4.7%) 
14 (5.5%) 
INFECTED.sup.c 
1 positive 174 152 
(87.4%) 
162 
(93.1%) 
159 (91.4%) 
167 
(96.0%) 
162 
(93.1%) 
__________________________________________________________________________ 
.sup.1 BPAT = buffered plate antigen test 
.sup.2 STAT = standard tube agglutination test 
.sup.3 CFT = complement fixation test 
.sup.4 HIGT = hemolysisin-gel test 
.sup.5 EIA = enzyme immunoassay 
.sup.a Free listed = cattle from herds certified in the previous year as 
being of brucellosis based on standard serological tests. 
.sup.b Reactor = cattle from herds containing two or more positive 
reactors based on standard serological tests, but herds in which no 
infected cattle have been indentified based on bacteriological culture. 
.sup.c Infected = cattle identified as infected by positive 
bacteriological culture 
Table 1 shows that the buffered plate antigen test (BPAT, which is 
currently used as a screening test) recorded 1.1% of non-vaccinated and 
7.8% of vaccinated cattle in Brucella-free herds as positive. The enzyme 
immunoassay (EIA, for further details see Nielsen and Wright, 1984, 
Agriculture Canada publication, ISBN 0-662-13421-4) greatly reduces the 
number of false-positive non-vaccinated cattle to 0.1% but the number for 
vaccinates remains high at 3.9%. The other serological tests show similar 
limitations. The complement fixation test (CFT) is an exception by having 
high specificity but its costs and technical manipulations make it 
unlikely to remain a routine method. Table 1 also shows that problem herds 
(those that persistently show reactors for no apparent cause) have a high 
number of false-positives for non-vaccinated and vaccinated cattle (i.e. 
0.6% and 5.5% respectively for the EIA). 
From the above data, it can be seen that present serodiagnostic tests are 
for the most part effective in discriminating Brucella-free from infected 
cattle. However, there is a small percentage of animals in the 
intermediate range (e.g. vaccinates with high titres or infected cattle 
with low titres of antibodies) that are impossible to classify reliably by 
standard serological methods. A small percentage of several million cattle 
is still a large population and since these animals may be highly valued, 
there is a great need for a test that will differentiate vaccinated cattle 
from B. abortus infected cattle. 
T. J. G. Raybould and Shireen Chantler, J. Immunol. Methods, 30 (1979) 
pages 37-46 describe an immunofluorescent procedure in which antigenic 
extracts of B. abortus 544/W were coupled to Sepharose.RTM. beads. The 
crude sodium dodecyl sulfate (SDS) extract contained polysaccharide, 
lipopolysaccharide, proteins, etc., and when coupled to the Sepharose.RTM. 
beads showed discrimination between vaccinated and infected cattle, with, 
however, some overlap of results. In subsequent work (Raybould et al., 
Infect. Immun. 29 (1980) pages 435-441), they determined from inspection 
of the SDS extract that the active group for the discrimination was an 
antigen "X". As it bound to oxidised Sepharose.RTM., they concluded that 
antigen "X" contained amino groups and hence was a basic protein. The test 
given in that report involved hemagglutination, gave some overlap similar 
to that seen in other tests and in some instances was inconclusive. 
It has also been reported (Diaz, R., P. Garatea, L. M. Jones, and I. 
Moriyon, 1979, Radial diffusion test with a Brucella polysaccharide 
antigen for differentiating infected from vaccinated cattle, J. Clin. 
Microbiol. 10:37-41; Jones, L. M., D. T. Berman, E. Moreno, B. L. Deyoe, 
M. J. Gilsdorf, J. D. Huber, and P. Nicoletti, 1980, Evaluation of a 
radial immunodiffusion test with polysaccharide B antigen for diagnosis of 
bovine brucellosis, J. Clin. Microbiol. 12:753-760), that there is a 
component of Brucella melitensis, termed "poly B" which can differentiate 
vaccinated from infected cattle, i.e. only the sera of the latter will 
precipitate "poly B" in agar gel immunodiffusion (or AGID). "Poly B" has 
been characterized by Moreno, E., S. L. Speth, L. M. Jones, and D. T. 
Berman, 1981, Immunochemical characterization of Brucella 
lipopolysaccharides and polysaccharides, Infect. Immun. 31:214-222, as a 
polysaccharide consisting primarily of glucose units. Our studies support 
these findings and we have now isolated "poly B" from B. melitensis 16M 
and found it to be essentially a polymer of glucose molecules. Some sera 
of infected cattle will react weakly with this component in AGID provided 
that 10% NaCl is added to the buffer (0.1 M sodium borate, pH 8.3). 
However, we have observed that while the principal component, "poly B" 
(the glucose polymer) is a poor antigen; the associated minor component, 
O-chain polysaccharide (containing 
4,6-dideoxy-4-formamido-D-mannopyranose), is the immunodominant or "active 
ingredient" in these preparations. We have also found that reports of 
immunological identify between the O-chain polysaccharide and "poly B", 
the glucose polymer, (Moreno et al., 1981, Infect. Immun. 31; 214-222, 
Fernandez-Lago et al., 1982, Infect. Immun. 38, 778-780) were likely due 
to the latter containing O-chain as an impurity. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide an improved 
method of differentiating between animals (e.g. cattle) which have been 
vaccinated and those infected with Brucella species based on serological 
activity of their sera on polysaccharide (e.g. O-chain). 
Another object of the invention is to provide a test kit for carrying out a 
discrimination between animals vaccinated or immunized against Brucella 
species by an indirect or competitive immunoassay. 
A further object is to provide methods of isolating the discriminating 
antigen, O-chain polysaccharide, which afford higher yields, within a 
relatively shorter time and in greater purity than conventional methods. 
Accordingly, one aspect of the invention provides a method of 
discriminating animals, for example cattle, vaccinated or immunized 
against a Brucella sp., such as B. abortus, from those infected with the 
Brucella sp., which comprises subjecting serum samples from the animals to 
an immunoassay using an antigen containing 
4,6-dideoxy-4-acylamido-D-mannopyranose repeating units, and thereafter 
differentiating the sera of the vaccinates from those of infected animals 
based on a negative or positive result, respectively, of said assay. The 
polysaccharide is preferably present as O-chain. 
The term "acylamido" includes, for example, formamido, acetamido, 
propionamido and butyramido. 
A particular aspect of the invention provides a method of discriminating 
between cattle vaccinated or immunized against Brucella abortus and those 
infected with B. abortus, which comprises subjecting sera from the cattle 
to enzyme immunoassay using a purified O-chain polysaccharide of B. 
abortus containing 4,6-dideoxy-4-formamido-D-mannopyranose repeating 
units, and thereafter differentiating the sera of vaccinates from those of 
infected animals based on a respective negative or positive result of the 
assay. 
Another aspect of the invention provides a test kit for carrying out an 
immunoassay for discriminating between animals, such as cattle, vaccinated 
or immunized against a Brucella species, such as B. abortus and those 
infected with the species, which comprises: 
(a) a support carrying antigenic polysaccharide (e.g. as O-chain) 
containing 4,6-dideoxy-4-acylamido-D-mannopyranose units; and 
(b) an immunological detection system for discriminating between the 
presence or absence of antibodies against Brucella sp. 
The kit operates in conjunction with a source of antibody, which can be a 
polyclonal source, i.e. animal sera, or a monoclonal source, i.e. from 
monoclonal hybridomas. 
The term "immunoassay" is intended to cover any assay format involving 
serological determination, including, for example, enzyme immunoassay 
(e.g. ELISA), competitive immunoassay, radioimmunoassay, immunofluorescent 
assay, and immunoassays using polystyrene plates, membranes, dipsticks or 
beads. 
The term "immunized" as used herein embraces immunization with whole cells 
of Brucella species, with cross-reacting organisms, extracts of said cells 
and organisms, synthetic antigens, derived antigens and gene products.

DETAILED DESCRIPTION OF THE INVENTION 
Analysis of a preparation of the "poly B" (prepared by the method of Diaz) 
referred to above reveals that it is predominantly the glucose polymer 
noted above. However, NMR-spectroscopy studies have shown that this 
preparation also contained small amounts of a polysaccharide containing 
4,6-dideoxy-4-formamido-D-mannopyranose units of the formula: 
##STR1## 
Polysaccharides of this type are described in copending Canadian Patent 
Application Ser. No. 448,768, filed March 2, 1984. 
Our AGID studies show that the preparation gave two precipitin lines (one 
strong and the other faint) with sera of infected animals. The prominent 
precipitin line gave a line of identity with the sLPS of B. abortus 
1119-3, B. melitensis 16M and Yersinia enterocolitica 0:9. The only 
component known to be common to these is their 0-chain polysaccharide 
which contains 4,6-dideoxy-4-formamido-D-mannopyranose repeating units. 
The precipitin line did not occur if the sera were cross-absorbed with Y. 
enterocolitica 0:9 cells. Therefore, the main antigenic component and the 
one giving discrimination of animals is the 
4,6-dideoxy-4-formamido-D-mannopyranose polymer. 
We have now determined that the purified O-chain containing 
4,6-dideoxy-4-formamido-D-mannopyranose units, from B. abortus (e.g. 
strains 1119-3 and 413) can differentiate sera of vaccinated from infected 
animals, e.g., cattle, by immunoassay techniques, for example by either 
AGID or ELISA (recommended O-chain concentrations are 1 mg/ml for AGID and 
2.5 .mu.g/ml for ELISA). The O-chains of B. melitensis 16M and Yersinia 
enterocolitica 0:9 are also effective for this purpose, although for the 
ELISA care must be exercised in preparing the solid phase for the assay. 
Both Y. enterocolitica 0:9 and B. melitensis (e.g. strain 16M) have 
O-chains of 4,6-dideoxy-4-formamido-D-mannopyranose as does B. abortus. 
Monoclonal antibodies (e.g. clones Ys-T9-3 and Ys-T9-7) as described in 
Canadian Patent Application No. 448,768 suggest that some small 
differences may exist between the O-chains (of B. abortus, B. melitensis 
and Y. enterocolitica), but this has not been rigorously proven. 
In the past, the O-chain polysaccharide of B. abortus has been difficult to 
purify, yielding at best about 100 mg of polysaccharide from 100 g wet 
weight of cells (Caroff et al., 1984, Infect. Immun. 46:384) and most 
often yielding far less than this amount. We have developed rapid methods 
which consistently give higher yields (e.g. about 500 mg of O-chain from 
100 g wet weight of cells by one method, and about 330 mg of purified 
O-chain in a few hours by another method). 
Accordingly, a further aspect of the invention provides a method of 
isolating polysaccharide containing 
4,6-dideoxy-4-formamido-D-mannopyranose units from Brucella sp. which 
comprises 
(a) heating a suspension of whole cells or a crude extract thereof in an 
acidic salt solution; 
(b) separating the supernatant from the cellular debris; and 
(c) extracting the O-chain polysaccharide therefrom by precipitating out 
contaminants and/or subjecting the supernatant to partitioning. 
Partitioning can be accomplished using phenol-water extraction in which the 
O-chain sequesters into the phenol layer. Increased yield of O-chain is 
obtained when the supernatant from step (b) is neutralized and dialyzed 
against buffer, then treated with enzymes that degrade contaminants. 
A preferred method of isolating the O-chain polysaccharide comprises: 
(a) suspending killed cells of B. abortus in TRIS-saline, washing and 
resuspending the cells in acetic acid and sodium chloride solution, 
(b) heating and centrifuging the suspension, 
(c) extracting the supernatant and treating it with lysozyme, ribonuclease, 
deoxyribonuclease and proteinase followed by dialysis against TRIS-saline, 
(d) extracting the product with phenol/water and dialyzing the phenol layer 
against TRIS-saline, and 
(e) extracting and separating the supernatant. 
A particular embodiment of this method is set out in tabular form in the 
following Table 2. 
TABLE 2 
PURIFICATION OF O-CHAIN POLYSACCHARIDE FROM 
BRUCELLA ABORTUS 1119-3 
1. Cells killed and suspended in TRIS-saline (1% NaCl, 0.02% NaN.sub.3, 
0.12% TRIS-HCl, pH 7) with 2% pheno. Wash and resuspend cells (100 g 
cells/500 ml) in 2% (vol/vol) acetic acid and 10% NaCl. 
2. Autoclave at 15 psi and 121.degree. C. for 30 minutes (sealed centrifuge 
bottles). 
3. Centrifuge at 20,000 X g for 30 minutes at 4.degree. C. Save 
supernatant, neutralize with NaOH. Precipitate with 5 volumes methanol 
with 1% sodium acetate. Redissolve and dialyze against TRIS-saline. 
4. Digest in 25 .mu.g/ml lysozyme, ribonuclease and deoxyribonuclease for 6 
hours Digest in 50 .mu.g/ml proteinase K for 48 hours Dialyze against 
TRIS-saline. 
5. Do phenol-water extraction (room temperature). Remove phenol layer, 
precipitate polysaccharide with methanol plus 1% sodium acetate, dialyze 
against TRIS-saline. 
6. Ultra-centrifuge 100,000 X g for 18 hours at 4.degree. C. Save 
supernatant (700 mg O-chain). 
7. Lyophilize, redissolve in 0.05 M pyridinium acetate (4 ml). 
Sephadex.RTM. G-50 column chromatography (column size 2.5 cm 
diameter.times.100 cm height). Lyophilize first peak (500 mg O-chain). 
Thus, for obtaining O-chain polysaccharide from cells of Brucella sp., the 
following general principles obtain: 
(a) The O-chain polysaccharide can be released from purified 
smooth-lipopolysaccharide with acid and heat (Caroff et al., 1984, Inf. 
Immun. 46; 384-388; Canadian Patent Application Ser. No. 448,768). We have 
now observed that such release will occur from whole cells as an initial 
step. If the cells (or crude extract thereof) are suspended in high salt 
concentrations (e.g. 10% NaCl) and weak acid (e.g. 2% acetic acid), then 
heated (e.g. autoclaved), a large amount of O-chain polysaccharide is 
released with reduced contamination (e.g. protein) than conventional 
methods. Cellular debris can be removed (e.g. by centrifugation). 
(b) A rapid yield of O-chain polysaccharide can be acquired from the above 
by 
(i) precipitating contaminants (e.g. proteins, oligonucleotides) with 
agents (such as trichloracetic acid) and removing these precipitates (e.g. 
by centrifugation); 
(ii) partitioning (e.g. by phenol-water extraction) in which the O-chain 
sequesters into one layer (phenol) while other contaminants sequester into 
the other layer (e.g. water); and 
(iii) removal of low-molecular contaminants (e.g. by the method of 
dialysis). 
(c) Increased yields of O-chain were observed when crude extracts (e.g. 
supernatant of part a, above) were neutralized and dialyzed against buffer 
then treated with enzymes that degrade contaminants. 
(d) precipitation of the O-chain (e.g. at least 5 volumes with methanol 
plus 1% sodium acetate) greatly conserved reagents and made purification 
more manageable. Precipitating O-chain or sLPS in phenol with the use of 
alcohol (e.g. at least 5 volumes methanol plus 1% sodium acetate), 
followed by resuspension and reprecipitation in this alcohol, removes the 
phenol without adversely affecting the antigen. 
Another embodiment of the above procedure is set out in the following Table 
3. 
TABLE 3 
RAPID PURIFICATION OF O-CHAIN POLYSACCHARIDE 
FROM BRUCELLA ABORTUS 1119-3 
1. Either killed or live cells are washed and resuspended (100 g wet weight 
of cells/500 ml) in 2% (vol/vol) acetic acid and 10% NaCl solution. 
2. Autoclave at 15 psi, 121.degree. C. for 20 minutes (in sealed 
Nalgene.RTM. centrifuge bottles). Open bottles, place in water-bath and 
cool. 
3. Centrifuge at 8,000.times.g for 20 minutes at 4.degree. C. Save the 
supernatant and neutralize with NaOH. To the remaining cell pellet, 
resuspend in 2% acetic acid, 10% NaCl (about 200 ml for 100 g cells), 
centrifuge and add this supernatant to the other. Neutralize with NaOH. 
4. Precipitate with 5 volumes pre-chilled (ice-bath) methanol plus 1% 
sodium acetate. Chill in an ice-bath, with occasional mixing, for about an 
hour. Centrifuge, save the pellet and redissolve this in 20 ml distilled 
water. Add 100 ml of 0.3 M trichloroacetic acid (pre-chilled at 4.degree. 
C. Stir in refrigerator (4.degree. C.) for 1 hour. Centrifuge and save the 
supernatant. Adjust pH to 7 with NaOH. 
5. Precipitate with 5 volumes pre-chilled (ice-bath) methanol plus 1% 
sodium acetate. Centrifuge and dissolve the pellet in about 100 ml 
distilled water. Add an equal volume of 95% phenol. Stir at room 
temperature for about 30 minutes. Centrifuge and save phenol layer. Add to 
the phenol layer another 100 ml of distilled water, stir at room 
temperature for 30 minutes, centrifuge and save phenol layer. 
6. Take the phenol layer, add 5 volumes pre-chilled methanol plus 1% sodium 
acetate, chill in an ice-bath for 1 hour with occasional mixing. 
Centrifuge, save pellet. Resuspend pellet in an equal volume of 
pre-chilled methanol plus 1% sodium acetate (i.e. high speed magnetic 
stirring so that suspension is complete and phenol is removed). 
Centrifuge, save pellet. Dissolve pellet in distilled water (if desired, 
remove sodium acetate by dialysis). Lyophilize. 
Time elapsed: 1 work day Yield: 330 mg O-chain from 100 g wet weight of 
cells. 
The method for the ELISA is described in the following Table 4. 
TABLE 4 
INDIRECT ELISA FOR DETECTION OF BOVINE SERUM 
ANTIBODY TO B. ABORTUS O-CHAIN POLYSACCHARIDE 
1. Prepare a 2.5 .mu.g/ml solution of O-chain polysaccharide (or 1 g/ml 
LPS) in coating buffer (0.06.mu.M sodium carbonate buffer, pH 9.6). 
Dispense 200 .mu.l volumes in all wells of polystyrene plates which have 
been previously rinsed with distilled water. Seal plates and incubate 
overnight (ca. 18h) at room temperature (ca. 22.degree. C). 
2. The following day, prepare dilutions of test samples and controls in 
wash/diluent buffer (0.85% NaCl, 0.01 M sodium phosphate, pH 7.2 plus 
0.05% Tween.RTM. 20). All dilutions should be prepared and maintained at 
room temperature. 
3. Immediately prior to sample application, wash with equal efficiency all 
wells of the antigen-coated plate with wash/diluent buffer. After four 
consecutive washes, discharge residual buffer by inverting plate and 
vigorously rapping onto a lint-free absorbent surface. 
4. Dispense 200 .mu.l volumes of serum samples in microtiter wells, seal 
the plate and incubate for 3 hours at room temperature. 
5. Prepare a working dilution of conjugate (e.g. rat monoclonal anti-bovine 
(H & L) IgG conjugated with horseradish peroxidase) in wash/diluent 
buffer. Wash polystyrene plate as described in Step 3, then dispense 200 
.mu.l volumes of conjugate to all wells, seal the plate and incubate for 1 
hour at room temperature. 
6. Prepare a working solution of enzyme substrate (0.015% H.sub.202, 1mM 
ABTS, in 0.05 M citrate, pH 5.0 with NaOH). Wash polystyrene plate as 
described in Step 3, dispense 2 volumes of substrate to all wells in the 
polystyrene plate and immediately begin timing the reaction with a stop 
watch. Incubation should be on a plate shaker at room temperature. 
7. At the end of incubation (e.g. 10 minutes), the A.sub.414nm by a 
photometric polystyrene plate scanner should be read Target control sera 
and mean A.sub.414nm of duplicate test samples are used to determine 
seropositive or seronegative status based on a threshold A.sub.414nm 
determined by seroepidemiological methods (e.g. positive is.gtoreq.0.300). 
The results for one immunoassay are shown by testing cattle sera by enzyme 
immunoassay using both the B. abortus sLPS and O-chain polysaccharide as 
shown in the following Table 5. 
TABLE 5 
______________________________________ 
Comparison by Enzyme Immunoassay of Cattle Sera 
Binding to Brucella abortus smooth- 
lipopolysaccharide and to 0-chain polysaccharide 
Antigen 
sLPS 0-chain 
Sera Dilution (1 .mu.g/ml) 
(2.5 .mu.g/ml) 
______________________________________ 
1 Negative 
Controls 
#280.sup.a 1:100 0.11.sup.b 
0.09 
#281 1:100 0.11 0.10 
#282 1:100 0.21 0.10 
Conjugate Control.sup.c 
0.10 0.10 
2 Vaccinates 
(bled 28 days post-vaccination) 
#679 1:100 2.03 0.15 
#743 1:100 2.31 0.21 
#1066 1:100 0.70 0.08 
1429 1:100 1.80 0.09 
3 Infected 
#25 1:12800 0.90 0.55 
#908 1:800 2.19 0.59 
#950 1:12800 1.43 0.45 
#CF80 1:200 2.29 0.59 
______________________________________ 
.sup.a Laboratory designation 
.sup.b OD.sub.414nm on an enzyme immunoassay (readings 0.3 are positive) 
.sup.c Conjugate control = rat antibovine antibody conjugated with 
horseradish peroxidase 
It can be seen that, upon testing cattle sera by using the whole SLPS 
complex from B. abortus as antigen on polystyrene plates in an enzyme 
immunoassay, there appeared to be little differentiation between high 
titre sera of vaccinated and infected cattle. However, when the O-chain 
polysaccharide of B. abortus prepared as described above is applied, 
considerable differentiation can be made. Table 5 shows that sera from 
either vaccinated or infected cattle with high antibody titres against 
Brucella abortus will both give positive readings with an enzyme 
immunoassay using polystyrene plates coated with B. abortus sLPS. However, 
if the above-obtained B. abortus O-chain polysaccharide is used as the 
antigen, then sera from infected cattle (identified as positive in the 
test) can be differentiated from the sera of vaccinated cattle (identified 
as negative). The mechanism for this differentiation is unclear but Table 
5 suggests that the antibodies of infected cattle have a different 
specificity or affinity than those of vaccinated cattle to the presented 
antigens. 
It can be noted that vaccinated-cattle serum #743 at a 1:100 dilution gives 
a considerably higher positive reading than infected-cattle serum #25 at a 
1:12800 dilution when Brucella abortus sLPS is used as antigen for the 
enzyme immunoassay. If these sera had antibodies of the same specificity 
for B. abortus O-chain as for sLPS, then one would expect to observe the 
same pattern with the results using O-chain. However, at the noted 
dilutions the reading for vaccinated-cattle serum #743 tested with O-chain 
should be considerably higher than for infected-cattle serum #25 on the 
same antigen (bearing in mind the dilution factor). This is not the case. 
Serum #743 gives instead a low or negative result, serum #25 gives a high 
or positive reading. Similar comparisons can be seen with other examples 
in Table 5 (compare vaccinate serum #679 with infected cattle sera #908 
and #CF80; vaccinate serum #1429 with infected serum #950). 
To date, 100 negative control sera, 50 vaccinates and 25 culture-positive 
cattle sera have been tested on this system and have given results as 
noted above. Therefore, it is likely that the antibodies of infected 
cattle have a higher affinity against O-chain polysaccharide than those of 
vaccinated cattle which react poorly to this antigen. Also tested were 
eight vaccinated adult cattle that had received "full dose" inoculation 
(rather than reduced dose) of B. abortus S19, with similar negative 
results on "O-chain". 
As stated previously, discrimination between sera of vaccinated and 
infected cattle was evident as in the examples of the serological tests of 
AGID and ELISA. For the former test, if a mixture of O-chain and 
smooth-lipopolysaccharide (each 1 mg/ml) is placed in a well cut in the 
agar, sera of infected cattle will precipitate both antigens, sera of 
vaccinated cattle will precipitate only sLPS (as judged by the appearance 
of the precipitin lines and lines of identity with a well containing only 
one antigen). Clearly the affinities or specificities of the two sera 
groups differ. 
For the ELISA, antigen adherence in our system may be due to: 
(i) O-chain polysaccharide adherence to polystyrene. 
(ii) our O-chain preparation may contain traces of LPS, the latter being 
the discriminating antigen. 
(iii) the antigenic polysaccharide is coupled to a component which in turn 
binds to polystyrene. 
For the first point, we have observed that the 0chain is both hydrophilic 
(readily dissolves in water) and hydrophobic (sequesters into the phenol 
phase of a phenol-water extraction). Such properties may account for 
adherence. 
For the second point, different concentrations (ranging from 2 ng/ml to 
12.5 .mu.g/ml) of O-chain, sLPS and alkaline-treated (0.2 M NaOH, 
70.degree. C., 15 min.) LPS were coated onto plates. Results were similar 
for 2.5 .mu.g/ml O-chain and 8 ng/ml LPS or alkaline-treated LPS (see the 
following Table 6). Since the use of low concentrations of LPS 
preparations in ELISA give some differentiation of vaccinated and infected 
cattle, we now present this technique as an alternative. However, Table 6 
shows that these dilutions of sLPS and alkaline-treated LPS as antigens in 
the ELISA test gave more false-positives for sera of vaccinated cattle 
than did O-chain as antigen. Therefore, we recommend the use of O-chain. 
It should also be noted that when O-chain preparations were tested with 
Limulus Amebocyte Lysate (Pyrotell.RTM., Associates of Cape Cod, Inc., 
Woods Hole, Mass.) there was less than 0.1% LPS detected. 
For the third point, samples of O-chain polysaccharide (1 mg/ml) were 
treated with 0.2 mg/ml lysozyme, deoxyribonuclease, ribonuclease, 
proteinase for 24 hours (room temperature). Such treatment did not affect 
ELISA test results and hence indicates that adherence by a contaminant is 
unlikely. 
It should also be noted that in the model of the use of a synthetic antigen 
(O-chain of Yersinia enterocolitica 0:9 coupled to a hydrophobic carrier, 
as described in Patent Application 448,678; used at 1 .mu.g/ml in the 
ELISA), similar discrimination of vaccinates and infected cattle occurred 
as with the use of O-chain. The existence of O-chain coupled to a 
hydrophobic carrier, as an active ingredient in our preparations, is a 
possibility. 
TABLE 6 
______________________________________ 
COMISON OF DIFFERENT ANTIGENS 
USED IN THE ELISA 
ANTIGENS 
Alkali- 
0-Chain SLPS Treated 
Sera (2.5 .mu.g/ml) 
(8 ng/ml) (8 ng/ml) 
______________________________________ 
1 Negative 
Control 
N1.sup.a 0.150.sup.b 
0.055 0.081 
N4 0.164 0.058 0.082 
N5 0.126 0.038 0.074 
N6 0.129 0.061 0.075 
2 Vaccinates 
170 0.168 0.170 0.236 
446 0.270 0.379 0.408 
679 0.185 0.190 0.182 
743 0.285 0.347 0.381 
1066 0.112 0.083 0.080 
1426 0.267 0.298 0.396 
1429 0.267 0.226 0.185 
1440 0.253 0.291 0.299 
3 Infected 
25 0.547 0.620 0.698 
950 0.553 0.538 0.824 
44/33 0.345 0.400 0.419 
______________________________________ 
.sup.a Laboratory designation 
.sup.b A.sub.414nm on an enzyme immunoassay (readings .gtoreq.0.3 are 
positive, falsepositive readings are underlined) 
Having regard to available sources of B. abortus O-chain polysaccharide, 
both B. abortus 1119-3 and 413 contain sLPS with O-chains of 
4,6-dideoxy-4-formamido-D-mannopyranose. Upon testing purified O-chains of 
these two strains, both were comparable in the differentiation of 
negative, vaccinated and culture-positive cattle sera. The advantage of 
using the O-chain of B. abortus 1119-3 is that from this strain more of 
this antigen can be isolated than from strain 413 (500 mg for the former 
and 170 mg for the latter from 100 g wet weight of cells). 
Similarly to the above, purified O-chains containing 
4,6-dideoxy-4-acylamido-D-mannopyranose, such as from B. melitensis 16M 
and Y enterocolitica 0:9 gave similar reactions. Results obtained using B. 
abortus 1119-3 O-chain and Y. enterocolitica 0:9 O-chain are set out in 
the following Table 7. 
TABLE 7 
______________________________________ 
Comparison of Different Antigens.sup.a 
Labora- ANTIGEN 
Cattle tory B. abortus 
B. abortus 
Y.enterocolitica 
Test Desig- 1119-3 1119-3 0:9 
Serum nation sLPS 0-chain 0-chain 
______________________________________ 
Negative 
CI .sup. 0.046.sup.b 
0.072 0.085 
CII 0.072 0.098 0.092 
Neg. 0.098 0.157 0.108 
Vaccinates 
679 0.155 0.092 0.064 
1429 0.245 0.077 0.100 
1066 0.065 0.071 0.074 
446 1.694 0.144 0.081 
743 0.609 0.097 0.087 
1426 1.411 0.160 0.122 
1440 0.370 0.145 0.170 
B. abortus 
J3 2.311 0.355 0.309 
Infected 
25 3.730 1.451 1.641 
44133 3.317 1.095 0.900 
______________________________________ 
.sup.a Stored 2 weeks in 0.06M sodium carbonate buffer (pH 9.6) 
.sup.b Readings are for A.sub.414nm .Data &lt;0.300 is negative, 
.gtoreq.0.300 is positive 
(note that there are no positives for vaccinate sera tested with 0chains 
as antigens, and that sera from infected cattle give comparable positive 
readings on these antigens) 
A suitable concentration of antigen for coating appears to be in the range 
of about 1 to 5 .mu.g/ml of O-chain for binding to polystyrene plates in 
the enzyme immunoassay, with an optimum concentration around 2.5.mu. g/ml. 
Lower concentrations cause decreased sensitivity, while higher 
concentrations give false positive results. 
In carrying out step 4 of Table 2 above, a crude autoclave preparation of 
O-chain from B. abortus 1119-3 was extracted with phenol-water. The 
O-chain was found in the phenol-phase (in the highly pure form as judged 
by optical rotation), interphase (contaminated with sLPS as judged by 
ultracentrifugation characteristics) and water-phase (contaminated by 
nucleic acids as judged by high A.sub.260nm readings). The phenol-phase 
antigen worked as before, the interphase antigen gave high readings for 
vaccinates and culture-positive cattle sera, and the water-phase antigen 
gave low or negative readings for all sera tested. Therefore, the use of 
only purified O-chain from B. abortus is recommended. 
The purified O-chain from B. abortus 1119-3 exhibits good shelf stability. 
For example, samples were stored for 1 year at -20.degree. C., 6 months at 
room temperature in a closed vial, and 3 months in saline at 4.degree. C. 
There were no discernible differences in results between these three 
antigens when applied to the test as described above. Recommended 
procedure is lyophilization, storage at -20.degree. C. or colder, and 
prepared fresh when needed. 
The procedures mentioned above are indirect tests, whereby the amount of 
antibody (bound to antigen coated polystyrene plates) is assayed by a 
colour change, e.g. anti-bovine antibodies conjugated with horseradish 
peroxidase act on hydrogen peroxide which reduces the chromogen ABTS 
(2,2'-azinodi-(3-ethylbenz-thiazolidine-sulfonic) acid). The colour change 
is time dependent, and optimal incubations for the test system have been 
found to be about 10 minutes. However, even with immunoassay absorbance 
readings taken at 70 minutes, or one hour past the optimum incubation, 
although both the negative sera, on either sLPS or O-chain antigens, and 
vaccinates sera, on the O-chain antigen, have given some reaction over 
this length of time, differentiation of these from B. abortus 
culture-positive sera is still easily made. Therefore, there is 
flexibility when using the present test system. 
Sera from cattle infected by cross-reacting organisms were also 
investigated. In a limited study using five cows, each was experimentally 
infected with Y. enterocolitica 0:9. Sera from these animals showed high 
titres against B. abortus as judged by standard serological methods. 
However, with the present enzyme immunoassay using sLPS/O-chain, these 
animals appeared as vaccinates or gave high reactions with sLPS and 
negative reactions with B. abortus O-chain. This increased specificity is 
a means of differentiating cattle infected with B. abortus or 
cross-reacting organisms. The results are shown in the following Table 8. 
TABLE 8 
______________________________________ 
Comparison by an Enzyme Immunoassay of Sera from Cattle 
Infected by Different Bacteria 
ANTIGEN 
B abortus B. abortus 
Cattle Laboratory 1119-3 1119-3 
Test Serum Designation 
sLPS 0-chain 
______________________________________ 
Negative Controls 
280 .sup. 0.11.sup.a 
0.09 
281 0.11 0.10 
282 0.21 0.10 
Cattle infected 
CF80 &gt;2.00 0.59 
with B. abortus 
25 &gt;2.00 1.45 
908 &gt;2.00 0.59 
44133 &gt;2.00 1.10 
Cattle experimentally 
518 &gt;2.00 0.08 
infected with Y. 
523 &gt;2.00 0.07 
enterocolitica 553 
&gt;2.00 0.11 
577 &gt;2.00 0.15 
582 &gt;2.00 0.09 
______________________________________ 
.sup.a Readings are for A.sub.414nm .Data &lt;0.30 is negative, &gt;0.30 is 
positive 
(note that only B. abortus infected cattle sera gives positive results on 
B. abortus 0chain as antigen) 
It should be noted that cattle that are either recently infected by B. 
abortus or have low numbers of the organism localized in a tissue may not 
have sufficient antibody titres to register as positive on this test. 
However, this is a limitation shared by other serological tests currently 
used to detect B. abortus. 
While the use of polystyrene plates has been described above, other 
technologies, such as dipsticks or beads coated with B. abortus O-chain, 
are also effective in the differentiation of sera and can be employed. 
Another example of an immunoassay is competitive binding assay utilizing 
the following principles. Polystyrene microtitre plates are coated with 
desired antigen (e.g. one set of wells with B. abortus 1119-3 O-chain). A 
mouse monoclonal antibody (e.g. Ys-T9-2) conjugated with an indicator 
enzyme (e.g. horseradish peroxidase) is then added. The plate is washed, 
test sera (e.g. from cattle) are added, incubated and then excess is 
removed by washing. If the test sera have antibodies of high affinity for 
the antigen, these will displace the mouse monoclonal antibody, decreasing 
the A.sub.414nm when substrate-chromogen (e.g. H.sub.202 -ABTS) is added. 
If the test sera lack antibodies with such affinity, no such displacement 
occurs and when substrate is added the A.sub.414nm will be high. 
Results showing the effectiveness thereof are shown in the following Table 
9. 
TABLE 9 
______________________________________ 
Comparison of the Indirect ELISA and the Competitive 
ELISA 
Cattle Indirect ELISA.sup.a 
Competive ELISA.sup.b 
Test Laboratory 
Using B. abortus 
Using B. abortus 
Serum Designation 
1119-3 sLPS 1119-3 0-chain 
______________________________________ 
Negative 
N1 .sup. 0.07.sup.c 
1.72.sup.c (0%).sup.d 
Control N4 0.13 1.73 (0%) 
N5 0.12 1.73 (0%) 
N6 0.10 1.65 (3%) 
Vaccinates 
446 &gt;2.00 1.54 (9%) 
542 0.66 1.61 (5%) 
679 1.56 1.64 (4%) 
743 &gt;2.00 1.61 (5%) 
1426 1.33 1.61 (5%) 
1429 1.19 1.70 (0%) 
1440 1.88 1.54 (9%) 
B. abortus 
25 &gt;2.00 0.16 (90%) 
Infected 
44133 &gt;2.00 0.21 (88%) 
950 &gt;2.00 0.19 (89%) 
TAR &gt;2.00 0.34 (80%) 
J2 &gt;2.00 0.51 (70%) 
J3 &gt;2.00 0.56 (67%) 
______________________________________ 
.sup.a The Indirect ELISA is that used in Tables 1 to 7 above. The above 
data gives insight into aniBrucella titres for &lt;0.30 is negative, 
.gtoreq.0.30 is positive. 
.sup.b For the Competitive ELISA, see preceding description. Mouse 
monoclonal antibody YsT9-2 was conjugated with the enzyme horseradish 
peroxidase. Data in brackets indicates percent inhibition (i.e. 
displacement of conjugated monoclonal antibody) by applied sera 
antibodies. Calculations were done by: 
##STR2## 
Note that only B. abortus infected sera give significant displacement of 
monoclonal antibody. 
.sup.c Readings are A.sub.414nm 
.sup.d Percent Inhibition 
Moreover, while the use of the polysaccharide O-chain to differentiate 
vaccinated animals from infected ones has been described above primarily 
with reference to cattle and B. abortus, the use of the polysaccharide 
O-chains of Brucella spp. (or of cross-reacting organisms) in swine, 
goats, sheep and humans may be expected to follow the same model, as might 
other animal or human diseases, and is considered to be within the scope 
of the invention.