Carbohydrate-based vaccine and diagnostic reagent for trichinosis

The present invention relates to Trichinella vaccines that include at least one tyvelose-containing oligosaccharide or functional equivalent thereof, to Trichinella vaccines that include at least one fucose-containing oligosaccharide or functional equivalent thereof, and to the use of such vaccines to protect animals from Trichinella infections, and particularly from trichinosis caused by Trichinella spiralis infection. Such vaccines can also be used to produce antibodies that are capable of protecting an animal from Trichinella infections and of diagnosing such infections. The present invention also relates to Trichinella diagnostic reagents that include at least one tyvelose-containing oligosaccharide, or functional equivalent thereof, and use of such reagents to detect Trichinella, and particularly Trichinella spiralis infections. The present invention also includes diagnostic kits based on such reagents and anti-Trichinella-spiralis drugs based on the knowledge that tyvelose is produced by Trichinella spiralis parasites.

FIELD OF THE INVENTION 
The present invention relates to novel carbohydrate-based vaccines and 
their use to protect animals from Trichinella infection. The present 
invention further relates to novel carbohydrate-based diagnostic reagents 
and their use to detect Trichinella infection in animals. The invention 
particularly relates to tyvelose-containing and fucose-containing vaccines 
to protect animals from trichinosis and to tyvelose-containing diagnostic 
reagents to detect Trichinella spiralis infection. 
BACKGROUND OF THE INVENTION 
Trichinosis is a disease of world-wide distribution that is primarily due 
to the ingestion of raw or undercooked meat (principally pork) containing 
the infective larval stage of Trichinella spiralis, the helminth parasite 
that causes the disease. After ingestion, Trichinella spiralis larvae 
infect the intestine where they mature within a few days. Female worms 
then bear newborn larvae which enter the general circulatory system and 
after several days accumulate in the striated muscles of the infected 
animal. Until recently, Trichinella spiralis infection has been detected 
by visual detection of larvae in muscle snips or digestion of muscle to 
liberate larvae (see, for example, U.S. Pat. No. 3,892,529 by Giles, 
issued Jul 1, 1975, and U.S. Pat. No. 3,918,818 by Giles, issued Nov. 11, 
1975). 
A group of protein (including glycoprotein) antigens extracted from 
Trichinella spiralis muscle stage larvae has been the subject of numerous 
studies in recent years, particularly in attempts to develop vaccines and 
diagnostic agents for trichinosis. These larval antigens are highly 
immunodominant and are apparently only present during the first muscle 
larval stage (L.sub.1) of Trichinella spiralis infection, being found on 
both the cuticular surface and excretory/secretory (ES) products of 
L.sub.1 larvae (see, for example, Denkers et al., pp. 241-250, 1990, Mol. 
Biochem. Parasitol., Vol. 41). These larval antigens, designated TSL-1 
antigens by Appleton et al., pp. 190-192, 1991, Parasitol. Today, Vol. 7, 
evoke a strong IgG.sub.1 antibody response in mice following oral 
infection (see, for example, Denkers et al., pp. 3152-3159, 1990, J. 
Immunol., Vol. 144) and induce substantial protection against challenge 
infections (see, for example, Silberstein et al., pp. 898-904, 1984, J. 
Immunol., Vol. 132; Silberstein et al., pp. 516-517, 1985, J. Parasitol., 
Vol. 71; Gamble, pp. 398-404, 1985, Exp. Parasitol., Vol. 59; Gamble et 
al., pp. 2396-2399, 1986, Am. J. Vet. Res., Vol. 47; Ortega-Pierres et 
al., pp. 563-567, 1989, Parasitol. Res., Vol. 75; Denkers et al., J. 
Immunol., ibid.; Jarvis et al., pp. 498-501, 1992, Parasite Immunol., Vol. 
14). 
TSL-1 antigens migrate under reducing conditions on SDS-PAGE (sodium 
dodecyl sulfate polyacrylamide gel electrophoresis) in a molecular weight 
range of 40-70 kilodaltons (kDa). Denkers et al., Mol. Biochem. 
Parasitol., ibid., have shown that at least six of the TSL-1 antigens 
share a common, highly immunodominant determinant. Use of monoclonal 
antibodies raised against the determinants indicated that the determinants 
are quite selective for Trichinella spiralis in that the monoclonal 
antibodies did not recognize other parasites, including the closely 
related species Trichuris muris (Denkers et al., pp. 403-410, 1991, Exp. 
Parasitol., Vol. 72). Moreover, the determinants appear to be ubiquitous 
among all Trichinella spiralis isolates tested so far (see, for example, 
Denkers et al., Exp. Parasitol., ibid.; Gamble et al., pp. 67-74, 1984, 
Am. J. Vet. Res., Vol. 46; Gamble et al., pp. 379-389, 1984, Vet. Immunol. 
Immunopath., Vol. 6). 
TSL-1 antigens are believed to have both protein and carbohydrate 
immunoreactive determinants, although there is some conflict in the 
literature about the importance of each. Denkers, et al., Mol. Biochem. 
Parasitol., ibid., demonstrated that the immunodominant determinants can 
be removed by treatment with trifluoromethanesulfonic acid, mild base, or 
N-glycanase, suggesting that the determinants are associated with both 
N-linked and O-linked carbohydrate groups. Denkers et al. also showed that 
the immunodominant determinants were not phosphorylcholine but did not 
further identify the composition of the carbohydrate moiety. Gold et al., 
pp. 187-196, Mol. Biochem. Parasitol., Vol. 41, isolated TSL-1 antigens of 
43 kDa and 45-50 kDa and treated them with N-glycanase. The deglycosylated 
antigens were no longer able to bind to polyclonal antibodies raised 
against the glycosylated versions of the proteins, again suggesting the 
importance of carbohydrate moieties, although Gold et al. do not exclude 
the possibility of peptide epitopes as well. 
In contrast, Jarvis et al., ibid., concluded that protein epitopes alone 
could induce protective immunity to Trichinella spiralis, having shown 
that ES antigens that had been deglycosylated using sodium periodate were 
as effective as native ES antigens in protecting mice from Trichinella 
spiralis infection in both active and passive immune assays. Su et al., 
pp. 331-336, 1991, Mol. Biochem. Parasitol., Vol. 45, reported that a 
recombinant fusion protein consisting of beta-galactosidase joined to the 
49-kDa TSL-1 antigen (P49) produced in Escherichia coli was bound by 
antibodies contained in serum isolated from swine infected with 
Trichinella spiralis, and by antibodies contained in serum isolated from 
mice immunized with native P49 antigen, but was not recognized by three 
monoclonal antibodies that bind selectively to native P49 antigen. Su et 
al. concluded that such results suggest that, at least the P49 antigen has 
both protein and carbohydrate immunoreactive determinants. 
Several investigators have developed enzyme-linked immunosorbent assays 
(ELISAs) to detect Trichinella spiralis infection using a variety of 
reagents, such as, crude Trichinella spiralis parasite preparations, 
partially purified ES antigen preparations, and monoclonal antibodies 
raised against, for example, the ES immunodominant determinants (see, for 
example, Ruitenberg et al., pp. 67-83, 1976, J. Immunol. Methods, Vol. 10; 
Gamble et al., 1983, pp. 349-361, Vet. Parasitol., Vol. 13; Gamble et al., 
Am. J. Vet. Res., ibid.; Gamble et al., Vet. Immunol. Immunopath., ibid.; 
U.S. Pat. No. 4,670,384, by Gamble et al., issued Jun. 2, 1987). Assays 
based on undefined crude or semi-defined antigen preparations are 
problematic due to false-positive and false-negative reactions as well as 
to cross-reactivity with antibodies corresponding to antigens of other 
parasites. Monoclonal antibody-based or purified protein-based assays, 
while often leading to fewer false-positive or false-negative reactions, 
can still have specificity and selectivity problems, in addition to 
difficulties of producing such components without batch-to-batch 
variation, and of maintaining the stability of the components. 
U.S. Pat. No. 4,795,633, by Murrell et al., issued Jan. 3, 1989, discloses 
a swine trichinosis vaccine consisting of an inert newborn larvae 
preparation emulsified with an adjuvant. GB 1,580,539, published Dec. 3, 
1989, discloses a trichinosis vaccine containing ES antigens of 
Trichinella spiralis muscle stage larvae. Several groups of investigators 
have reported the cloning of at least portions of certain Trichinella 
spiralis antigen genes with the goal of developing defined diagnostics 
reagents and/or vaccines (see, for example, Su et al., Mol. Biochem. 
Parasitol., ibid.; Sugane et al., pp. 1-8, 1990, J. Helminth., Vol. 64; 
Zarlenga et al., pp. 165-174, 1990, Mol. Biochem. Parasitol., Vol. 42). 
Problems with protein-based vaccines, and particularly with recombinant 
protein-based vaccines, include difficulty of preparation (particularly 
with respect to removal of harmful contaminants), lack of stability, 
potential reduced antigenicity compared to the native protein, and 
potential autoimmune reactions due to similarities between parasite and 
animal host proteins (e.g., Brugia pahangi glutathione peroxidase and 
Dirofilaria immitis superoxide dismutase; see, for example, Callahan et 
al., 235-252, 1991, Mol. Biochem. Parasitol., Vol. 49). 
A number of anthelminthic drugs have been developed to treat trichinosis 
(see, for example, U.S. Pat. No. 5,140,042 by Arison et al., issued Aug. 
18, 1992; U.S. Pat. No. 5,089,530 by Tsipouras et al., issued Feb. 18, 
1992; U.S. Pat. No. 5,073,567, by Maeda et al., issued Dec. 17, 1991; U.S. 
Pat. No. 5,008,250, by Fisher et al., issued Apr. 16, 1991; and U.S. Pat. 
No. 4,833,168, by Wyvratt, issued May 23, 1989). Such drugs, however, 
apparently cannot be used to prevent trichinosis, are expensive to 
produce, usually have undesirable side effects, and are not always 
effective. 
There remains a need for both diagnostic reagents to detect Trichinella 
spiralis infection and for vaccines and other drugs to protect animals 
from trichinosis that have improved specificity, selectivity, stability, 
consistency, and ease of use. 
SUMMARY OF THE INVENTION 
The present invention relates to a vaccine capable of protecting an animal 
from Trichinella infection, and preferably from trichinosis caused by 
Trichinella spiralis infection, when the vaccine is administered to the 
animal in an effective amount. One embodiment is a vaccine that includes 
at least one tyvelose-containing oligosaccharide or functional equivalent 
thereof, the tyvelose-containing oligosaccharide being selected from the 
group consisting of tyvelose and tyvelose joined through glycosidic 
linkage to at least one monosaccharide. Another embodiment is a vaccine 
that includes at least one fucose-containing oligosaccharide or functional 
equivalent thereof, the fucose-containing oligosaccharide being selected 
from the group consisting of fucose and fucose joined through glycosidic 
linkage to at least one monosaccharide. Vaccines of the present invention 
can also include both tyvelose-containing oligosaccharides and 
fucose-containing oligosaccharides. Preferred monosaccharides to join to 
either tyvelose or fucose include tyvelose, fucose, mannose, 
N-acetylgalactosamine, and N-acetylglucosamine. Tyvelose-containing 
oligosaccharides preferably have at least one tyvelose terminal residue, 
and fucose-containing oligosaccharides preferably have at least one fucose 
terminal residue. Preferred vaccines contain tyvelose-containing 
disaccharides and/or fucose-containing disaccharides. 
Vaccines of the present invention preferably include tyvelose-containing 
oligosaccharides and/or fucose-containing oligosaccharides conjugated to 
an effective carrier. Vaccines of the present invention can also include 
at least one immunopotentiator. 
Vaccines of the present invention can protect animals by, for example, 
preventing infection by Trichinella or by ameliorating or treating disease 
caused by the parasite. In one embodiment, the vaccines are also capable 
of protecting the animal from infection by other organisms containing 
tyvelose antigenic epitopes such as Salmonella serogroup D and Yersinia 
pseudotuberculosis serogroup IV microorganisms. Preferred animals to 
vaccinate include mammals, preferably pigs and humans, and particularly 
pigs. 
The present invention also includes a method to protect an animal from 
Trichinella infection, and preferably trichinosis, by administering to the 
animal an effective amount of a vaccine of the present invention. 
The present invention furthermore relates to a diagnostic reagent effective 
in detecting Trichinella infection, and preferably Trichinella spiralis 
infection. The diagnostic reagent includes at least one 
tyvelose-containing oligosaccharide or functional equivalent thereof, the 
tyvelose-containing oligosaccharide being selected from the group 
consisting of tyvelose and tyvelose joined through glycosidic linkage to 
at least one monosaccharide, which preferably is selected from the group 
consisting of tyvelose, fucose, mannose, N-acetylgalactosamine, and 
N-acetylglucosamine. Tyvelose-containing oligosaccharides preferably have 
at least one tyvelose terminal residue, and preferably are disaccharides. 
One embodiment of the present invention is a diagnostic reagent that 
includes a tyvelose-containing oligosaccharide conjugated to an effective 
carrier. 
Another embodiment of the present invention is a method to determine 
Trichinella, and preferably Trichinella spiralis, infection in an animal 
which includes: (a) applying serum collected from the animal onto a 
surface coated with a diagnostic reagent of the present invention under 
conditions such that selective binding of an antibody from the serum 
indicative of Trichinella infection to the reagent-coated surface results 
in the formation of a selective binding complex on the reagent-coated 
surface; (b) removing non-bound serum material under conditions that 
retain the selective binding complex on the reagent-coated surface; and 
(c) determining Trichinella infection by detecting the selective binding 
complex. The step of detecting preferably includes (a) contacting the 
selective binding complex with an identifying labeled compound capable of 
binding selectively to the antibody or to the complex; (b) removing 
substantially all of the identifying labeled compound that does not 
selectively bind to the antibody or to the complex; and (c) detecting the 
identifying labeled compound, wherein presence of the labeled compound is 
indicative of Trichinella infection. Also disclosed is a method to 
discriminate between Trichinella infection and infection caused by 
Salmonella serogroup D microorganisms and/or Yersinia pseudotuberculosis 
serogroup IV microorganisms that includes the use of fucose-containing 
oligosaccharides of the present invention. 
The invention also provides a diagnostic kit for detecting Trichinella, and 
preferably Trichinella spiralis, infection in an animal that includes a 
diagnostic reagent of the present invention. The kit can also include a 
surface capable of being coated with the reagent. Preferably the surface 
is pre-coated the reagent. The kit can also include a means for detecting 
the binding of an antibody indicative of Trichinella infection to the 
reagent. The kit can also include an agent capable of discriminating 
between Trichinella infection and an infection caused by Salmonella 
serogroup D and/or Yersinia pseudotuberculosis microorganisms. 
Animals that can be diagnosed using diagnostic reagents and diagnostic kits 
of the present invention include mammals, preferably humans and pigs, and 
particularly pigs. 
One embodiment of the present invention is a vaccine including at least one 
tyvelose-containing oligosaccharide or functional equivalent thereof, the 
tyvelose-containing oligosaccharide being selected from the group 
consisting of tyvelose and tyvelose joined through glycosidic linkage to 
at least one monosaccharide, the oligosaccharide having at least one 
tyvelose terminal residue that is joined through glycosidic linkage to a 
monosaccharide other than mannose. 
Another embodiment of the present invention is an antibody, or functional 
equivalent thereof, capable of selectively binding to a Trichinella 
parasite, the antibody being produced by a process comprising: (a) 
administering to an animal an effective amount of a vaccine of the present 
invention; and (b) recovering the antibody. Antibodies of the present 
invention can be either polyclonal or monoclonal antibodies. Such 
antibodies or functional equivalents thereof can be used to protect an 
animal from Trichinella infection, such as from trichinosis, by 
administering to the animal an amount of the antibody or functional 
equivalent thereof effective to protect the animal from trichinosis. Such 
antibodies or functional equivalents thereof can also be used to diagnose 
Trichinella infection in an animal. 
Yet another embodiment is an antibody of the present invention that is 
conjugated to a cytotoxic agent that can be used to protect an animal from 
Trichinella infection, and preferably from trichinosis. 
An additional embodiment is an anti-Trichinella spiralis drug capable of 
substantially inhibiting tyvelose production by Trichinella spiralis or 
other Trichinella parasites, the drug being capable of substantially 
inhibiting at least one enzyme essentially specific for tyvelose 
biosynthesis, thereby protecting an animal from Trichinella infection, and 
preferably from trichinosis.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention includes vaccines and diagnostic reagents, preferably 
directed against Trichinella, and even more preferably directed against 
Trichinella spiralis, infection that contain tyvelose (i.e., 
3,6-dideoxy-D-arabinohexose) predicated upon the inventors' surprising 
discovery that the immunodominant determinants of TSL-1 antigens contain 
significant amounts of tyvelose, an extremely rare sugar in nature. As 
such, vaccines and diagnostic reagents of the present invention are 
advantageous because they are highly selective, particularly for 
Trichinella spiralis (i.e., they specifically target Trichinella spiralis 
and do not substantially recognize other organisms). 
Tyvelose has, to the inventors' knowledge, not previously been found in 
eukaryotes and has not previously been found associated with a protein 
(i.e., as a component of a glycoprotein). In fact, 3,6-dideoxyhexoses as a 
class are quite rare, having only been identified in ascaryl alcohols 
(i.e., ascarosides) of the eggs of the worm parasite Ascaris and in 
certain gram negative bacterial lipopolysaccharides (see, for example, 
Fairbairn, pp. 491-554, 1957, Exp. Parasitol., Vol. 6; Jezyk et al., pp. 
691-705, 1967, Comp. Biochem. Physiol., Vol. 23; Lindberg et al., pp. 
83-118, 1983, in Bacterial Lipopolysaccharides-Structure, Synthesis and 
Biological Activities, L. Anderson and F. M. Unger, eds., ACS Symposium 
Series, Washington, D.C.; and references cited therein). Ascaris eggs 
contain ascarylose, or 3,6-dideoxy-L-arabinohexose, which is 
immunologically distinct from tyvelose. The 3,6-dideoxyhexoses contained 
in bacterial lipopolysaccharides have been particularly well studied in 
Salmonella, a bacterial genus that has been serotyped essentially 
according to the nature of its 3,6-dideoxyhexose determinants. For 
example, the lipopolysaccharides of Salmonella serogroup A contain 
paratose, those of serogroup B contain abequose, and those of serogroup D 
contain tyvelose. Although the 3,6-dideoxyhexoses are part of a repeating 
unit of 3 to 5 monosaccharides, it has been shown that immune responses to 
Salmonella lipopolysaccharide antigens are dominated by 
3,6-dideoxyhexose-based epitopes (see, for example, Lindberg et al., 
ibid.; Luderitz et al., pp. 192-255, 1966, Bacteriol. Rev., Vol. 30). 
Similarly, Yersinia (i.e., Pasteurella) pseudotuberculosis microorganisms 
have been serotyped based on whether the bacteria contain paratose 
(serogroups I and III), abequose (serogroup II), tyvelose (serogroup IV), 
or ascarylose (serogroup V) (see, for example, Luderitz et al., ibid.). 
That investigators would not have expected tyvelose to be present on 
Trichinella spiralis antigens is underscored by the study of Jarvis et 
al., ibid., in which Jarvis et al. concluded that peptide epitopes, rather 
than carbohydrate epitopes, were responsible for the antigenicity of 
periodate-treated ES antigens. This conclusion was reached in spite of 
reports in the literature that 3,6-dideoxyhexoses are resistant to 
cleavage by periodate, a characteristic that has been used to verify 
Salmonella serotyping (see, for example, Kabat, pp. 176-179, 1976, 
Structural Concepts in Immunology and Immunochemistry, Holt, Rinehart and 
Winston, New York). Clearly, Jarvis et al. did not recognize that tyvelose 
was a component of Trichinella spiralis immunodominant determinants. 
The inventors have also identified four other monosaccharides that comprise 
a substantial proportion of Trichinella spiralis TSL-1 immunodominant 
determinants: fucose, mannose, N-acetylgalactosamine, and 
N-acetylglucosamine, with the amount of fucose present being surprisingly 
high. Fucose, although prevalent in nature, is not usually a dominant 
sugar in the overall composition. Fucose has also been shown to have 
immunological relevance in mammals and parasites, having been found 
associated with several particular parasites and with glycosphingolipids 
in certain mammalian tumor tissues. As such, fucose-based vaccines of the 
present invention are also believed to be advantageous for protecting an 
animal from trichinosis. 
One embodiment of the present invention is a vaccine that includes at least 
one tyvelose-containing oligosaccharide or functional equivalent thereof, 
the vaccine being capable of protecting an animal from Trichinella 
infection, and preferably from trichinosis caused by Trichinella spiralis 
infection when administered to the animal in an effective amount. As used 
herein, a vaccine "capable of protecting an animal from Trichinella 
infection" refers to the ability of the vaccine to treat (e.g., as an 
immunotherapeutic agent), ameliorate, and/or prevent Trichinella infection 
caused by a Trichinella parasite that contains tyvelose antigenic epitopes 
(i.e., epitopes that are able to bind to antibodies produced upon 
administration of a tyvelose-containing oligosaccharide vaccine of the 
present invention). As used herein, a vaccine "capable of protecting an 
animal from trichinosis" refers to the ability of the vaccine to treat 
(e.g., as an immunotherapeutic agent), ameliorate, and/or prevent 
Trichinella spiralis infection that otherwise would lead to trichinosis in 
the animal. Preferably the vaccine protects the animal by eliciting an 
immune response. A preferred vaccine is one that, when administered to an 
animal, is able to elicit (i.e., stimulate) the production of high 
antibody titers as well as a high-level cellular immune response capable 
of protecting the animal from trichinosis. 
As used herein, a "tyvelose-containing oligosaccharide" can be a single 
tyvelose or tyvelose joined through glycosidic linkage to at least one 
monosaccharide. Preferred monosaccharides include tyvelose, fucose, 
mannose, N-acetylgalactosamine, and N-acetylglucosamine. Preferably, the 
tyvelose-containing oligosaccharide has at least one tyvelose terminal 
residue. As used herein, "tyvelose joined through glycosidic linkage to at 
least one of the following monosaccharides" denotes an oligosaccharide in 
which tyvelose is joined to one or more monosaccharides according to 
standard carbohydrate chemistry (i.e., by glycosidic linkages). As such, 
the oligosaccharide can be either linear or branched. The inventors have 
found that tyvelose is apparently almost always located at the 
non-reducing terminal position of TSL-1 immunodominant determinants; i.e., 
that tyvelose is believed to be principally a terminal residue of 
naturally-occurring oligosaccharides on TSL-1 antigens. As used herein, a 
"terminal residue" is located either at an end (terminus) of or at a 
branch-point of an oligosaccharide such that the residue is exposed to 
elicit an immune response capable of protecting an animal from trichinosis 
and/or to bind (i.e., adsorb) selectively to an antibody indicative of 
Trichinella spiralis infection. 
As used herein, a functional equivalent of a tyvelose-containing 
oligosaccharide is a molecule (e.g., a carbohydrate or other organic 
molecule) that has an epitope that can essentially mimic the 
tyvelose-containing oligosaccharide's epitope by eliciting a substantially 
similar immune response. Preferably the functional equivalent has a 
similar tertiary structure to the oligosaccharide's epitope. As such, 
functional equivalents of tyvelose may be determined, for example, either 
by analyzing molecules sharing a substantially similar epitope structure 
as that of naturally occurring tyvelose or by selecting those molecules 
with substantially similar functional identifying characteristics. 
Another embodiment of the present invention is a vaccine that includes at 
least one fucose-containing oligosaccharide or functional equivalent 
thereof, the vaccine being capable of eliciting an immune response capable 
of protecting an animal from Trichinella infection, and preferably from 
trichinosis caused by Trichinella spiralis infection, when administered in 
an effective amount. As used herein, a "fucose-containing oligosaccharide" 
can be a single fucose or fucose joined through glycosidic linkage to at 
least one monosaccharide. Preferred monosaccharides include tyvelose, 
fucose, mannose, N-acetylgalactosamine, and N-acetylglucosamine. 
Preferably, the fucose-containing oligosaccharide has at least one fucose 
terminal residue. The inventors have found that the fucose moieties 
present on Trichinella spiralis immunodominant determinants are primarily 
non-reducing terminal residues. As used herein, a functional equivalent of 
a fucose-containing oligosaccharide is a molecule (e.g., a carbohydrate or 
other organic molecule) that has an epitope that can essentially mimic the 
fucose-containing oligosaccharide's epitope by eliciting a substantially 
similar immune response. Preferably the functional equivalent has a 
similar tertiary structure to the oligosaccharide's epitope. As such, 
functional equivalents of fucose may be determined, for example, either by 
analyzing molecules sharing a substantially similar epitope structure as 
that of naturally occurring fucose or by selecting those molecules with 
substantially similar functional identifying characteristics. 
Preferred tyvelose-containing oligosaccharides of the present invention 
include tyvelose, and tyvelose joined to from about one to about three 
monosaccharides, with disaccharide oligosaccharides being more preferred. 
Similarly, preferred fucose-containing oligosaccharides of the present 
invention include fucose, and fucose joined to from about one to about 
three monosaccharides, with disaccharide oligosaccharides being more 
preferred. It is however, within the scope of the invention that larger 
oligosaccharides having at least one tyvelose terminal residue and/or at 
least one fucose terminal residue can also be effective vaccines. 
Particularly preferred vaccine contain the disaccharide 
tyvelose--tyvelose, tyvelose--fucose, tyvelose--mannose, 
tyvelose--N-acetylgalactosamine, tyvelose--N-acetylglucosamine, 
fucose--fucose, fucose--mannose, fucose--N-acetylgalactosamine, 
fucose--N-acetylglucosamine, or mixtures of those disaccharides. As used 
herein "--" in this context indicates the glycosidic linkage that forms 
the disaccharide. Without being bound by theory, the inventors believe 
that disaccharide vaccines against trichinosis can be equally effective as 
disaccharide vaccines against Salmonella, which are described, for 
example, in Svenungsson et al., pp. 1-11, 1977, Med. Microbiol. Immunol., 
Vol. 163; in Lindberg et al., ibid.,; and in references cited therein. The 
inventors further believe that tyvelose particularly is likely to make an 
effective vaccine since tyvelose, like other 3,6-dideoxyhexoses, is a 
dominant antigenic determinant. 
A preferred vaccine of the present invention comprises a 
tyvelose-containing oligosaccharide conjugated to an effective carrier. 
Another preferred vaccine has a fucose-containing oligosaccharide 
conjugated to an effective carrier. As used herein, an "effective carrier" 
is a compound that enables the oligosaccharide to function as a vaccine or 
diagnostic agent and that is conjugated to the oligosaccharide in such a 
manner as to not substantially interfere with the oligosaccharide's 
desired function. Preferably, the carrier is able to augment, or enhance, 
the oligosaccharide's activity as a vaccine or diagnostic reagent. As used 
herein, "conjugated" refers to joining the carbohydrate moiety and carrier 
together, preferably by a covalent attachment. For example, in fucose or 
tyvelose vaccines, the carrier is attached to tyvelose or fucose in such a 
manner that the tyvelose or fucose epitope maintains the capacity to 
elicit an immune response capable of protecting an animal from trichinosis 
or to selectively bind to an antibody indicative of Trichinella spiralis 
infection. When the vaccine is a disaccharide or larger oligosaccharide, 
the carrier is typically attached to a monosaccharide other than the 
tyvelose or fucose epitope in order to reduce potential interference with 
the ability of the oligosaccharide to function as an effective vaccine or 
diagnostic reagent. Particularly preferred vaccines include 
tyvelose::carrier, tyvelose--tyvelose::carrier, tyvelose--fucose::carrier, 
tyvelose--mannose::carrier, tyvelose--N-acetylgalactosamine::carrier, 
tyvelose--N-acetylglucosamine::carrier, fucose::carrier, 
fucose--fucose::carrier, fucose--mannose::carrier, 
fucose--N-acetylgalactosamine::carrier, 
fucose--N-acetylglucosamine::carrier, or mixtures thereof, wherein "::" 
indicates the attachment of the carrier to the disaccharide. 
A preferred carrier is a compound of sufficient size and immunogenicity 
capable of augmenting the immune response of the vaccine. Suitable 
carriers include, but are not limited to: proteins, such as toxoids, serum 
proteins, keyhole limpet hemocyanin, or Trichinella spiralis muscle stage 
larval antigens; polymerized sugars, such as polydextrans; other polymers; 
viruses or viral subunits; and lipid-containing compounds, such as 
liposomes. Preferred carriers of the present invention include bacterial 
toxoids, such as tetanus toxoid, diphtheria toxoid, and cholera toxoid; 
bovine serum albumin; ovalbumin; and Trichinella spiralis muscle stage 
larval antigens. A particularly preferred carrier is tetanus toxoid, which 
has been shown to be safe in vaccine applications (see, for example, 
Herrington et al., pp. 257-259, 1987, Nature, Vol. 328). 
Another particularly preferred class of carriers consists of Trichinella 
spiralis antigen carriers, defined herein as Trichinella spiralis muscle 
stage larval antigens (as heretofore described), recombinant protein 
antigens corresponding to those antigens, and functional equivalents of 
the larval antigens or corresponding recombinant protein antigens (e.g., 
that elicit at least some immunogenic response against trichinosis). A 
vaccine comprising at least one Trichinella spiralis antigen carrier 
conjugated to a tyvelose-containing or fucose-containing oligosaccharide 
of the present invention may afford animals enhanced protection compared 
to either the larval antigen or oligosaccharide alone. 
One embodiment of the present invention is a vaccine containing more than 
one tyvelose- or fucose-containing oligosaccharide. Although a single type 
of oligosaccharide is believed capable of eliciting an immune response, it 
is likely that a mixture of various types of oligosaccharides of the 
present invention may be more efficacious. Preferably the various types of 
oligosaccharides of the present invention are conjugated to effective 
carriers, as heretofore described. 
Another embodiment of the present invention is the inclusion of at least 
one fucose-containing oligosaccharide in tyvelose-containing 
oligosaccharide vaccines of the present invention. Yet another embodiment 
of the present invention is the inclusion of at least one 
tyvelose-containing oligosaccharide in fucose-containing oligosaccharide 
vaccines of the present invention. Mixtures of fucose-containing 
oligosaccharides and tyvelose-containing oligosaccharides are believed to 
enhance the ability of such a vaccine to protect an animal from 
trichinosis. While not being bound by theory, it is believed that the 
prevalence of fucose and tyvelose moieties as non-reducing terminal 
residues in Trichinella spiralis immunodominant determinants suggests that 
each structure is likely to possess a dominant epitope that is able to 
elicit an immune response that is capable of protecting an animal from 
trichinosis. 
Another embodiment of the present invention is a vaccine including at least 
one tyvelose-containing oligosaccharide having at least one tyvelose 
terminal residue joined through glycosidic linkage to a monosaccharide 
other than mannose. 
Yet another aspect of the present invention is the realization that the 
claimed tyvelose-containing oligosaccharide vaccines of the present 
invention are also capable of protecting animals from infection by 
Salmonella or Yersinia pseudotuberculosis microorganisms that have 
lipopolysaccharides containing tyvelose, e.g., Salmonella serogroup D or 
Yersinia pseudotuberculosis serogroup IV microorganisms. Thus, vaccines of 
the present invention may be used to simultaneously protect animals from 
Salmonella serogroup D, Yersinia pseudotuberculosis serogroup IV and 
Trichinella spiralis infections. 
Furthermore, it is within the scope of the present invention that 
tyvelose-containing oligosaccharide vaccines of the present invention can 
be used to protect an animal against infection by any parasite of the 
genus Trichinella, and even more broadly against infection by any 
organism, that contains tyvelose antigenic epitopes (i.e., epitopes that 
can be bound by antibodies produced upon administration of a 
tyvelose-containing oligosaccharide vaccine of the present invention). 
Similarly, it is within the scope of the present invention that 
fucose-containing oligosaccharide vaccines of the present invention can be 
used to protect an animal against infection by any parasite of the genus 
Trichinella, and even more broadly against infection by any organism, that 
contains fucose antigenic epitopes (i.e., epitopes that are able to bind 
to antibodies produced upon administration of a fucose-containing 
oligosaccharide vaccine of the present invention). 
Vaccines of the present invention can also include additional antigenic 
compounds effective in eliciting an immune response against, for example, 
other stages of the Trichinella life cycle. Vaccines of the present 
invention can also be components of multiple vaccine preparations that 
include antigens targeted against more than one disease. 
Vaccines of the present invention can be produced using standard techniques 
of carbohydrate and protein linkage technologies (see, for example, 
Lindberg et al., ibid.; Russell et al., pp. 95-114, 1990, Carbohydrate 
Research, Vol. 201; Svenson and Lindberg, pp. 323-335, 1979, J. Immunol. 
Methods, Vol. 25; McBroom et al., pp. 212-219, 1972, Methods in 
Enzymology, Vol. 28B). Briefly, the monosaccharides are produced and, as 
necessary for specific vaccine embodiments, joined by glycosidic linkage 
to form disaccharides and larger oligosaccharides. For preferred 
embodiments, the carbohydrate moieties are conjugated to effective 
carriers, preferably using reactive group linking agents. For example, one 
method to produce a vaccine containing tyvelose--mannose::tetanus toxoid 
includes the steps of (a) synthesizing tyvelose precursors, (b) joining 
the precursors to derivatized mannose residues, (c) joining the 
synthesized disaccharide to a suitable aglycone-containing reactive group, 
and (d) conjugating the modified disaccharide to a tetanus toxoid. One 
advantage of the present invention is the ease with which such 
carbohydrate-based vaccines can be produced on a consistent basis, 
particularly as compared with the time and effort required to produce 
recombinant protein-based vaccines. In addition, it may be particularly 
difficult to produce recombinant Trichinella spiralis proteins having 
tyvelose-containing epitopes using conventional recombinant techniques 
since (a) bacteria do not glycosylate proteins and (b) no eukaryotic cells 
are known to the inventors that are capable of producing tyvelose, except 
Trichinella spiralis. 
Tyvelose- and fucose-containing oligosaccharide vaccines of the present 
invention are preferably recovered in "substantially pure" form. As used 
herein, "substantially pure" refers to a purity that allows for the 
effective use of the vaccine without substantial negative side effects. 
For example, substantially pure vaccines would not elicit undesired 
biological reactions when administered to animals to be treated. 
Vaccines of the present invention can be administered to any animal, 
preferably to mammals, more preferably to humans and pigs, and 
particularly to pigs. 
Vaccines can be formulated in an aqueous balanced salt solution that the 
animal to be vaccinated can tolerate. The vaccine can also include an 
immunopotentiator, such as an adjuvant or other agent that enhances the 
immune response of the vaccine. Suitable immunopotentiators include, but 
are not limited to, polymeric controlled release formulations, 
biodegradable implants, liposomes, bacterial preparations (such as 
bacterial coat proteins), viruses or viral proteins (such as coat 
proteins), oils, esters, glycols, Freund's adjuvant, aluminum-based salts, 
calcium-based salts, silica, polynucleotides, gamma interferon, Ribi 
adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, 
Mont.), and saponins and their derivatives, such as Quil A (available from 
Superfos Biosector A/S, Denmark). 
In order to protect animals from trichinosis, a vaccine of the present 
invention is administered in an effective amount, wherein an "effective 
amount" is an amount that allows the animal to produce a sufficient immune 
response to protect itself from trichinosis. Vaccines of the present 
invention can be administered to animals prior to infection by Trichinella 
spiralis to prevent trichinosis. Vaccines of the present invention can 
also be administered to animals after infection by Trichinella spiralis in 
order to treat the disease, in which case the vaccine is acting as an 
immunotherapeutic agent. Vaccines of the present invention are 
advantageous because they are stable and are easy to use, particularly in 
the field. Acceptable administration protocols include individual dose 
size, number of doses, frequency of dose administration, and mode of 
administration. A suitable single dose of the vaccine is a dose that is 
capable of protecting an animal from trichinosis when administered one or 
more times over a suitable time period. A preferred single dose of the 
vaccine is from about 1 microgram (.mu.g) to about 1 milligram (mg) of the 
vaccine per kilogram (kg) body weight of the animal. Booster vaccinations 
can be administered from about 2 weeks to several years after the original 
vaccination. Preferably booster vaccinations are administered when the 
immune response of the animal becomes insufficient to protect the animal 
from trichinosis. A preferred administration schedule is one in which from 
about 1 .mu.g to about 1 mg of the vaccine per kg body weight of the 
animal are administered from about one to about two times over a time 
period of from about 2 weeks to about 12 months. Modes of administration 
can include, but are not limited to, subcutaneous, intradermal, 
intravenous, nasal, oral, transdermal and intramuscular routes. 
The efficacy of a vaccine of the present invention to protect an animal 
from trichinosis can be tested in a variety of ways including, but not 
limited to, detection of protective antibodies (using, for example, 
diagnostic reagents of the present invention), detection of cellular 
immunity within the vaccinated animal, or challenge of the vaccinated 
animal with Trichinella spiralis or antigens thereof to determine whether 
the vaccinated animal is resistant to trichinosis. 
Another embodiment of the present invention relates to the production and 
use of antibodies, or functional equivalents of such antibodies, that are 
capable of selectively binding to Trichinella spiralis muscle stage larvae 
produced by an animal in response to administration of a vaccine of the 
present invention. Such antibodies can be either polyclonal or monoclonal 
antibodies. As used herein, functional equivalents of such antibodies are 
antibodies, including fragments of any size, that have similar selective 
epitope binding characteristics as the antibodies produced in response to 
vaccination. Antibodies produced by animals vaccinated with a vaccine 
containing tyvelose-containing oligosaccharides can bind selectively to 
tyvelose-containing epitopes. Similarly, antibodies produced by animals 
vaccinated with a vaccine containing fucose-containing oligosaccharides 
can bind selectively to fucose-containing epitopes. Antibodies of the 
present invention, including functional equivalents thereof, have a 
variety of potential uses that are within the scope of the present 
invention. For example, such antibodies can be used (a) as vaccines to 
passively immunize an animal in order to protect the animal from 
trichinosis, (b) as reagents in assays to detect Trichinella spiralis 
larvae or antigens thereof, and/or (c) as tools to recover Trichinella 
spiralis antigens having immunodominant determinants from a mixture of 
proteins and other contaminants. 
Furthermore, antibodies of the present invention, including functional 
equivalents thereof, can be used to target cytotoxic agents to Trichinella 
spiralis larvae and larval antigens in order to directly kill the larvae 
or cells expressing larval antigens on their cell surface. Targeting can 
be accomplished by conjugating (i.e., stably joining) such antibodies to 
the cytotoxic agents. Suitable cytotoxic agents include, but are not 
limited to: double-chain toxins (i.e., toxins having A and B chains), such 
as diphtheria toxin, ricin toxin, Pseudomonas exotoxin, modeccin toxin, 
abrin toxin, and shiga toxin; single-chain toxins, such as pokeweed 
antiviral protein, .alpha.-amanitin, and ribosome inhibiting proteins; and 
chemical toxins, such as melphalan, methotrexate, nitrogen mustard, 
doxorubicin and daunomycin. Preferred double-chain toxins are modified to 
include the toxic domain and translocation domain of the toxin but to lack 
the toxin's intrinsic cell binding domain. 
One embodiment of the present invention is a diagnostic reagent effective 
in detecting Trichinella, and particularly Trichinella spiralis, infection 
in an animal. A diagnostic reagent of the present invention includes at 
least one tyvelose-containing oligosaccharide that is capable of 
selectively binding to an antibody indicative of infection by the 
parasite. As heretofore defined, a "tyvelose-containing oligosaccharide" 
can be tyvelose or tyvelose joined through glycosidic linkage to at least 
one monosaccharide, preferably such that the tyvelose-containing 
oligosaccharide has at least one tyvelose terminal residue. Preferred 
monosaccharides include tyvelose, fucose, mannose, N-acetylgalactosamine, 
and N-acetylglucosamine. 
Diagnostic reagents of the present invention, being based on the rare sugar 
tyvelose, are particularly advantageous because they exhibit great 
selectivity for Trichinella spiralis and other Trichinella parasites 
having tyvelose-containing epitopes in that they selectively bind to 
antibodies indicative of infection by such Trichinella parasites, and 
preferably by Trichinella spiralis. Preferred diagnostic reagents bind to 
antibodies raised by the animal in response to Trichinella spiralis 
infection but do not appreciably bind to antibodies directed against 
agents that do not have substantial amounts of tyvelose-containing 
epitopes. Thus, diagnostic reagents of the present invention are much less 
likely to give false-positive or false-negative reactions than are known 
diagnostic reagents, such as those heretofore described. Two possible 
exceptions are antibodies produced in response to infection by Salmonella 
serogroup D microorganisms or Yersinia pseudotuberculosis serogroup IV 
microorganisms since the antigens of these bacterial serogroups are the 
only antigens, other than TSL-1 antigens, known by the inventors to 
include tyvelose. It should be noted that all diagnostic reagents based on 
Trichinella spiralis TSL-1 immunodominant determinants, regardless of 
whether they are antigen- or antibody-based, are vulnerable to the same 
potential complications (e.g., false-positive reactions). Until the 
present invention, such a concern was unappreciated by those 
skilled-in-the art. Agents including fucose-containing oligosaccharides 
that are capable of eliciting an immune response to protect an animal 
against trichinosis may be used to discriminate between Trichinella 
spiralis and Salmonella serogroup D or Yersinia pseudotuberculosis 
serogroup IV infections, since neither Salmonella serogroup D nor Yersinia 
pseudotuberculosis serogroup IV lipopolysaccharides contain fucose. 
Alternatively, an antibody, preferably a monoclonal antibody, raised 
against the protein portion of Trichinella spiralis muscle larval antigens 
could be used to discriminate between Trichinella spiralis and Salmonella 
serogroup D or Yersinia pseudotuberculosis serogroup IV infections. 
It is also within the scope of the present invention that diagnostic 
reagents of the present invention could be used to detect Salmonella 
serogroup D or Yersinia pseudotuberculosis serogroup IV infections, or any 
infections caused by organisms having tyvelose antigenic epitopes. 
Diagnostic reagents of the present invention are also advantageous because, 
as described above for vaccines, the tyvelose-containing oligosaccharides 
and tyvelose-containing oligosaccharides conjugated to effective carriers 
are stable, are easy to produce on a consistent basis, and are easy to 
use, particularly in field tests. Previous assays for trichinosis, 
regardless of whether they were competitive or non-competitive in nature, 
and whether they were based on crude or partially purified Trichinella 
spiralis larval antigen preparations, polyclonal antibodies, and/or 
monoclonal antibodies have been hampered by several problems, including 
selectivity (i.e., large numbers of false-positive or false-negative 
reactions), ease of preparation, and/or usefulness in field tests 
(reviewed in, for example, Su et al., pp. 76-82, 1991, J. Parasitol., Vol. 
77). 
Tyvelose-containing oligosaccharides used in vaccines of the present 
invention are also suitable for use as diagnostic reagents. As such, the 
method to produce tyvelose-containing oligosaccharides for diagnostic 
reagents is similar to that heretofore disclosed for the production of 
such oligosaccharide for vaccines. Preferred tyvelose-containing 
oligosaccharides include tyvelose, and tyvelose joined to from about one 
to about three monosaccharides, with disaccharide oligosaccharides being 
more preferred. Particularly preferred diagnostic reagents contain the 
disaccharide tyvelose--tyvelose, tyvelose--fucose, tyvelose--mannose, 
tyvelose--N-acetylgalactosamine, tyvelose--N-acetylglucosamine, or 
mixtures of such disaccharides. 
One embodiment of the present invention is a diagnostic reagent in which 
the tyvelose-containing oligosaccharide is conjugated to an effective 
carrier in such a manner as to not substantially interfere with the 
ability of the reagent to selectively bind to antibodies indicative of 
Trichinella spiralis infection. Such a carrier may be useful in coating a 
diagnostic reagent to a surface for use in a diagnostic assay for 
trichinosis. The method and manner in which carriers are attached to 
diagnostic reagent oligosaccharides is similar to that heretofore 
disclosed for vaccine oligosaccharides. Preferred diagnostic reagents 
conjugated to a carrier include tyvelose::carrier, 
tyvelose--tyvelose::carrier, tyvelose--fucose::carrier, 
tyvelose--mannose::carrier, tyvelose--N-acetylgalactosamine::carrier, 
tyvelose--N-acetylglucosamine::carrier, or mixtures thereof. 
Suitable and preferred carriers for tyvelose-containing 
oligosaccharide-based diagnostic reagents are as heretofore disclosed for 
tyvelose-containing oligosaccharide-based vaccines. 
One embodiment of the present invention is a diagnostic reagent containing 
more than one tyvelose-containing oligosaccharide. Although a single 
oligosaccharide is capable of selectively binding to an antibody 
indicative of Trichinella spiralis infection, it is likely that a mixture 
of oligosaccharides may be more efficacious. Preferably the 
oligosaccharides are conjugated to effective carriers, as heretofore 
described. 
Another embodiment of the present invention is the use of a diagnostic 
reagent of the present invention to detect Trichinella spiralis infection 
in an animal (i.e., a method to determine Trichinella spiralis infection 
in an animal using such a diagnostic reagent). Any animal susceptible to 
Trichinella spiralis infection can be tested, including, but not limited 
to humans and pigs. The detection method of the present invention is 
particularly useful in field tests, such as those conducted on pigs. 
Any suitable assay can be used in which at least one diagnostic reagent of 
the present invention can be contacted with animal serum under conditions 
that allow for selective binding of the diagnostic reagent to at least one 
antibody in the serum that is indicative of Trichinella spiralis 
infection. As used herein, "under conditions that allow for selective 
binding" refers to reaction conditions, such as appropriate buffers, 
temperatures, and reaction times that enable selective binding of an 
antibody to an antigen that the antibody recognizes. Such conditions are 
known to those skilled in the art as are methods to optimize such 
conditions for a specific antigen-antibody interaction (see, for example, 
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Labs Press, Cold Spring Harbor, N.Y.; Su et al., J. Parasitol., 
ibid.). Suitable assays can include, but are not limited to, solution 
assays as well as solution assays including a solid phase, and can be 
either competitive or non-competitive. That any such assay system is 
suitable for determining infection is due to the advantages of the 
diagnostic reagent per se: that the reagent is specific (i.e., can bind to 
anti-Trichinella spiralis antibodies with high affinity), selective, easy 
to prepare, and easy to use. 
A preferred method to determine Trichinella spiralis infection in an animal 
includes the following steps: (a) applying serum collected from the animal 
onto a surface coated with a diagnostic reagent of the present invention 
under conditions such that selective binding of an antibody indicative of 
Trichinella spiralis infection to the reagent-coated surface is 
accomplished (i.e., occurs) in order to form a selective binding complex 
on the reagent-coated surface, (b) removing non-bound serum material under 
conditions that retain the selective binding complex on the reagent-coated 
surface, and (c) determining Trichinella spiralis infection by detecting 
the selective binding complex. As used herein, a "reagent-coated surface" 
is a surface to which a diagnostic reagent of the present invention is 
bound (i.e., adsorbed). As used herein, a "selective binding complex" 
refers to the complex formed when an antibody indicative of Trichinella 
spiralis infection binds to a diagnostic reagent of the present invention. 
Suitable surfaces on which to coat a diagnostic reagent of the present 
invention include any surface to which a tyvelose-containing 
oligosaccharide and/or an effective carrier can bind in an essentially 
stable configuration (i.e., such that the oligosaccharide or carrier 
adsorbs to the surface and is not substantially removed from the surface 
during the assay). Preferably, a suitable plastic, glass, cell, or 
celluloid surface is used. In addition, the oligosaccharide and/or carrier 
should be able to bind to the surface without substantially interfering 
with the ability of the oligosaccharide to selectively bind to an antibody 
indicative of Trichinella spiralis infection. Examples of suitable 
surfaces include, but are not limited to, plate wells (e.g., in microtiter 
dishes), plates, dishes, tubes, beads, dip-sticks, filters (e.g., nylon, 
nitrocellulose, or derivatives thereof), and suitable celluloid-type 
matrices. Suitable assays to conduct using such surfaces include, but are 
not limited to, competitive or noncompetitive ELISAs (enzyme-linked 
immunosorbent assays), Western blots, dot blots, radioimmunoassays, 
immunoprecipitation assays, agglutinin assays, Ouchterlony assays, and 
Mancini assays. Methods to coat antigens onto surfaces are well known in 
the art (see, for example, Carpenter, pp. 2-9, 1992, The Manual of 
Clinical Laboratory Immunology, 4th Edition, Rose et al., eds., American 
Society of Microbiology, Washington, D.C.; Su et al., J. Parasitol., 
ibid.). 
Conditions for removing non-bound serum material that retain the selective 
binding complex on the surface are known to those skilled in the art as 
are methods to optimize such conditions for a specific antigen-antibody 
complex (see, for example, Sambrook et al., ibid.; Su et al., J. 
Parasitol., ibid.). 
A number of methods known to those skilled in the art can be used to detect 
antigen-antibody binding interactions indicative of selective binding 
complexes of the present invention. For example, the actual binding 
reaction can be monitored by following changes in the configurations of 
the antigen and antibody, for instance by noting changes in electrical 
potential. The complex can also be identified using a compound, preferably 
labeled (i.e., an "identifying labeled compound"), which can selectively 
bind to the selective binding complex. Alternatively, an identifying 
compound, preferably labeled, that selectively binds to the antibody 
indicative of Trichinella spiralis infection while the antibody is 
attached to the diagnostic reagent can be used. Such a compound generally 
binds primarily to a non-binding epitope of the antibody. As used herein, 
a "non-binding epitope of the antibody" is a portion of the antibody that 
does not include the site at which the antibody binds selectively to the 
diagnostic reagent. Non-binding epitopes can include, for example, the 
constant regions of the antibody. Examples of compounds that can be used 
to detect selective binding complexes include secondary antibodies, such 
as antibodies that target antibodies of the species being tested (e.g., 
anti-pig antibodies in a pig assay); bacterial surface proteins that bind 
to antibodies, such as Protein A and Protein G; cells that interact with 
antibodies, such as T cells, B cells, and macrophages; eukaryotic cell 
surface proteins that bind to antibodies, such as Fc receptors; and 
complement proteins. Preferred compounds include secondary antibodies, 
Protein A and Protein G. 
A variety of tags can be used to label compounds used to detect selective 
binding complexes of the present invention, including radioactive, 
enzymatic, or fluorescent labels. A preferred labeled compound of the 
present invention is an enzyme-linked compound capable of selectively 
binding to a non-binding site epitope of the antibody indicative of 
Trichinella spiralis infection. Depending on the label used, assays to 
determine Trichinella spiralis infection can be either qualitative or 
quantitative. Detection can be accomplished using a variety of well-known 
techniques, depending on the assay. For example, an enzymatic assay often 
yields a colorimetric product that can be detected visually or by a 
machine such as a densitometer or a spectrophotometer. 
In a preferred embodiment, selective binding complexes are detected by a 
method including (a) contacting the selective binding complex with a 
labeled compound capable of binding selectively to the antibody indicative 
of infection or to the complex, (b) removing substantially all of the 
labeled compound that does not selectively bind to the complex, and (c) 
detecting the labeled compound, wherein presence of the labeled compound 
is indicative of Trichinella spiralis infection. 
A particularly preferred assay system is an ELISA. In one embodiment, wells 
of a microtiter dish are coated with a diagnostic reagent of the present 
invention to form a reagent-coated surface. Effective coating can be 
accomplished by, for example, adding the diagnostic reagent, preferably 
contained in a buffer, to the wells and allowing the reagent-containing 
buffer to incubate in the wells at about 4.degree. C. for several hours 
(e.g., overnight). The buffer is then removed and a blocking agent (e.g., 
milk or bovine serum albumin) is added to the reagent-coated wells in 
order to prevent non-selective and non-specific binding. The 
reagent-coated wells are washed, for example with phosphate buffered 
saline (PBS) containing small amounts of a detergent (e.g., about 0.05% 
Tween) to remove excess blocking agent. The serum to be tested for 
antibodies indicative of Trichinella spiralis infection is then added to 
the reagent-coated wells and incubated at about room temperature for about 
1 hour to allow antibodies indicative of infection, if present in the 
serum, to bind to the reagent coating the wells (i.e., to form selective 
binding complexes). The wells are then washed using, for example, PBS 
containing Tween, to remove unbound serum material under conditions that 
retain the selective binding complexes attached to the wells. An 
enzyme-labeled secondary antibody conjugate (e.g., goat anti-pig IgG 
conjugated to horse radish peroxidase) is added to the wells and incubated 
under conditions to allow for binding between the secondary antibody and 
any selective binding complexes present in the wells. Excess secondary 
antibody is then removed (e.g., by washing with PBS containing Tween), 
enzyme substrate is added (e.g., 5'aminosalicyclic acid and hydrogen 
peroxide if the enzyme is horse radish peroxidase), and color change is 
monitored either visually or using, for example, a spectrophotometer or 
densitometer. 
Another embodiment of the present invention is a diagnostic kit which 
includes at least one diagnostic reagent of the present invention. 
Suitable diagnostic reagents are heretofore disclosed. Preferably, the 
diagnostic reagent comprises a tyvelose-containing oligosaccharide 
conjugated to an effective carrier, such as a carrier heretofore 
disclosed. The kit can furthermore include a surface capable of being 
coated by the reagent. Preferably the surface is pre-coated by the 
reagent. Suitable surfaces are heretofore disclosed. A preferred surface 
is a dip-stick, particularly for field use. The kit can also include a 
means for detecting the binding of an antibody indicative of Trichinella 
spiralis infection (i.e., an indicative antibody) to the reagent. Suitable 
means for detection are heretofore disclosed. One example of a means 
(e.g., compound) to detect an indicative antibody is a secondary antibody 
that is raised against the constant regions of antibodies of the species 
being tested and that is conjugated to an enzyme that effects a color 
change in the presence of a suitable substrate. 
In accordance with the present invention, anti-Trichinella spiralis drugs 
can be designed that are much safer and more effective than anthelminthic 
drugs currently available for use in treating trichinosis. Furthermore, 
apparently unlike current anthelminthic drugs, anti-Trichinella spiralis 
drugs of the present invention can be used for prophylaxis as well as 
treatment. Design of such drugs is based upon the discovery that tyvelose 
is found on Trichinella spiralis larvae and upon the assumption that the 
presence of tyvelose on Trichinella spiralis larvae and ES products is 
important physiologically to the parasite. In other words, if the parasite 
were unable to produce tyvelose, the parasite would die or fail to 
prosper. 
Without being bound by theory, it is believed that tyvelose may play an 
important role in the physiology of the host-parasite relationship since 
the tyvelose-containing epitope is conserved among Trichinella spiralis 
isolates despite significant differences in nucleic acid sequences between 
isolates. Furthermore, it has been reported that ascarosides, which are 
composed of the 3,6-dideoxyhexose ascarylose joined to an alcohol, are 
important in maintaining the toughness and impermeability of Ascaris eggs 
to, for example, chemicals and in preventing eggs from desiccating (see, 
for example, Fairbairn et al., pp. 130-134, 1955, Can. J. Biochem. 
Physiol., Vol. 33; Fairbairn, pp. 491-554, 1957, Exp. Parasitol., Vol. 6). 
Anti-Trichinella spiralis drugs of the present invention preferably inhibit 
the biosynthesis of tyvelose, preferably by being targeted against 
Trichinella spiralis enzymes that are essentially specific to tyvelose 
biosynthesis (i.e., enzymes involved in the biosynthesis of tyvelose but 
essentially not involved in the synthesis of compounds produced by 
mammals). Since mammals do not produce tyvelose and, thus, would be 
unlikely to produce proteins specific to tyvelose biosynthesis, it is 
believed that such drugs will be selectively targeted to Trichinella 
spiralis and, as such, will have substantially insignificant, if any, 
negative side effects. 
Preferred anti-Trichinella spiralis drugs of the present invention can be 
produced in a variety of ways, including the following method: (a) enzymes 
that are essentially specific to tyvelose biosynthesis in Trichinella 
spiralis are identified; and (b) drugs are identified and/or synthesized 
that inhibit the activity of such enzymes, thereby inhibiting tyvelose 
production. 
Several reports describe the pathways by which tyvelose and other 
3,6-dideoxyhexoses are synthesized by, for example, Salmonella and 
Yersinia pseudotuberculosis (see, for example, Matsuhashi et al., pp. 
4267-4274, 1966, J. Biol. Chem., Vol. 241; Matsuhashi, pp. 4275-4282, 
1966, J. Biol. Chem., Vol. 241; Matsuhashi et al., pp. 4283-4287, 1966, J. 
Biol. Chem., Vol. 241; Hey et al., pp. 5473-5478, 1966, J. Biol. Chem., 
Vol. 241; and references included therein), and genes encoding at least 
these of enzymes have been isolated from Salmonella strains (see, for 
example, Wyk et al., pp. 5687-5693, 1989, J. Bacteriol., Vol. 171; Verma 
et al., pp. 5694-5701, 1989, J. Bacteriol., Vol. 171). Furthermore, 
ascarylose appears to be synthesized in a similar manner by Ascaris (Jezyk 
et al., pp. 707-719, 1967, Comp. Biochem. Physiol., Vol. 23). Thus, at 
least some Trichinella spiralis enzymes specific to tyvelose synthesis may 
be identified, for example, by isolating enzymes similar to those used by 
bacteria and Ascaris to produce 3,6-dideoxyhexoses. Furthermore genes 
encoding Trichinella spiralis enzymes specific for tyvelose synthesis may 
be identified by homology with reported genes encoding certain steps of 
tyvelose synthesis in Salmonella serogroup D strains. 
Once Trichinella spiralis enzymes essentially specific to tyvelose 
biosynthesis have been identified, potential drugs can be identified and 
produced that inhibit tyvelose biosynthesis, for example, by a screening 
program of organic molecules to identify those that specifically inhibit 
activity of the enzyme or by rational drug design in which, for example, 
the active site of the enzyme is identified and a drug designed that would 
interfere with the active site. 
Anti-Trichinella spiralis drugs of the present invention can be 
administered to animals in effective amounts in order to protect animals 
from trichinosis. Effective amounts and dosing regimens can be determined 
using techniques known to those skilled in the art. It is also within the 
scope of the present invention that anti Trichinella spiralis drugs of the 
present invention can also be used to treat infection by any organism, 
including any Trichinella parasite, having tyvelose-containing antigenic 
epitopes. 
The following examples are provided for the purposes of illustration and 
are not intended to limit the scope of the invention. 
EXAMPLES 
Example 1. Carbohydrate analysis of Trichinella spiralis muscle stage 
larval antigens 
This example describes the identification of monosaccharide compositions of 
Trichinella spiralis muscle larval antigens, including TSL-1 antigens, 
L.sub.1 larval homogenates, and L.sub.1 ES (excretory/secretory) products. 
Trichinella spiralis L.sub.1 larval homogenates, TSL-1 antigens (i.e., Tsp 
130 immunoaffinity-purified group II antigens), preparations enriched for 
a 43 kDa TSL-1 antigen, and ES antigens were prepared as described in 
Denkers et al., J. Immunol., ibid., and Denkers et al., Mol. Biochem. 
Parasitol., ibid. 
Total monosaccharide compositions of the larval homogenate, TSL-1 antigens, 
and ES antigens were determined by gas chromatography/mass spectrometry 
(GC/MS) of both the trimethylsilyl (TMS) ethers of methyl glycosides and 
of the alditol acetate derivatives of the glycosyl residues. TMS methyl 
glycosides were prepared by acidic methanolysis, re-N-acetylation, and 
trimethylsilylation. The general procedure for analysis of carbohydrate 
components of glycoproteins described by Chaplin, pp. 336-341, 1982, Anal. 
Biochem., Vol. 123, was used, with the following modifications: (a) 
scyllo-inositol (2 nanomoles) was used as the internal standard; (b) 
samples were dried (e.g., in a SpeedVac Concentrator SVC100H, available 
from Savant Instruments Inc., Farmingdale, N.Y. in 1.0 milliliter (ml) 
Reacti-Vials (available from Pierce, Rockford, Ill.); (c) acidic 
methanolysis was conducted by adding 40 microliters (.mu.l) of 3M 
methanolic HCl (available from Supelco, Bellefonte, Pa.) and 10 .mu.l 
methyl acetate (available from Aldrich, Milwaukee, Wis.), sealing the vial 
with a teflon-lined septa in an open-top screw cap (available from 
Pierce), vortexing, and heating to about 70.degree. C. for about 4 hours; 
(d) trimethylsilylation was achieved by adding 20 .mu.l Sylon HTP 
(available from Supelco), vortexing, and heating to about 70.degree. C. 
for about 20 minutes. 
Dry derivatized samples were dissolved in HPLC-grade hexanes and the 
insoluble salts were allowed to settle. A portion of the clear 
hexane-extracted sample was analyzed by GC/MS on a gas chromatograph 
connected to a mass selective detector (e.g., Hewlett-Packard (HP) 5980 
gas chromatograph and HP 5970 mass selective detector, each available from 
Hewlett-Packard, Palo Alto, Calif.). Samples were injected in the spitless 
mode, using, for example an HP 12-m HP-1 column and dry oxygen-free helium 
as the carrier gas. The oven was programmed to hold at about 80.degree. C. 
for about 1 minute followed by an about 30.degree. C. per minute rise to 
about 100.degree. C., an about 10.degree. C. per minute rise to about 
265.degree. C., an about 5 minute hold at about 265.degree. C., and a 
final about 2 minute hold at about 280.degree. C. The mass spectrometer 
was set to scan from mass to charge ratio (m/z) about 50 to about 800 
atomic mass units (amu) at about 0.81 scans/second. TMS-derivatives were 
identified by both characteristic retention times and mass spectra 
electron impact fragmentation patterns compared to those of authentic 
standards. Quantitation was achieved by integration of specific ion peak 
areas (m/z 204--pentoses and hexoses; m/z 173--hexosamines; m/z 
318--inositols) with response factors calculated from known concentrations 
of standards prepared under identical conditions as the samples. Standards 
included: xylose, rhamnose, fucose, mannose, galactose, glucose, 
scyllo-inositol, myo-inositol, N-acetylgalactosamine (galNAc), 
N-acetylglucosamine (glcNAc), N-acetylneuraminic acid, Salmonella 
typhimurium lipopolysaccharide (LPS), Salmonella enteritidis LPS, and 
Escherichia coli LPS serotype O55:B5, all of which are available from 
Sigma Chemical Co., St. Louis, Mo.; chemically synthesized methyl 
tyvelose, methyl abequose, and methyl paratose were obtained from Dr. D. 
R. Bundle, Division of Biological Sciences, National Research Council of 
Canada, Ottawa, Ontario, Canada; and Ascaris suum eggs containing 
ascarylose were obtained from infected pigs. 
Alditol acetate derivatives were prepared by trifluoroacetic acid 
(available from Pierce) hydrolysis of the antigen samples, followed by 
sodium borohydride or borodeuteride (each available from Sigma) reduction 
and acetylation. The general procedure for formation of the alditol 
acetate derivatives described in York et al., pp. 3-40, 1986, Methods 
Enzymol., Vol. 118, as modified by Waeghe et al., pp. 281-304, 1983, 
Carbohyd. Res., Vol. 123, for analysis of small amounts of samples, was 
used. Further modifications included: (a) scyllo-inositol (2 nanomoles) as 
the internal standard; (b) O-acetylation of the alditols by addition of 
100 .mu.l acetic anhydride (available from Supelco) and heating to about 
121.degree. C. for about 1 hour; and (c) partitioning of the 
per-O-acetylated alditols between about 1 ml chloroform and about 1 ml 
water. Dry samples were dissolved in acetone, and a portion was applied to 
the GC/MS as above. The oven was programmed to hold at about 50.degree. C. 
for about 1 minutes, followed by an about 30.degree. C. per minute rise to 
about 165.degree. C. and an about 10.degree. C. per minute rise to about 
280.degree. C., with a final about 2 minute hold at about 280.degree. C. 
The mass spectrometer was set to scan from m/z about 80 to about 450 amu 
at about 1.48 scans/second. 
A 3,6-dideoxyhexose, which was not seen as the TMS-methyl glycoside, was 
identified by alditol acetate derivation. To quantitate this sugar, equal 
amounts of fucose and chemically synthesized methyl tyvelose were 
subjected to alditol acetate derivatization, and the peak area ratio was 
calculated. This response factor was used to quantitate the amount of 
3,6-dideoxyhexose in the Trichinella spiralis samples based on the amount 
of fucose in both the trimethylsilyl and alditol acetate preparations. 
The glycosyl compositions of the Tsp 130 immunoaffinity-purified TSL-1 
antigen, ES antigens and muscle stage larval homogenate are shown in Table 
1. 
TABLE 1 
______________________________________ 
Glycosyl compositions of Trichinella spiralis muscle stage 
larval antigens 
Larval 
TSL-1 antigens.sup.a 
ES antigens.sup.a 
homogenate.sup.a 
______________________________________ 
tyvelose.sup.b 
24 21 8 
fucose 36 19 12 
xylose 0 1 1 
mannose 22 17 19 
galactose 
0.5 2 2 
glucose 1 4 19 
galNAc 9 15 13 
glcNAc 7 21 25 
myo-inositol 
0.5 0 1 
sialic acid 
0 0 0 
______________________________________ 
.sup.a mean of 4 values obtained from 2 separate GC/MS analyses on each o 
2 different antigen preparations of TSL1 antigens, ES antigens, and larva 
homogenate. Values are molar percentages of total glycosyl residues found 
.sup.b 3,6dideoxy-D-arabinohexose 
All sugars listed, with the exception of the 3,6-dideoxyhexose, were 
identified by retention time and mass spectra following methanolysis, 
re-N-acetylation, and trimethylsilylation, and quantitated based on peak 
area. The 3,6-dideoxyhexose was detected only as the alditol acetate 
derivative. It was not found as the trimethylsilyl derivatized methyl 
glycoside, nor as any other TMS derivative (i.e., trimethylsilyl butyl 
glycoside). 
The glycosyl composition of the TSL-1 fraction was surprising in two 
respects: (a) fucose accounted for about 36 molar percent of the total 
glycosyl residues; and (b) a 3,6-dideoxyhexose was identified, which 
accounted for at least about 24 molar percent of the glycosyl residues. 
The 3,6-dideoxyhexose also was found in preparations greatly enriched for 
the 43-kDA TSL-1 glycoprotein antigen. Similar to the TSL-1 antigens, the 
glycosyl composition of the ES antigens was shown to have large amounts of 
fucose (about 19%) and 3,6-dideoxyhexose (about 21%). In addition, the ES 
antigens were comprised largely of hexosamines (about 15% 
N-acetylgalactosamine and about 21% N-acetylglucosamine). The crude larval 
homogenate also had relatively high amounts of fucose (about 12%) and 
hexosamines (about 13% N-acetylgalactosamine and about 25% 
N-acetylglucosamine), while the 3,6-dideoxyhexose was found in lower 
amounts (about 8%) compared to the TSL-1 and ES antigens. 
Example 2. Determination of the 3,6-dideoxyhexose relative configuration 
This example indicates that the Trichinella spiralis 3,6-dideoxyhexose 
identified in Example 1 is 3,6-dideoxyarabinohexose. 
Identification of the Trichinella spiralis 3,6-dideoxyhexose relative 
configuration was achieved by comparing GC retention times of various 
per-O-acetylated bacterial and parasitic 3,6-dideoxyhexoses after 
conversion to alditol acetate derivatives. Chemically synthesized 
standards included methyl tyvelose (3,6-dideoxy-D-arabinohexose), methyl 
abequose (3,6-dideoxy-D-xylohexose), and methyl paratose 
(3,6-dideoxy-D-ribohexose). In addition, acid hydrolysates of biological 
materials containing 3,6-dideoxyhexoses were used, including colitose 
(3,6-dideoxy-L-xylohexose) released from Escherichia coli LPS, abequose 
(3,6-dideoxy-D-xylohexose) released from Salmonella typhimurium LPS, 
tyvelose (3,6-dideoxy-D-arabinohexose) released from Salmonella 
enteritidis LPS, and ascarylose (3,6-dideoxy-L-arabinohexose) released 
from decoated Ascaris suum eggs. The identification of relative 
configuration was verified by co-injection with authentic standards. 
The alditol acetate derivative of the TSL-1 and ES 3,6-dideoxyhexose showed 
the same chromatographic mobility and mass spectrum as the alditol acetate 
derivatives of standards containing 3,6-dideoxyarabinohexose. On the 
non-chiral GC column, D- and L- alditol acetate enantiomers necessarily 
co-elute. In contrast, the alditol acetate derivatives prepared from 
chemically synthesized methyl paratose, chemically synthesized methyl 
abequose, from abequose released from Salmonella typhimurium, and from 
colitose released from Escherichia coli all had identical mass spectra but 
were chromatographically distinguishable. Both the ribo and xylo 
3,6-dideoxyhexose derivatives eluted later than the arabino 
3,6-dideoxyhexose derivatives, suggesting that the TSL-1 3,6-dideoxyhexose 
was of the arabino configuration. 
Example 3. Determination of the 3,6-dideoxyhexose absolute configuration 
This example indicates that the Trichinella spiralis 3,6-dideoxyhexose 
identified in Example 1 is 3,6-dideoxy-D-arabinohexose (i.e., tyvelose). 
Assignment of the absolute configuration of the TSL-1 and ES 
3,6-dideoxyhexose was achieved by GC/MS analysis of the acetylated 
glycosides formed from chiral 2-octanol. 3M HCl in both (-)-2 and (+)-2 
octanol (available from Sigma) were prepared by the dropwise addition of 
about 256 .mu.l acetyl chloride (available from Mallinkrodt, Inc., Paris, 
Ken.) to about 1.2 ml octanol. Derivatizations of TSL-1 and ES antigens 
were achieved by: (a) hydrolysis in 2M TFA at about 121.degree. C. for 
about 1 hour (hr); (b) octanolysis in either (-)-2 or (+)-2 3M octanol HCl 
at about 80.degree. C. for about 3 hr; (c) addition of sodium acetate; and 
(d) acetylation in acetic anhydride at about 100.degree. C. for about 1 
hr. The acetylated octyl glycosides were partitioned into the organic 
phase between about 1 ml chloroform and about 1 ml water, dried, and 
extracted into acetone (see, for example, Leontein et al., pp. 359-362, 
1978, Carbohyd. Res., Vol. 62). Samples were analyzed by GC/MS as above 
for alditol acetates (total ion chromatogram) or in the selected ion 
monitoring mode (selecting m/z's of about 83, 85, 112, 145, and 215). 
Methyl tyvelose (3,6-dideoxy-D-arabinohexose) and Ascaris suum eggs 
(containing 3,6-dideoxy-L-arabinohexose) were also subjected to 
hydrolysis, (-)-2 and (+)-2 octanolysis, and acetylation. GC/MS data from 
the resulting acetylated 3,6-dideoxyarabinohexose octyl glycoside 
derivatives were compared to those obtained from the TSL-1 and ES antigen 
samples. Verification of absolute configuration was achieved by 
co-injection. 
The absolute configuration of the Trichinella spiralis 
3,6-dideoxyarabinohexose was identified as D- on the basis of retention 
time and mass spectra of the acetylated, optically pure, 2-octyl 
glycosides. Both the acetylated (-)-2 and (+)-2 octyl glycosides of TSL-1 
3,6-dideoxyarabinohexose from TSL-1 and ES co-eluted with the 
corresponding acetylated (-)-2 and (+)-2 octyl glycosides derived from 
chemically synthesized tyvelose (3,6-dideoxy-D-arabinohexose). 
Correspondingly, as required, the acetylated (-)-2 and (+)-2 octyl 
glycosides of TSL-1 3,6-dideoxyarabinohexose co-eluted with their 
respective enantiomers, namely the acetylated (+)-2 and (-)-2 octyl 
glycoside derivatives of ascarylose (3,6-dideoxy-L-arabinohexose) derived 
from Ascaris suum eggs. Therefore, the Trichinella spiralis sugar was 
designated as 3,6-dideoxy-D-arabinohexose (tyvelose) on the basis of the 
determination of relative configuration by alditol acetate derivatization 
and of the determination of absolute configuration by acetylation of the 
chiral octyl glycosides. 
Example 4. Methylation analysis 
This example demonstrates determination of the glycosyl composition of 
TSL-1 carbohydrates. 
TSL-1 antigens (670 .mu.g protein) were buffer exchanged from PBS/NaN.sub.3 
into 18 megaohm Milli-Q (available from Millipore Corp., Bedford Mass.) by 
centrifugation at 5000 x g in a BSA-passivated Centriprep C-10 (available 
from Amicon, Danvers, Mass.). The carbohydrates were then treated with 
about 100 .mu.l 1M NaBD.sub.4 in 50 mM NaOH at about 45.degree. C. for 
about 20 hr to .beta.-eliminate and reduce O-glycosidically-linked 
oligosaccharides. Following addition of glacial acetic acid and 
evaporation, the sample was redissolved and evaporated in 10% acetic acid 
in methanol (about 3 times) and in absolute methanol (about 3 times). The 
sample was then desalted by cation exchange column chromatography (330 
.mu.l BioRad (Hercules, Calif.) AG50W-X8 resin, H+ form, 1.7 meq/ml, 
packed on a 5 mm silanized glass wool plug in a 53/4 inch silanized 
Pasteur pipet). The carbohydrates were eluted with Milli-Q water until the 
pH of the eluate became neutral. The eluate was concentrated to about 0.5 
ml, transferred to a 1.0 ml Reacti-Vial, and dried to completion. TSL-1 
carbohydrate antigens were methylated by the Hakomori procedure (see, 
Hakomori, pp. 205-208, 1964, J. Biochem. (Tokyo), Vol. 55), as adapted by 
Sandford and Conrad (see, Sandford et al., pp. 1508-1517, 1966, Biochem., 
Vol. 5) and as modified for microanalysis by Waeghe et al., ibid. The 
sample was initially dissolved in about 250 .mu.l dry dimethylsulfoxide 
(available from Pierce), with continuous stirring for about 2 hr at room 
temperature. About 20 .mu.l of 4.5M sodium dimethylsulfinyl carbanion 
(see, for example, York et al., ibid.; Stellner et al., pp. 464-472, 1973, 
Arch. Biochem. Biophys., Vol. 155) was added, and the reaction mixture was 
stirred for about 2 hr at room temperature. About 35 .mu.l of methyl 
iodide (available from Aldrich) was added dropwise, and the mixture was 
stirred for 12 hr at room temperature. The reaction mixture was diluted 
with water to obtain a 1:1 (v:v) dimethyl sulfoxide:water solution, and 
the pre-reduced, per-O-methylated carbohydrates were recovered and 
purified by reverse-phase chromatography on a Sep-Pak C-18 cartridge 
(available from Waters Associates, Inc., Milford, Mass.) (see Waeghe et 
al., ibid.). The final two elution fractions (2 ml 100% acetonitrile for 
per-O-methylated alditols d.p. 2-10; 4 ml 100% EtOH for per-O-methylated 
alditols of larger oligosaccharides d.p.&gt;10 and polysaccharides) were 
collected in 13.times.100 mm test tubes and the solvent was evaporated to 
dryness using a stream of filtered air at room temperature. The 
per-O-methylated carbohydrates were converted into their partially 
O-acetylated, partially O-methylated alditols by hydrolysis in 2M TFA, 
reduction with NaBD.sub.4, and acetylation with acetic anhydride (York et 
al., ibid.). Glycosyl linkage composition was determined by GC/MS of the 
partially O-acetylated, partially O-methylated alditols using the 
temperature program as described for alditol acetates. The hexosamine 
residues were identified by comparing retention times and mass spectra to 
undermethylated N-acetylglucosamine and N-acetylgalactosamine standards. 
Verification of these designations was achieved by co-injection. 
Results from the characterization of TSL-1 glycosyl linkages as determined 
by Hakomori methylation of the TSL-1 antigens are shown in Table 2. 
TABLE 2 
______________________________________ 
Glycosyl linkage composition of TSL-1 
carbohydrates 
Sugar Mole percent 
______________________________________ 
t-tyvelose.sup.a,b 
8.8 
t-fucose.sup.b 13.8 
t-mannose 1.9 
3,4-fucose 2.7 
2-mannose 3.9 
2,4-mannose 7.6 
2,6-mannose 5.5 
3,6-mannose 10.1 
4-glcNAc.sup.c 9.7 
3-galNAc.sup.c 14.5 
3,4-glcNAc.sup.c 
21.4 
______________________________________ 
.sup.a 3,6dideoxy-D-arabinohexose 
.sup.b the yields of ttyvelose and tfucose were lower than would be 
expected, presumably due to their acidliability and/or volatility 
.sup.c hexosamines identif ied by comparing retention times and mass 
spectra to undermethylated Nacetylglucosamine and Nacetylgalactosamine 
standards 
The TSL-1 3,6-dideoxy-D-arabinohexose was found to be present entirely as 
non-reducing terminal residues. Approximately 83% of the fucose was also 
present as non-reducing terminal residues, with the remaining fucose found 
as 3,4-linked branched residues. The mannosyl derivatives found included 
terminal, 2-linked, 2,4-linked, 2,6-linked, and 3,6-linked residues. 
Because the entire TSL-1 sample was methylated without first separating N- 
and O-linked sugars, it is probable that these residues may be 
constituents of both N-linked and O-linked glycoproteins. 
While various embodiments of the present invention have been described in 
detail, it is apparent that modifications and adaptations of those 
embodiments will occur to those skilled in the art. It is to be expressly 
understood, however, that such modifications and adaptations are within 
the scope of the present invention, as set forth in the following claims: