Merozoite proteins for use in detection of Babesia equi in horses using immunological techniques

The present invention relates to a purified and isolated merozoite protein which is a specific indicator of infection by Babesia equi (B. equi) in horses. This protein contains a conserved region found in all strains of B. equi. It has a molecular weight of approximately 28 KDa and has been successfully purified and sequenced. The isolated and purified merozoite protein is used to prepare antibodies which can then be used in a competitive inhibition enzyme linked immunosorbent assay for the diagnosis of B. equi infection in horses.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a purified and isolated merozoite protein 
derived by either conventional or recombinant means useful for the 
detection of Babesia equi in horses by means of a competitive inhibition 
enzyme-linked immunosorbent assay (CI ELISA). The instant invention 
likewise relates to antibodies to the protein as well as cell lines which 
produce the antibodies. 
2. Description of the Related Art 
Equine babesiosis, caused by Babesia equi or Babesia caballi, is a 
tick-borne hemoprotozoan disease of horses (Schein, E., 1988. Equine 
Babesiosis, pp. 197-208. In M. Ristic (Ed.), Babesiosis of Domestic 
Animals and Man. CRC Press, Boca Raton, Fla.). Clinical disease is 
characterized by fever, anemia, and icterus, most likely arising from 
hemolysis caused by merozoites, the intraerythrocytic stage of equine 
Babesia infection. Mortality rate is high during initial infection of 
horses introduced into enzootic regions, and horses which survive initial 
infection are protected from clinical disease upon subsequent challenge. 
It is hypothesized that this immunity acquired by horses in enzootic areas 
is the result of persistent infection. 
The complement fixation test (CFT) is presently the official United States 
Department of Agriculture test for detecting antibody to B. equi and B. 
caballi. Horses with antibody to either parasite are restricted from 
importation into the United States. Three problems with the CFT are that 
(i) sera with anticomplement activity are not testable by the CFT; (it) 
sera which react with CFT control erythrocyte antigen cannot be evaluated 
by the GFT; and (iii) sera containing specific immunoglobulin G(T) 
[IgG(T)] antibody may yield false-negative results because IgG(T) does not 
fix complement by the classical pathway. 
Merozoite surface proteins are known to be important in the pathogenesis of 
hemoprotozoan diseases because of their role in parasite recognition of, 
attachment to, and penetration of host erythrocytes. Antigens recognized 
by antibody from hosts demonstrating immunity to clinical disease during 
Plasmodium spp., B. rhodhaini, B. bovis, and B. bigemina infection include 
surface proteins of merozoites, the only blood stage of the parasite that 
is extracellular and directly accessible to serum antibody. It has 
previously been demonstrated that cattle immune to infection with B. bovis 
had high-titered antibody preferentially directed against four 
immunodominant merozoite surface proteins (Hines et al., Mol. Biochem. 
Parasitol. 37:1-9; 1989). Invasion of erythrocytes by merozoites of 
Plasmodium knowlesi was inhibited by immune sera, and inhibition of P. 
falciparum merozoite invasion of erythrocytes in vitro required high 
concentrations of specific antibodies. These observations suggest that 
antibody to merozoite surface proteins may block erythrocyte invasion in 
vivo and that these proteins should be tested as potential immunogens. 
Detection of antibodies has been the method of choice for diagnosis of 
infection with equine Babesia spp.; however, the specificity or role of 
antibodies in the acquired protective immunity against clinical disease 
following equine Babesia infection has not thus far been determined. 
Applicants have now developed a competitive inhibition enzyme-linked 
immunosorbent assay (CI ELISA) based on the use of a merozoite protein for 
detection of antibody to B. equi. The formatting of the CI ELISA overcomes 
the above three problems related to use of the CFT. Furthermore, a high 
concordance was found to exist between the CI ELISA and CFT in detecting 
antibody to B. equi. 
SUMMARY OF THE INVENTION 
The present invention relates to the discovery and use of a novel merozoite 
protein of Babesia equi which has been isolated and purified. This protein 
contains a conserved epitope that is diagnostically useful as a sensitive 
and specific indicator of infection by Babesia equi in horses. The 
isolated protein has a molecular weight of approximately 28 kDa, with the 
amino acid sequence having been determined as follows: 
__________________________________________________________________________ 
1 RPPVKMISKS 
FAFVFASIAI 
SSILAEEEKP 
KASGAVVDFQ 
LESIDHVTID 
51 KQSEEHIVYT 
AHEGYAVEKV 
KEGDSVIKTF 
DLKEQTPKTV 
VRHIKDNKPY 
101 
VVIAVESALH 
LVLKKDGDKW 
VELEVAEFYQ 
EVLFKGFEAV 
SVDLAAAVSD 
151 
KFTETTFGSG 
KKHTFKAPGK 
RVLKVVDGKT 
ELIDGDNEVV 
LDLELFVSSD 
201 
NKVARVVYLY 
KGDGRIKEIF 
LKLVEKAWKR 
VEVKDAAETL 
HGINSTFPAD 
251 
YKVVYDGFSV 
YGALLAVAAI 
AFSTLFY 277 
__________________________________________________________________________ 
The isolated and purified merozoite protein is used to prepare antibodies 
which are useful in immunoassays for the diagnosis of B. equi in horses. A 
molecular clone of the protein expressing the conserved epitope has been 
obtained and shown to likewise be useful in such immunoassays. This 
recombinant merozoite protein is designated SEQ ID NO:1. 
It is an object of this invention to provide an immunological assay for B. 
equi in horses based upon the antigenicity of a conserved epitope of a 
novel merozoite protein of 
It is also an object of this invention to provide hybridomas for the 
production of antibodies to the conserved epitope of the merozoite 
protein. 
It is a further object of this invention to provide antibodies as 
immunochemical reagents for the diagnosis of B. equi in horses. 
Other objects and advantages of this invention will become readily apparent 
from the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION 
In the present invention a novel protein isolated and purified from the 
merozoite of B. equi has been discovered and proven to be a sensitive and 
specific indication of the presence of antibodies to B. equi in horses. 
The novel protein of the invention is effective for use in immunoassays 
such as the competitive inhibition enzyme-linked immunosorbent assay (CI 
ELISA). Samples used in the test may be obtained from the serum of the 
horse to be tested. Immunoprecipitation of B. equi merozoite proteins 
recovered from an infected horse were found to have apparent molecular 
masses of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28 and 25 kDa. The descri 
36/133.97 was found to react with a protein epitope on the 44-, 36-, 34-, 
and 28-kDa merozoite antigens. This monoclonal antibody, 36/133.97, has 
been deposited under the Budapest Treaty in the American Type Culture 
Collection (12301 Parklawn Drive, Rockville, Md., 20852, USA) on Jan. 10, 
1995, and has been assigned Deposit Number ATCC HB11788. Applicants found, 
through a competitive binding assay, that horses infected with B. equi 
throughout the world consistently produce antibodies to the antigens 
associated with this epitope. The 28-kDa antigen was found to be of 
particular interest due to its immunodominance in infected horses as 
recognized by MAb 36/133.97 in serum dilution studies. This protein was 
subsequently determined to possess the amino acid sequence: 
__________________________________________________________________________ 
1 RPPVKMISKS 
FAFVFASIAI 
SSILAEEEKP 
KASGAVVDFQ 
LESIDHVTID 
51 KQSEEHIVYT 
AHEGYAVEKV 
KEGDSVIKTF 
DLKEQTPKTV 
VRHIKDNKPY 
101 
VVIAVESALH 
LVLKKDGDKW 
VELEVAEFYQ 
EVLFKGFEAV 
SVDLAAAVSD 
151 
KFTETTFGSG 
KKHTFKAPGK 
RVLKVVDGKT 
ELIDGDNEVV 
LDLELFVSSD 
201 
NKVARVVYLY 
KGDGRIKEIF 
LKLVEKAWKR 
VEVKDAAETL 
HGINSTFPAD 
251 
YKVVYDGFSV 
YGALLAVAAI 
AFSTLFY 277 
__________________________________________________________________________ 
hereby designated as SEQ ID NO:1. 
The mRNA associated with the 28 kDa merozoite protein of B. equi, as 
isolated in Example II, may be used as a template in the synthesis of cDNA 
by conventional techniques such as those described by Maniatis (1982, 
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y.), herein incorporated by reference. 
The selected vector may be cut with an appropriate restriction enzyme, 
ligated via conventional techniques to the ends of the fragments of B. 
equi cDNA, and inserted into a host microorganism resulting in the 
synthesis of the 28 kDa protein referred to hereinabove. Without being 
limited thereto, suitable techniques for the preparation of vectors and 
transformed microorganisms are described by Drummond et al., U.S. Pat. No. 
5,041,378, issued Aug. 20, 1991; the contents of which are herein 
incorporated by reference. 
The antigen may be employed for the generation of hybrid cell lines 
producing MAb's specific thereto. Establishing the antibody-secreting cell 
lines for use in the invention is a multistep procedure which includes 
hyperimmunizing an animal to induce a proliferation of antibody-producing 
cells, promoting fusion between the primed cells and cells of an immortal 
cell line, selecting for antibody-secreting hybridomas, screening the 
hybridomas for selectability in a subsequent fusion stage, and then 
cloning the antibody producing hybrids. The practitioner skilled in the 
art would recognize that hybrid cell lines could be produced by 
conventional techniques. Suitable techniques for the generation of hybrid 
cell lines include those described by Kohler and Milstein (Nature; Vol. 
256; pp. 495-497; 1975); herein incorporated by reference; and Stites 
(Clinical Laboratory Methods for Detection of Antigens and Antibodies. In 
Basic and Clinical Immunology; Stites et al. (Ed.) Lang Medical 
Publications, Los Altos, Calif., 1984, pp. 350-351). Without being limited 
thereto, particularly preferred is, the hybrid cell line producing MAb 
36/133.97 discussed in Example IV below. The resultant MAb produced from 
the cell line binds selectively with B. equi. 
It is envisioned that the monoclonal antibody (MAb) specific for the 28 kDa 
merozoite protein of this invention may be employed for the detection of 
infection by B. equi in clinical specimens, particularly serum, by use of 
conventional immunoassay techniques. Such an immunoassay would comprise 
the steps of: A) collecting serum from a horse to be tested; B) contacting 
the serum with antibodies specific for a conserved epitope of a merozoite 
protein of B. equi; and C) detecting the presence of the antigen-antibody 
complex. While the skilled practitioner will recognize that suitable 
immunoassay techniques include IFA, immunoelectrophoresis and Western 
Blot; enzyme-linked immunosorbent assays (ELISA) are preferred, with 
competitive inhibition enzyme-linked immunosorbent assays (CI ELISA) as 
described in Knowles et al. (Infect. Immun. 59:2412-2417, 1991), herein 
incorporated by reference, being most preferred. 
The present invention is not limited to any specific separation or 
identification methodology. Rather, all modifications obvious to one 
skilled in the art are envisioned and encompassed by the present 
invention. The following examples are offered to illustrate the present 
invention and are not intended to limit its scope. 
EXAMPLE I 
Babesia equi isolates 
A B. equi isolate was obtained in 1976 from a horse in Florida and 
cryopreserved as a blood stabilate containing 10% dimethyl sulfoxide in 
liquid nitrogen. A nonsplenectomized horse (H5) was infected with 30 ml of 
the Florida B. equi first-passage stabilate containing 5.6.times.10.sup.6 
viable organisms per ml. Viability was determined by incubating merozoites 
with fluorescein diacetate (FDA) as described by Rotman et al. (Proc. 
Natl. Acad. Sci. USA 55:134-141; 1966). This horse was monitored for 
clinical disease and parasitemia. During ascending parasitemia, 200 ml of 
whole blood was passaged to a splenectomized horse. At peak parasitemia 
(49%), infected erythrocytes were collected and stored in liquid nitrogen 
as a blood stabilate containing packed erythrocytes 1:1 with a 
cryopreservant of 20% (wt/vol) polyvinylpyrrolidone and 2% (wt/vol) 
glucose in Puck's saline G (GIBCO Laboratories, Chagrin Falls, Ohio); see 
Palmer et al. (Parasitology 84:567-572; 1982). Aliquots (25 ml) of washed 
packed infected erythrocytes were frozen at -70.degree. C. 
The Europe isolate of B. equi was obtained from a mare from Georgia, USSR; 
gee Kutler et al. (Am. J. Vet. Res. 47:1668-1670; 1986). A splenectomized 
pony was infected with the Europe isolate, and blood smears for indirect 
immunofluorescence assay (IFA) were prepared. 
EXAMPLE II 
In vitro translation of B. equi mRNA 
B. equi merozoite mRNA was isolated from infected erythrocytes by 
modification of methods previously described by Maniatis et al. (Molecular 
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring 
Harbor, N.Y.; 1982). A 25 ml aliquot of washed packed infected 
erythrocytes was thawed in the presence of equal volumes of guanidinium 
isothiocyanate (4.0 M guanidinium isothiocyanate [Bethesda Research 
Laboratories, Gaithersburg, Md.], 0.1 M Tris-HCl [pH 7.5], 1% 
2-mercaptoethanol, 2% SARKOSYL.RTM. (N-Lauroylsarcosine, sodium salt) 0.01 
M EDTA [pH 7.6]). Lysates were sequentially extracted with buffered 
phenol, phenol-chloroform-isoamyl alcohol, and ether before nucleic acids 
were ethanol precipitated. Polyadenylated mRNA was isolated by 
POLY(U)SEPHADEX.RTM. (Bethesda Research Laboratories) chromatography. In 
parallel, mRNA was isolated from 25 ml of washed packed uninfected 
erythrocytes. Stained smears of washed infected erythrocytes revealed less 
than 1 leukocyte per 10.sup.4 erythrocytes. Integrity of mRNA was 
evaluated by the migration of rRNA species in 1% agarose gel. Merozoite 
mRNA was translated in vitro (Promega, Madison, Wis.), using 2 .mu.g of 
polyadenylated mRNA per reaction and a nuclease-treated rabbit 
reticulocyte lysate; see Jackson et al. (Methods Enzymol. 96:50-71; 1983) 
and Pelham et al. (Eur. J. Biochem. 67:247-256; 1976). The rabbit 
reticulocyte lysate was chosen because it lacks microsomal membranes 
necessary for processing events such as signal peptide cleavage and core 
glycosylation. 
EXAMPLE III 
Radiolabeling of B. equi proteins 
Defibrinated blood from a splenectomized horse infected with the Florida 
isolate of B. equi was collected when ascending parasitemia reached 5%. 
Erythrocytes were washed twice in Puck's saline G to remove the majority 
of buffy coat cells. A final wash was made in serum- and amino acid-free 
medium 199 (Hazleton Laboratories, Lenexa, Kans.). Short-term cultures 
were established in 2.5-cm.sup.2 flasks at a 10% erythrocyte suspension in 
amino acid-free medium 199 containing 40% autologous, preinoculation horse 
serum, 1% penicillin G, streptomycin, amphotericin B, 25 .mu.Ci (500 
.mu.Ci total) each of tritiated isoleucine, lysine, tyrosine, valine, and 
arginine per ml (respective specific activities, 110.8, 97.4, 46.7, 64.6, 
and 53.3 Ci/mmol; Dupont-New England Nuclear, Boston, Mass.) and buffered 
with 10 mM 3-[N-tris-(hydroxymethyl)methylamino]-2-hydroxy propanesulfonic 
acid, pH 7.35. Metabolic labeling proceeded during an 18-h incubation 
period at 37.degree. C. in 5% CO.sub.2 and ambient air. The labeled cells 
were then washed and solubilized as described by McElwain et al. (J. 
Immunol. 138:2298-2304; 1987). In vitro translation products were labeled 
with [.sup.35 S] methionine at 0.8 mCi/ml per reaction. 
EXAMPLE IV 
Production of monoclonal antibody (Mab) 
Eight-week-old BALB/c mice were immunized subcutaneously with 10.sup.7 
viable merozoites in 0.1 ml of phosphate-buffered saline (PBS) emulsified 
in an equal volume of Freund's complete adjuvant. Merozoites for MAb 
production were prepared from stabilates containing a 49% parasitemia. The 
stabilates were diluted with 2 volumes of PBS and centrifuged at 
2,500.times.g for 5 min. Pellets were lysed for 30 s with an equal volume 
of distilled water, diluted with 3 ml of PBS, vortexed gently, and 
centrifuged at 400.times.g for 5 min. The supernatant was centrifuged at 
2,500.times.g to pellet the merozoites. Two additional immunizations 
consisting of the same number of parasites in incomplete Freund's adjuvant 
were given subcutaneously at 10-day intervals. The mice were then 
immunized intravenously with 10.sup.7 viable merozoites in 0.1 ml of PBS 
72 h prior to fusion. Cell fusions and cloning by limiting dilution were 
performed utilizing x63-A68.653 murine myeloma cells utilizing methods 
described by Riggs et al. (J. Immunol. 143:1340-1345; 1989). The CI ELISA 
used an IgG1 MAb (36/133.97) which reacts with a protein epitope on the 
surface of B. equi merozoites as disclosed by Knowles et al. (Infect. 
Immun. 59:2412-2417; 1991). Heavy-chain isotypes were identified by 
enzyme-linked immunosorbent assay (ELISA), and concentrations of 
antibodies were determined by immunodiffusion; as described by Johnstone 
et al. (1982, Precipitation Techniques in Agar and Agarose, pp. 120-140. 
In A. Johnstone and R. Thorpe (Ed.), Immunochemistry in Practice. 
Blackwell Scientific Publications, Boston). Supernatants from the initial 
fusion and from limiting-dilution clones were screened by IFA with 
acetone-fixed B. equi organisms. 
EXAMPLE V 
Immune sera from horses experimentally and naturally infected with B. equi 
Serum was obtained from an adult horse (H5) infected intravenously twice at 
a 2-month interval with a Florida isolate of B. equi. After 50 ml of serum 
was obtained, the initial inoculation of H5 was with 30 ml of a 
first-passage stabilate of a Florida isolate of B. equi. This stabilate in 
10% dimethyl sulfoxide contained 5.6.times.10.sup.6 viable merozoites per 
ml. The second inoculation was with a 2.0-ml stabilate containing a 49% 
parasitemia prepared as described for B. equi isolates. Equine sera that 
tested positive for antibodies to B. equi by the complement fixation test; 
see Hirato et al. (Jpn. J. Vet. Sci. 7:197-205; 1945) were obtained from 
the National Veterinary Services Laboratory, U.S. Department of 
Agriculture, Ames, Iowa. These sera were obtained from horses in 19 
countries, the data regarding such are herein presented as Table 1. 
TABLE I 
______________________________________ 
CI ELISA for assessment of antibodies to B. equi merozoite 
proteins recognized by MAb 36/133.97 
Country of 
OD at serum dilution of .sup.a : 
origin 10.sup.-1 
10.sup.-2 
10.sup.-3 
10.sup.-4 
CI titer.sup.b 
______________________________________ 
Argentina 0.252 0.483 1.130 1.027 
10.sup.-2 
Austria 0.563 0.703 0.826 0.948 
10.sup.-2 
Brazil 0.126 0.236 0.641 0.824 
10.sup.-3 
Chile 0.650 0.866 1.241 1.315 
10.sup.-1 
Colombia 0.180 0.713 1.259 1.191 
10.sup.-2 
Ecuador 0.247 0.543 1.055 1.263 
10.sup.-2 
England 0.292 0.816 1.233 1.237 
10.sup.-1 
France 0.238 0.608 1.110 1.229 
10.sup.-2 
Italy 0.378 0.804 1.181 1.292 
10.sup.-1 
Netherlands 
0.148 0.266 0.740 1.093 
10.sup.-2 
North Yemen 
0.663 0.851 1.166 1.193 
10.sup.-1 
Panama 0.240 0.484 1.066 1.139 
10.sup.-2 
Peru 0.185 0.540 1.012 1.077 
10.sup.-2 
Poland 0.601 1.000 1.247 1.185 
10.sup.-1 
Saudi Arabia 
0.420 0.771 1.218 1.266 
10.sup.-1 
Spain 0.295 0.607 0.687 0.733 
10.sup.-3 
Trinidad 0.269 0.594 1.143 1.227 
10.sup.-2 
United States 
0.202 0.377 1.012 1.264 
10.sup.-2 
Venezuela 0.325 0.771 1.244 1.324 
10.sup.-1 
______________________________________ 
.sup.a OD of MAb 36/133.97 reaction with B. equi merozoites with equine 
serum at the specified dilution. OD for isotype control MAb with B. equi 
merozoites = 0.153 .+-. 0.05 (n = 8). 
.sup.b Dilution of serum reducing OD values to less than 3 standard 
deviations below the mean for control horses (&lt;0.73) in CI ELISA with MAb 
36/133.97. OD for control horses at a 1/2 dilution = 0.97 .+-. 0.08 (n = 
68). Controls included preinoculation sera of H5 and SN76N8401 (control 
serum from the National Veterinary Services Laboratory, Ames, Iowa). 
EXAMPLE VI 
Immunoprecipitation and SDS-PAGE 
Immunoprecipitation of radiolabeled antigen was performed as previously 
described by McElwain et al. (J. Immunol. 138:2298-2304; 1987). A total of 
1.times.10.sup.6 to 2.times.10.sup.6 trichloroacetic acid-precipitable 
counts of antigen and 10 .mu.g of MAb or 10 .mu.l of equine immune serum 
were used in each precipitation. Immune complexes were precipitated with 
protein A (Pansorbin; Calbiochem, San Diego, Calif.) or protein G 
(Immu-Bind; Genex, Gaithersburg, Md.). Metabolically radiolabeled antigen, 
in vitro-translated proteins, or immunoprecipitates were boiled for 3 min 
in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) 
sample buffer (final concentrations of 25 mM Trio [pH 6.8], 2% [wt/vol] 
SDS, 15% [vol/vol] glycerol, 2.5% 2-mercaptoethanol, and a few crystals of 
bromophenol blue) and were electrophoresed in a 7.5 to 17.5% 
SDS-polyacrylamide gradient slab gel with a 5% stacking gel; (see Takacs, 
B. 1979. Blectrophoresis of Proteins in Polyacrylamide Slab Gels, pp. 
81-105. In I. Lefkovits and B. Pernis (Ed.), Immunological Methods. 
Academic Press, Inc., N.Y.). SDS-polyacrylamide gels were processed for 
autoradiography as described previously. 14C-labeled standards used for 
molecular weight comparisons (Amersham, Arlington Heights, Ill.) consisted 
of myosin (200,000), phosphorylase b (92,500), bovine serum albumin 
(69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lysozyme 
(14,300). 
EXAMPLE VII 
Western immunoblotting 
Western blotting was performed on a miniblotter 25 (Immunetics, Cambridge, 
Mass.) by modification of the techniques of Towbin et al. (J. Immunol. 
Methods 72:313-340). Merozoite antigen was prepared from stabilates 
containing a 49% parasitemia as described for MAb production. Control 
erythrocyte antigen was prepared identically to merozoite antigen and was 
obtained from stabilates prepared from an uninfected horse. Pelleted 
merozoites were added to equal volumes of SDS-PAGE sample buffer and 
boiled for 10 min. Merozoite proteins separated in SDS-PAGE (as previously 
described) were electrophoretically transferred overnight to 
nitrocellulose filters in 25 mM Tris-190 mM glycine buffer containing 20% 
(vol/vol) methanol. Filters were blocked for 2 h in 0.17 M NaCl-0.01 M 
Trts-0.1 mM phenylmethylsulfonyl fluoride-1.0% (wt/vol) bovine hemoglobin 
(buffer A). Serum (50 .mu.l) or MAb (10 .mu.g) was diluted in buffer A 
with the addition of 0.1% (wt/vol) SDS-0.1% (vol/vol) TRITON 
X-100.RTM.-(polyethylene glycol p-isooctylphenyl ether) 1.0 mM EDTA 
(buffer B). Bound antibodies were detected by incubation for 1 h each in 
second antibody (rabbit anti-horse or rabbit anti-murine immunoglobulin) 
and .sup.125 I-protein A in buffer B. Filters were washed three times in 
buffer B after incubation with equine serum or MAb, second antibody, and 
.sup.125 I-protein A, followed by three washes in buffer B without 
hemoglobin before drying and autoradiography. .sup.14 C-labeled molecular 
weight standards were the same as for SDS-PAGE previously described. 
EXAMPLE VIII 
IFA 
(i) Fixed B. equi 
IFA of acetone-fixed B. equi was performed as described previously by 
McGuire et al. (Infect. Immun. 45:697-700; 1984). Bound murine or equine 
antibodies were detected with fluorescein isothiocyanate-conjugated rabbit 
anti-mouse immunoglobulin or goat anti-horse immunoglobulin. 
(ii) Live B. equi 
Merozoites for live IFA were prepared from stabilates containing a 49% 
parasitemia as described for MAb production. Live IFA was performed by 
minor modification of methodology previously described by Goff et al. 
(Infect. Immun. 56:2363-2368; 1988). Merozoite pellets resuspended in 100 
.mu.l of PBS were incubated with 25 .mu.g of MAb 36/133.97. After a 30-min 
incubation at room temperature, the cells were washed three times with 10% 
normal goat serum in PBS, diluted to 975 .mu.l with normal goat serum-PBS, 
and added to 12.5 .mu.g of goat anti-mouse antibody conjugated with 
tetramethylrhodamine isothiocyanate (Kirkegaard & Perry Laboratories, 
Gaithersburg, Md.). Samples were incubated for 30 min, washed three times 
with PBS, and mixed with 2.0 .mu.l of a 5-mg/ml solution of FDA. Samples 
were incubated for 15 min, washed once with PBS, resuspended in 100 .mu.l 
of PBS, and examined in a wet mount by phase and fluorescence microscopy. 
A total of 757 FDA-positive merozoites were examined for reactivity to MAb 
36/133.97. 
EXAMPLE IX 
CI ELISA 
A competitive inhibition (CI) ELISA was established to test for a direct 
relationship between proteins recognized by immune equine sera and MAb 
36/133.97. Merozoites were prepared as described for MAb production. 
Merozoite preparations were diluted to 40 .mu.g/.mu.l in PBS containing 20 
mM MgGl.sub.2 and treated with an equal volume of lysis buffer (50 mM Tris 
[pH 8.0], 5 mM EDTA, 5 mM iodoacetamide, 0.1 mM 
N-C.alpha.-p-tosyl-L-lysine chloromethyl ketone, and 1.0 mM 
phenylmethylsulfonyl fluoride in 1.0% NONDET-P-40.RTM.). 
(octylphenol-ethylene oxide condensate containing an average of nine moles 
ethylene oxide per mole of phenol) Lysates were placed on ice for 15 min 
and then centrifuged at 1,500.times.g for 15 min, and the supernatant was 
collected. Four microliters of supernatant adjusted to 0.20 .mu.g of 
protein per .mu.l was added to individual wells of IMMULON-2.RTM. (96 
well, non-reactive plastic flat-bottom plates (Dynatech Laboratories, 
Chantlily, Va.) and incubated overnight at room temperature. Each well was 
blocked for 2-h with 350 .mu.l of 20% milk in PBS containing 0.2% TWEEN 
20.RTM. (polyoxyethylenesorbitan monolaurate) (buffer A). Equine sera were 
diluted in buffer A to a final volume of 290 .mu.l and added to the wells. 
Samples were incubated for 30 min, 0.125 .mu.g of MAb 36/133.97 in 10 
.mu.l of buffer A was added, and the reaction mixture was incubated for 
1-h at room temperature. Wells were washed three times with PBS containing 
0.2% TWEEN 20 (buffer B). Biotinylated equine anti-murine immunoglobulin G 
(IgG; Vector Laboratories, Burlingame, Calif.) in buffer A was added, 
incubation was continued for 30 min, and the wells were washed three times 
with buffer B. Addition of avidin-conjugated alkaline phosphatase (Vector 
Laboratories) in buffer B was followed by a 30-min incubation. Wells were 
washed three times with buffer B, and 100 .mu.l of a 1.0-g/.mu.l solution 
of p-nitrophenyl phosphate in 100 mM NaHCO.sub.3 (pH 9.5) with 10 mM 
MgCl.sub.2 (Sigma Laboratories, St. Louis, Mo. ) was added to each well. 
Following a 30-min incubation, reactions were stopped with 50 .mu.l of 0.2 
M EDTA and the optical density (OD) was read at 405 nm on a Dynatech 
MR-5000 ELISA plate reader. 
EXAMPLE X 
Immunoprecipitation of B. equi merozoite proteins with equine serum 
FIG. 1 shows immunoprecipitation of B. equi merozoite proteins with pre- 
and postinoculation serum from horse H5 infected with a Florida isolate of 
B. equi. The major B. equi merozoite proteins recognized by antibodies 
from this horse have apparent molecular masses of 210, 144, 108, 88, 70, 
56, 44, 36, 34, 28, and 25 kDa. Immunoprecipitations with sera from 10 
additional experimentally infected and 2 naturally infected horses 
provided similar results. 
EXAMPLE XI 
Immunoprecipitation of B. equi antigens with MAb 36/133.97 
An autoradiograph comparing immunoprecipitation of merozoite proteins with 
MAb 36/133.97 and equine immune serum is shown in FIG. 2. MAb 36/133.97, 
isotyped as IgGl, immunoprecipitated proteins with approximate molecular 
masses of 44, 36, 34, and 28 kDa which comigrated with proteins 
immunoprecipitated by serum from infected horse H5. 
EXAMPLE XII 
IFA of fixed and live merozoites with MAb 36/133,97 
The epitope recognized by MAb 36/133.97 is conserved on at least two 
isolates of B. equi, as determined by reactivity in IFA. MAb 36/133.97 
reacted with both the Florida and Europe; see Kutler et al. (Am. J. Vet. 
Res. 47:1668-1670, 1986), isolates of B. equi at a final concentration of 
0.66 .mu.g/ml. Up to 100% of merozoites from the Florida and Europe 
isolates of B. equi reacted with MAb 36/133.97 in fixed IFA. MAb 36/133.97 
did not react with uninfected erythrocytes or B. caballi in IFA. At the 
same concentrations, IgG1 isotype control MAb and rabbit anti-mouse second 
antibody did not react with B. equi-infected erythrocytes. The surface 
reactivity of MAb 36/133.97 was demonstrated by its binding to viable 
(FDA-positive) merozoites. Approximately 80% of isolated merozoites 
stained with FDA and 64% (482 of 757) of FDA-positive merozoites reacted 
diffusely with MAb 36/133.97. 
EXAMPLE XIII 
Protein character of the epitope and immunodominance of the protein 
recognized by MAb 36/133.97 
Equal volumes of washed packed erythrocytes from infected and uninfected 
horses yielded 5.7 and 0.22 .mu.g of polyadenylated RNA. The small amounts 
of polyadenylated RNA isolated from uninfected erythrocytes provided 
insufficient incorporation of [.sup.35 S]methionine from in vitro 
translation for use in immunoprecipitations. Immunoprecipitation of in 
vitro-translated B. equi mRNA with serum from infected horse H5 and with 
MAb 36/133.97 is shown in FIG. 3A and 3B. MAb 36/133.97 immunoprecipitated 
proteins at 38, 28, 26, and 23 kDa (FIG. 3B, arrowheads) which comigrated 
with proteins immunoprecipitated by serum from horse H5 at 10.sup.-3 to 
10.sup.-4 dilutions (FIG. 3A). In vitro translation products derived from 
rabbit reticulocyte lysate are not glycosylated. Therefore, 
immunoprecipitation of these products by MAb 36/133.97 indicates that the 
binding site recognized by this antibody is a protein epitope. 
Immunoprecipitation of in vitro-translated B. equi mRNA with sera from 
four naturally infected horses provided similar results. 
In Western blot analysis, MAb 36/133.97 did not react with antigen from 
uninfected erythrocytes; however, it recognized proteins of 44, 36, 34, 
and 28 kDa prepared from stabilates of infected erythrocytes (FIG. 4, 
arrowheads). Evaluation of diluted horse sera demonstrated reactivity with 
a 28-kDa protein at a dilution of 10.sup.-4 as also seen in FIG. 4. 
EXAMPLE XIV 
Relatedness of proteins recognized by sera from B. equi infected horses and 
MAb 36/133,97 
Relatedness of proteins recognized by MAb 36/133.97 and sera B. 
equi-infected horses was investigated by a CI ELISA. Sera from 34 
noninfected horses allowed MAb 36/133.97 to bind in the CI ELISA with 0D 
values of 0.97.+-.0.08. Thus, inhibition of MAb binding to B. equi 
merozoites was considered significant at OD values of &lt;0.73, corresponding 
to mean OD minus 3 standard deviations. Table I shows that sera from 
infected horses from 19 countries significantly inhibited the binding of 
MAb 36/133.97 to isolated merozoites. At a 10.sup.-1 dilution, sera from 
all infected horses uniformly inhibited binding in the CI ELISA. Some of 
these sera also inhibited the binding of MAb 36/133.97 at dilutions of 
10.sup.-2 and 10.sup.-3. 
EXAMPLE XV 
One hundred fifty-four equine serum samples from 19 countries in North 
America (6 samples), South America (113 samples), Europe (28 samples), and 
the Middle East (7 samples) were obtained from the National Veterinary 
Services Laboratory, USDA-APHIS, Ames, Iowa. Each serum was tested for 
antibody to B. equi by the CFT as described by Frerichs et al. (Am. J. 
Vet. Res. 30:697-702, 1337-1341; 1969). Three anticomplement serum samples 
and one serum sample reactive with the CFT erythrocyte antigen control 
were also obtained from the National Veterinary Services Laboratory. H5 
serum is from a horse experimentally infected with stabilate of a Florida 
B. equi isolate as disclosed by Knowles et al. (Infect. Immun. 
59:2412-2417; 1991) and SN76N8401 is a GFT-negative control serum obtained 
from the National Veterinary Services Laboratory. One hundred and four 
equine serum samples submitted to Washington State University for equine 
infections anemia testing were used as control sera. 
A CI ELISA was performed on all samples utilizing applicants' recombinant 
antigen preparation of Example in conjunction with the protocol set forth 
in Example IX. Serum samples were tested by CI ELISA in groups of 5 to 15 
per day without knowledge of the CFT results. Duplicates of each serum 
sample were tested at dilutions of 1:2 and 1:10. Five to 10 different 
control serum samples were tested at a 1:2 dilution in duplicate each day. 
A mean and standard deviation of the OD for the control serum samples was 
calculated following each test day. A serum sample was considered positive 
for antibody to B. equi if it inhibited the binding of MAb 36/133.97 such 
that the mean duplicate OD value for that dilution of test serum as at 
least 3 standard deviations below the mean OD value of the control serum 
samples for that test day. Sample data from the CI ELISA and CFT for a 
test day are given in Table 2. 
TABLE II 
______________________________________ 
Sample data from CI ELISA and CFT.sup.a 
CI ELISA, OD.sup.b CFT titer.sup.c 
Serum 1:2 1:10 B. equi B. caballi 
______________________________________ 
224 0.381, 0.389 
0.382, 0.441 
1:5 1:40 
225 0.471, 0.486 
0.732, 0.721 
1.5 Negative 
226 1.489, 1.470 
1.717, 1.672 
Negative 
1:5 
227 1.337, 1.369 
1.146. 1.619 
Negative 
1:40 
228 0.217, 0.156 
0.229, 0.236 
1:40 1:20 
229 0.301, 0.298 
0.336, 0.363 
1:5 1:40 
230 1.374, 1.362 
1.560, 1.528 
Negative 
1:5 
231 0.356, 0.356 
0.439, 0.426 
1:40 1:5 
232 0.219, 0.254 
0.334, 0.313 
1:5 Negative 
233 0.246, 0.260 
0.351, 0.389 
1:5 1:10 
234 0.521, 0.486 
0.761, 0.736 
1:10 Negative 
235 0.189, 0.198 
0.314, 0.383 
1:40 1:10 
236 1.380, 1.351 
1.535, 1.384 
Negative 
1:40 
237 0.347, 0.277 
0.465, 0.345 
1:5 1:40 
238 0.314, 0.308 
0.461, 0.470 
* * 
H5 0.293, 0.303 
ND.sup.d Negative 
Negative 
______________________________________ 
.sup.a CI ELISA and CFT were performed as described in the text. 
.sup.b Serum samples reducing mean of duplicate OD values to less than 3 
SD below mean of control horses (&lt;1.17) were considered positive. OD for 
control horses at a 1:2 dilution on this test day = 1.47 .+-. 0.10 (SD) ( 
= 9). OD for isotype control MAb = 0.145, 0.142. 
.sup.c CFT titers are presented at the highest dilution yielding a 
positive result. 
*, serum sample which reacted with CFT erythrocyte control antigen. 
.sup.d ND, not done. 
Of the 154 serum samples testable by CFT, 126 were both CFT and CI ELISA 
positive [CFT(+) CI ELISA(+)] for antibody to B. equi. Eighteen serum 
samples were negative in both tests, and CFT and CI ELISA results differed 
in the remaining 10 serum samples. Sixteen of the 18 serum samples 
negative by both the CFT and CI ELISA for antibody to B. equi were CFT(+) 
for B. caballi. 
The ten serum samples in which the CI ELISA and CFT results differed were 
retested in both assays and analyzed by immunoprecipitation. CI ELISA, 
CFT,and immunoprecipitation results for the 10 discrepant serum samples 
are summarized in Table 3. Upon retesting, four of the CFT(+) CI ELISA(-) 
serum samples had decreased CFT titers. Two of these serum samples which 
were originally CFT(+) were negative in the repeat CFT. The decreasing CFT 
titers of these serum samples may reflect, at least in part, multiple 
freeze-thaw cycles. 
TABLE III 
______________________________________ 
CI ELISA, CFT, and immunoprecipitation results of 
sera differing on initial testing.sup.a 
CFT CI ELISA Immunopre- 
Serum Original Repeat Original 
Repeat cipitation 
______________________________________ 
8 Negative Negative Positive 
Positive 
Positive 
17 Negative Negative Positive 
Positive 
Positive 
113 Negative Negative Positive 
Positive 
Positive 
175 Negative Negative Positive 
Positive 
Positive 
H5 Negative Negative Positive 
Positive 
Positive 
18 1:10 Trace Negative 
Negative 
Inconclusive 
22 1:10 1:5 Negative 
Negative 
Inconclusive 
126 1:5 Negative Negative 
Negative 
Inconclusive 
167 1:40 1:40 Negative 
Negative 
Inconclusive 
171 1:5 Negative Negative 
Negative 
Inconclusive 
______________________________________ 
.sup.a CI ELISA, CFT, and immunoprecipitation results were determined as 
described in the text. Serum samples which differed in the CI ELISA and 
CFT at original testing were retested by CI ELISA, CFT, and 
immunoprecipitation. CFT titers are presented as the highest dilution 
yielding a positive result. 
Serum samples at a 1:10 dilution were evaluated for their ability to 
immunoprecipitate .sup.35 S-labeled in vitro translation products of B. 
equi merozoite mRNA as described by Knowles et al. (Infect. Immun. 
59:2412-2417; 1991). FIG. 5 displays immunoprecipitation data from the 
five serum samples which were CFT(-) CI ELISA(+), the five serum samples 
which were CFT(+) CI ELISA(-), and two serum samples which were negative 
in both tests. Five serum samples which were CI ELISA(+) CFT(-) clearly 
immunoprecipitated multiple B. equi proteins that comigrated with proteins 
immunoprecipitated by positive control serum H5 (FIG. 5, lanes 1 to 5). 
Interestingly, serum H5, from a horse experimentally infected with B. equi 
and used as positive reference serum in the CI ELISA and 
immunoprecipitations, was one of the serum samples consistently negative 
by the CFT. 
While B. equi-specific IgG(T) antibody was not measured in the five CI 
ELISA(+) CFT(-) serum samples, IgG(T) remains a likely explanation for the 
false-negative CFT results. It has been previously shown that IgG(T) 
specific for equine infectious anemia virus inhibits the CFT for detecting 
antibody to equine infectious anemia virus because IgG(T) does not fix 
complement by the classical pathway disclosed by McGuire et al. (J. 
Immunol. 107:1738-1744; 1971). 
Immunoprecipitation results with the five serum samples which were GI 
ELISA(-) CFT(+) were inconclusive (FIG. 5, lanes 7 to 11). Fewer proteins 
were immunoprecipitated by these serum samples than by H5 serum. However, 
proteins not present in the negative control serum samples (FIG. 5, lanes 
6 and 12) were immunoprecipitated by the CI ELISA(-) CFT(+) serum samples. 
The results obtained from the five CI ELISA(-) CFT(+) serum samples may 
represent false-positive GFT results; however, the immunoprecipitation 
results show reactivity with B. equi merozoite proteins (FIG. 5, lanes 7 
to 11). Three of these serum samples (22, 126, 171) also had CFT titers to 
B. caballi, and t immunoprecipitation results with these serum samples may 
reflect serological cross-reactivity between B. equi and B. caballi 
merozoite proteins as previously recognized by Frerichs et al. (Am. J. 
Vet. Res. 30:697-702; 1969). 
If the five CI ELISA(-) CFT(+) serum samples are true positives, there are 
at least three possible explanations: (i) a genetic inability of those 
horses to produce antibody to the epitope defined by MAb 36/133.97; (ii) 
absence of the epitope on B. equi isolates which infected those horses; 
and (iii) insufficient CI ELISA sensitivity. The third explanation does 
not seem likely since 32 of the CFT(+) CI ELISA(-) serum samples had CFT 
titers of only 1:5. 
Three anticomplement serum samples and one serum sample which reacted with 
the CFT erythrocyte control antigen were tested by the CI ELISA and 
immunoprecipitation are shown in FIG. 6. Immunoprecipitations with these 
serum samples were compared with immunoprecipitations with H5 serum, four 
randomly selected CI ELISA(+) CFT(+) serum samples, and two serum samples 
negative by both tests (FIG. 2). One of three anticomplement serum samples 
and the serum sample reactive with CFT erythrocyte control antigen were 
positive by both CI ELISA and immunoprecipitation (FIG. 6, lanes 7 and 
11). Two anticomplement serum samples were CI ELISA(-), and one of these 
serum samples was clearly negative by immunoprecipitation (FIG. 6, lane 
9). Lane 8 of FIG. 6 represents immunoprecipitation with the additional 
anticomplement serum which was CI ELISA(-). Data obtained from this 
immunoprecipitation were inconclusive. The proteins in lane 8 not found in 
the control serum samples (lanes 2 and 10) may signify cross-reactivity 
between antigens of B. equi and B. caballi. Also, this serum may represent 
a false CI ELISA(-). 
The collective data of this report indicate a high (94%) concordance 
between the CI ELISA and CFT for detecting antibody to B. equi. Since 16 
of 18 serum samples in this study which were CI ELISA(-) CFT(-) for 
antibody to B. equi were CFT(+) for antibody to B. caballi, the CI ELISA 
is clearly specific for B. equi. Furthermore, the formatting of the CI 
ELISA overcomes the aforementioned limitations associated with the CFT, 
and as the data clearly indicate, the geographic conservation of the 
epitope recognized by MAb 36/133.97 allows reliable use of the CI ELISA to 
detect B. equi antibody in sera from horses worldwide. 
EXAMPLE XVI 
Construction and expression of cDNA library 
B. equi merozoite mRNA was isolated from infected erythrocytes as 
previously described in Example II. cDNA library construction was 
performed utilizing the methods of Maniatis et al. (1982. Molecular 
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring 
Harbor, N.Y.). In this process 2 .mu.g of merozoite mRNA were copied by 
reverse transcriptase followed by T4 polymerase (pharmacia). The resultant 
DNA was blunted, EcoRl linkers were applied and ligation into the 
EcoRl-site of LAMBDA ZAPII.RTM. (Stratagene Cloning Systems) was carried 
out. The lambda library in E. coli XL-blue was screened with MAb 36/133.97 
by immunoblot assay. Transfer of bacteria to nitrocellulose was done by 
standard procedures and recombinant protein was detected using MAb 
36/133.97 (2 .mu.g/ml), rabbit anti-mouse second antibody and .sup.125 I 
protein A. Positive plaques were isolated, replated and rescreened to 
achieve purity. Recombinant plasmids were excised from the bacteriophages 
and following induction with 5 mM isopropyl-1-B-D-thiogalactopyranoside 
(IPTG), tested for expression by immunoblot assay. 
EXAMPLE XVII 
Plasmid purification via cesium chloride 
The bacterial suspension containing pBluescript/10E3; which has been 
deposited under the Budapest Treaty in the American Type Culture 
Collection (12301 Parklawn Drive, Rockville, Md., 20852, USA) on Jan. 10, 
1995, and has been assigned Deposit Number ATCC 97016; was transferred to 
250 ml proplyene bottles and pelletized by centrifugation in a Beckman at 
7000 rpm for 20 min at 4.degree. C. As a separate step 37.5 mg of lysozyme 
was dissolved in 7.5 ml of an aqueous reagent solution comprising 50 mM 
glucose, 25 mMTris and an HCl adjusted pH of 8.0 (Solution A). The 
pelleted bacteria are then suspended in the lysozyme-containing Solution A 
and transferred to a 50 ml polypropylene centrifuge tube. The suspension 
was allowed to stand at room temperature for 5 min, at which point 15 ml 
of an aqueous reagent solution comprising 0.2 N NaOH and 1% SDS (Solution 
B) was added and thoroughly mixed. The tube was then incubated on ice for 
10 min before adding 11 ml of an aqueous reagent solution composed of 60 
ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid and 22.5 ml of 
distilled water (Solution C), and thoroughly mixed. The contents of the 
tube were then centrifuged at 17,000 rpm for 40 min at 4.degree. C. in a 
polyallomer SW28 centrifuge tube. The resultant liquid phase of 32 ml was 
then distributed evenly between two SW28 tubes. The plasmid DNA present in 
the tubes was then precipitated by the addition of 9.6 ml of isopropanol 
to each tube and being allowed to stand at room temperature for 20 min. 
The plasmid DNA was then pelleted by centrifugation at 12,000 rpm for 30 
min at 4.degree. C. The pellets were then lyophilized, combined and 
suspended in precisely 4.3 ml of TE Buffer (pH 8.0) in a SW28 tube. 4.63 
grams of CsCl was added to the sample. After equilibration at room 
temperature 80 .mu.l of Ethictrium Bromide (10 mg/ml) was added and mixed 
thoroughly. The sample was then placed in a Ti65 polyallomer tube and 
heat-sealed. The tube was then centrifuged with a Vti65 rotor at 48,000 
rpm for 16 hours at 20.degree. C. After centrifugation, the plasmid band 
was then extracted by syringe as part of a 1.2 ml sample and expelled into 
a polyallomer SW41 centrifuge tube. The DNA was then precipitated by the 
addition of 2.4 ml of water and 7.2 ml of 95% ethanol to the 1.2 ml 
sample. The sample was then stored at -20.degree. C. for one hour and then 
centrifuged with a SW41 rotor at 15,000 rpm for 30 min at 4.degree. C. The 
pellet was then lyophilized and transferred to a 1.5 ml microcentrifuge 
tube where it was solubilized in approximately 300 .mu.l of TE Buffer (pH 
7.2). Approximately 30 .mu.l of RNAse A Solution was added to the sample 
which was then incubated at 37.degree. C. for 30 min. Approximately 2 
.mu.g of Proteinase K was then added to the sample which was then 
incubated again at 37.degree. C. for 30 min. The sample was then extracted 
with phenol/chloroform. A reextraction with phenol/chloroform was then 
performed using 200 .mu.l of TE Buffer (pH 7.2). The sample was then 
extracted with water-saturated ether. After removal of the ether phase the 
sample was precipitated by adding 1/10 th volume of 5 M NaCl and 3 volumes 
of the combined sample plus salt of 95% ethanol. The sample was then 
stored at -20.degree. C. 
EXAMPLE XVIII 
Banahan Std. high frequency transformation 
A sample of DH5 bacteria (from BRL Product Profile-see Hanahan, D., 1983, 
J. Mol. Biol. 166:557-580) was streaked onto SOB(Mg++).sup.+ agar and 
incubated at 37.degree. C. for about 18 hours. The colonies were 
transferred into 1 ml SOB broth per colony. Each 1 ml cell suspension was 
used to inoculate a flask containing 10 ml of SOB broth. The flasks were 
incubated at 37.degree. C. and 250 rpm until a cell density of at least 
4.times.10.sup.7 viable cells/ml was reached. The cell suspension was then 
cooled on ice for 10-15 min. The suspension was then centrifuged at 2000 
rpm for 12 min at 4.degree. C. to pelletize the cells. The cells were then 
resuspended in 3.3 ml of TFB and incubated on ice for 10 min. The cells 
were then repelletized by centrifugation at 2000 rpm, for 12 min at 
4.degree. C. The cells were then resuspended in 0.8 ml of SOB with TFB, 
adding 28 .mu.l of DMSO and DTT to make a 3.5% concentration. The sample 
was then incubated on ice for 10 min and a second 28 .mu.l portion of DMSO 
and DTT was added to make a final concentration of 7%. The sample was then 
incubated on ice for 10 min. 210 .mu.l of the cell suspension was then 
combined with less than 20 .mu.l of the DNA solution (plasmid) resulting 
from Example XVII and incubated on ice for 20 min. The reaction was then 
heat shocked in a 42.degree. C. water bath for 90 seconds and immediately 
chilled on ice for 2 min. 800 .mu.l of SOC broth was then added to the 
sample and allowed to incubate at 37.degree. C. for 30 min. 500 .mu.l of 
the reactant was then spread on a YT/Amp.sup.++ plate and allowed to dry 
before incubation at 37.degree. C. 
______________________________________ 
.sup.+ S.O.C./S.O.B. Preparation 
Amt./ 485H.sub.2 O/ 
Reagent Conc. 100 ml 500 ml 
______________________________________ 
BactoTryptone 
2.0% 2.0 gm 10 gm 
Yeast Extract 
0.5% 0.5 gm 2.5 gm 
NaCl 10 mM 1.0 ml 1M NaCl 5.0 ml 
KCl 2.5 mM 0.25 ml 1M KCl 1.25 m. 
MgCl2.MgSO.sub.4 
20 mM 1.0 ml 2M Mg Stock 
5.0 ml 
(10 mM each) 
Glucose 20 mM 1.0 ml 2M Glucose 
5.0 ml 
Distilled H.sub.2 O 
qs to 100 ml total volume 
______________________________________ 
S.O. Broth 
Bactotryptone, yeast extract, NaCl and KCl were added to 97 ml of distilled 
water, dissolved and then autoclaved. MgCl.sub.2 and MgSO.sub.4 were then 
added at a rate of 1/100 to the solution. 
S.O.B. Plates 
Same procedure for S.O. broth but with the additional inclusion of agar to 
the solution at a rate of 15 g/l prior to autoclaving. 
S.O.C. Broth 
Same procedure for S.O. broth but with the additional inclusion of 2M 
glucose at a rate of 1/100 after autoclaving. 
______________________________________ 
.sup.++ YT/Amp Plate Agar 
Reagent 1 liter 500 ml 250 ml 
______________________________________ 
NaCl 5.0 gm 2.5 gm 1.25 gm 
Bacto Yeast Extract 
5.0 gm 2.5 gm 1.25 gm 
Bacto Tryptone 8.0 gm 4.0 gm 3.0 gm 
Bacto Agar 15 gm 7.5 gm 3.75 gm 
______________________________________ 
The first three ingredients are dissolved in the desired volume of 
distilled water. The agar is then suspended and autoclaved for 20-30 min. 
After the agar has cooled to about 50.degree. C., 50 mg/L of ampicillin are 
added to the agar Just before it is poured into the plates. 
EXAMPLE XIX 
Production of recombinant antigen 
The purified and isolated plasmid pBluescrtpt/lOE3 resulting from the 
cesium chloride purification process of Example XVI (see Maniatis et al., 
1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y.; herein incorporated by reference) 
was used in an amount of 1 .mu.g to transform E. coli strain DH 5 
(Bethesda Research Laboratories) using the Hanahan Standard High Frequency 
Transformation of Example XVIII (see Hanahan, D., 1983, J. Mol. Biol. 
166:557-580; herein incorporated by reference). The entire transformation 
reaction was added to 250 ml of YT broth containing 12.5 mg ampicillin and 
59.5 mg IPTG. The culture was incubated overnight at 37.degree. C. and 250 
rpm. The cells from the culture were then pelletized by centrifugation at 
1000.times.g for 10 min at 4.degree. C. The resultant pellet was then 
resuspended in 40 ml of Proteinase Inhibition Buffer.sup.+ and 
recentrifuged at 1000.times.g for 10 min at 4.degree. C. The resultant 
pellet was then resuspended in 20 ml of Proteinase Inhibition Buffer 
containing 1 mg/ml lysozyme and incubated on ice for 20 min. NP40 (Sigma 
Chemicals, #N3516) was added to 1% (200 .mu.l) was added, and the solution 
was incubated on ice for 10 min. The solution was then sonicated twice at 
100 watts with the probe in the solution; with each event lasting 20 min 
and pausing 15 min on ice between the two events. The solution was then 
centrifuged at 12,000.times.g for 10 min at 4.degree. C. The supernatant, 
representing the recombinant antigen, was then recovered and stored at 
4.degree. C. 
______________________________________ 
.sup.+ Proteinase Inhibitor Buffer 
100 ml 500 ml 
______________________________________ 
50 mM Tris ph 8.0 606 mg 3.03 gm 
5 mM EDTA 186 mg 0.93 gm 
5 mM lodcacetamide 
92.5 mg 0.46 gm 
0.1 Mm TLCK 3.69 mg 18.45 
gm 
1 mM PMSF 2.27 ml 11.35 
ml 
______________________________________ 
EXAMPLE XX 
Coating a plate with recombinant antigen 
The recombinant antigen of Example XIX was slowly vortexed then made into a 
1:10 dilution by addition of 50 .mu.l of antigen to 450 .mu.l of coating 
buffer.sup.+. Two tubes of 1:100 dilution were then prepared by adding 50 
.mu.l of the 1:10 dilution to 450 .mu.l of the coating buffer. The coating 
buffer was then added to the wells of a Dynatech Immulon 2 plate, skipping 
columns 1 and 12--the total volume of the coating buffer being added 
equaling 100 ml less the volume of the diluted antigen. The appropriate 
amount of the 1:100 dilution was then added to the coating buffer in each 
well. The plate was sealed and stored at room temperature overnight. 
______________________________________ 
.sup.+ Coating Buffer 
______________________________________ 
To make 100 ml: 
Add to 75 ml d H.sub.2 O: 
0.88 gm NaCl 
0.02 gm KCl 
0.158 gm Na.sub.2 HPO.sub.4 
0.02 gm KH.sub.2 PO.sub.4 
Mix and adjust pH to 7.4 with 1M HCl 
0.446 gm MgCl.sub.2.6H.sub.2 O 
Add H.sub.2 O to bring volume to 100 ml 
______________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 277 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Babesia equi 
(B) STRAIN: Florida 
(D) DEVELOPMENTAL STAGE: merozoite 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: pEma1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ArgProProValLysMetIleSerLysSerPheAlaPheValPheAla 
151015 
SerIleAlaIleSerSerIleLeuAlaGluGluGluLysProLysAla 
202530 
SerGlyAlaValValAspPheGlnLeuGluSerIleAspHisValThr 
354045 
IleAspLysGlnSerGluGluHisIleValTyrThrAlaHisGluGly 
505560 
TyrAlaValGluLysValLysGluGlyAspSerValIleLysThrPhe 
65707580 
AspLeuLysGluGlnThrProLysThrValValArgHisIleLysAsp 
859095 
AsnLysProTyrValValIleAlaValGluSerAlaLeuHisLeuVal 
100105110 
LeuLysLysAspGlyAspLysTrpValGluLeuGluValAlaGluPhe 
115120125 
TyrGlnGluValLeuPheLysGlyPheGluAlaValSerValAspLeu 
130135140 
AlaAlaAlaValSerAspLysPheThrGluThrThrPheGlySerGly 
145150155160 
LysLysHisThrPheLysAlaProGlyLysArgValLeuLysValVal 
165170175 
AspGlyLysThrGluLeuIleAspGlyAspAsnGluValValLeuAsp 
180185190 
LeuGluLeuPheValSerSerAspAsnLysValAlaArgValValTyr 
195200205 
LeuTyrLysGlyAspGlyArgIleLysGluIlePheLeuLysLeuVal 
210215220 
GluLysAlaTrpLysArgValGluValLysAspAlaAlaGluThrLeu 
225230235240 
HisGlyIleAsnSerThrPheProAlaAspTyrLysValValTyrAsp 
245250255 
GlyPheSerValTyrGlyAlaLeuLeuAlaValAlaAlaIleAlaPhe 
260265270 
SerThrLeuPheTyr 
275 
__________________________________________________________________________