U.S. patent application number 11/187736 was filed with the patent office on 2007-06-21 for plasmodium vivax blood stage antigens, pvesp-1, antibodies and diagnostic assays.
This patent application is currently assigned to New York University. Invention is credited to John W. Barnwell.
Application Number | 20070141075 11/187736 |
Document ID | / |
Family ID | 22108714 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141075 |
Kind Code |
A1 |
Barnwell; John W. |
June 21, 2007 |
Plasmodium vivax blood stage antigens, PVESP-1, antibodies and
diagnostic assays
Abstract
This invention is directed to novel species-specific P. vivax
malarial peptide antigens which are proteins or fragments of
proteins secreted into the plasma of a susceptible mammalian host
after infection, and to monoclonal or polyclonal antibodies
directed against those antigens. The peptide antigens, monoclonal
antibodies, and/or polyclonal antibodies are utilized in assays
used to diagnose malaria, as well as to determine whether
Plasmodium vivax is the species responsible for the infection.
Inventors: |
Barnwell; John W.; (New
York, NY) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
New York University
New York
NY
|
Family ID: |
22108714 |
Appl. No.: |
11/187736 |
Filed: |
July 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09667130 |
Sep 21, 2000 |
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11187736 |
Jul 22, 2005 |
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08719821 |
Sep 30, 1996 |
6706872 |
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09667130 |
Sep 21, 2000 |
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08478417 |
Jun 7, 1995 |
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08719821 |
Sep 30, 1996 |
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08072610 |
Jun 2, 1993 |
5532133 |
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08478417 |
Jun 7, 1995 |
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Current U.S.
Class: |
424/191.1 ;
530/350; 530/388.1 |
Current CPC
Class: |
A61K 39/015 20130101;
G01N 2333/445 20130101; C12Q 1/6888 20130101; A61K 2039/55566
20130101; G01N 33/56905 20130101; Y02A 50/30 20180101; C07K 16/205
20130101; C07K 14/445 20130101 |
Class at
Publication: |
424/191.1 ;
530/350; 530/388.1 |
International
Class: |
A61K 39/002 20060101
A61K039/002; C07K 14/445 20060101 C07K014/445 |
Goverment Interests
[0001] The U.S. Government has rights in this invention by virtue
of Grant Nos. RO1 AI 24710 from The National Institute's of Health
and DPE-5979-A-00-0006 from the Agency for International
Development.
Claims
1-21. (canceled)
22. An isolated and purified nucleic acid hybridizable to the
polynucleotide of SEQ. ID NO. 1 under stringent conditions.
23. The isolated and purified nucleic acid of claim 22, wherein
said stringent conditions are 1.times.SSC and 65.degree. C.
Description
SUBJECT AREA OF THE INVENTION
[0002] This invention is directed to novel species-specific
malarial polypeptides which are secreted into the plasma of a
susceptible mammalian host after infection, and to antibodies
directed against those proteins. The polypeptides and/or antibodies
are utilized in assays used to diagnose malaria, as well as to
determine whether Plasinodium vivax is the species responsible for
the infection.
BACKGROUND OF THE INVENTION
[0003] Malaria is transmitted by the bite of the Anopheles
mosquito. Minutes after infection, sporozoites (the mosquito-hosted
stage of the malarial parasite) enter hepatocytes of the
susceptible mammal where they multiply by schizogony and develop
into merozoites. Rupture of the infected cells releases the
merozoites into the blood, where they enter erythrocytes to begin a
phase of asexual reproduction. During acute infections, malarial
parasite protein antigens are known to be released, accumulate, and
circulate in the plasma of infected individuals (Wilson et al., The
Lancet, Jul. 26, 1969; Wilson et al., International Journal for
Parasitology 3:511-520, 1973; Wilson et al., Parasitology,
71:183-192; Wilson, Nature, 284:451-452, 1980). The release of
these antigens of parasitic origin can occur at the time that
infected erythrocytes rupture to allow invasive merozoites to
invade new red blood cells. The antigens that spill into the host
plasma are those that have accumulated in the host cell cytoplasm
and internal membranous structures.
[0004] Additionally, release of antigen can occur during the
intraerythrocytic growth of the parasite as it matures from the
ring stage, the stage which invades the erythrocyte, through the
trophozoite stage, and into schizogony when the parasite
differentiates into merozoites. Release of antigens at this time
involves transport of the protein from the parasite across the
parasitophorous vacuole and its membrane, across the host cell
cytoplasm to the infected erythrocyte membrane, and then secretion
as an intact soluble protein into the plasma of the host. One P.
falciparum protein, PfHRP-2 (Histidine Rich Protein-2) has been
described that follows this route of transport and is secreted into
the culture supernatant or found in plasma (Wellems et al., Proc.
Natl. Acad. Sci. USA, 83:6065-6069, 1986; Howard et al., J. of
Cell. Biol., 103:1269-1277, 1986; Rock et al., Parasitology,
95:209-227, 1987; Panton et al., Mol. and Biochem. Parasitology,
35:149-160, 1989). A search for HRP analogues in P. vivax using
PfHRP gene probes and HRP-antisera gave only negative results (Rock
et al., Parasitology, 95:209-227, 1987; J. Barnwell, unpublished
results).
[0005] There is a need in the field for antibodies specific for a
P. vivax blood stage protein in a diagnostic assay. The prior art
assays based on antibodies specific for blood stage proteins have
been specific only for P. falciparum (Khusmith, Southeast Asian J
Trop Med Public Health (THAILAND), 19:21-6, 1988) or have involved
the use of panspecies-specific antibodies, so no existing assays
are specific for P. vivax (Gao et al., Southeast Asian J Trop Med
Public Health (THAILAND), 22:393-6, 1991 and James, M A et al.,
American Society of Tropical Medicine and Hygiene, Seattle, Wash.,
Nov. 16-19, 1992, Abs. #135, pp. 145-146). P. vivax has latent
liver stages, termed hypnozoites, which are reactivated and
reinitiate blood stage parasitemias. Hypnozoites are eliminated by
treatment with primaquine, but are not affected by chloroquine,
which acts only on blood stage parasites. As P. falciparum does not
produce hypnozoites, it is important to identify correctly the
Plasmodium species responsible for infection in order to provide
the appropriate course of chemotherapy for complete cure. The
increased prevalence of drug resistant strains in certain species
also makes it important to identify the species involved so correct
chemotherapy can be given. Thus, there is a need for a method and
reagents adapted for differential diagnosis of P. vivax
malaria.
[0006] However, a number of criteria should be met by a particular
protein antigen considered as a potential diagnostic target. First,
it should be soluble and relatively stable and not rapidly degraded
and/or rapidly removed from circulation. Second, the antigen should
contain epitopes unique to a species to allow specific diagnosis
and preferably be well-conserved within all or most isolates of a
species. Additionally, it should be relatively abundant to allow
detection at low parasitemia. As discussed below, the proteins of
this invention fulfill most or all of these requirements.
SUMMARY OF THE INVENTION
[0007] Secreted species-specific blood stage antigens have now been
identified from a major human malaria parasite species, P. vivax.
Two particular such proteins are designated P. vivax Erythrocyte
Secreted Protein-1 (PvESP-1) and P. vivax Erythrocyte Secreted
Protein-2 (PvESP-2). These antigens and fragments thereof have
unique P. vivax-specific epitopes which permits their use in
differential determination of P. vivax merozoites. Antibodies can
be and have been elicited against unique epitopes of such P. vivax
proteins and used in assays which not only diagnose malaria, but
also selectively identify P. vivax as the species having caused the
infection.
DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A and 1B are Western immunoblots of P. vivax
trophozoite infected erythrocytes probed with antibodies specific
for PvESP-1 and PvESP-2. They show that mAb 1D11.G10 reacts with a
225 KD protein, while mAb 3D4.E2 and 1A3.B4 react with a 70 KD
protein.
[0009] FIGS. 2A and 2B are Western immunoblots of P. vivax infected
erythrocytes and supernatant from cultures which were matured from
ring stage to late-staged trophozoites in vitro. The blots are
probed with mAbs specific for PvESP-1 (2A) and PvESP-2 (2B). They
show that both PvESP-1 and PvESP-2 are present in isolated infected
erythrocytes and in the culture medium.
[0010] FIG. 3A is a schematic representation of the P. vivax ESP-1
gene and structural features of the deduced protein. FIG. 3B is a
partial restriction map of the P. vivax ESP-2 gene.
[0011] FIG. 4A is an immunoblot of P. vivax culture supernatants
and plasma from P. vivax infected squirrel (Saimiri) monkeys. 4B is
an immunoblot of multiple species of Plasmodium in multiple stages
probed with PvESP-1 specific antibodies. FIGS. 4C and 4D are
immunoblots of plasma from individuals infected with P. falciparum,
P. vivax or both, and also probed with PvESP-1 specific antibodies.
This group of figures shows the selective reaction of these
antibodies with P. vivax and with proteins in the plasma of those
infected with P. vivax. Similar results can be obtained with
PvESP-2 antibodies using immunoblot procedures. (Example 5) Similar
results for malaria specificity are also obtained for PvESP-1 or
PvESP-2 antibodies on smears of different species of malaria
parasites by indirect immunofluorescence assay.
[0012] FIG. 5 is the DNA sequence and deduced amino acid sequence
of P. vivax ESP-1 (a sequence listing is provided separately).
[0013] FIGS. 6A and B are the immunoelectron micrographs of P.
vivax infected erythrocytes probed with mAb 1D11.G10 and mAb
3D4.A2, respectively.
[0014] FIGS. 7A and B are immunofluorescent assays of P. vivax
infected erythrocytes reacted with fluorescence-conjugated mAb
1D11.G10 and mAb 3D4.A2, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0015] All U.S. patents and references referred to herein are
hereby incorporated by reference in their entirety. In case of
conflict, the present disclosure controls.
[0016] The following definitions apply to the terms as used in this
application only and should not be construed to necessarily apply
to uses of the terms in other art.
[0017] "Stringent conditions" are as defined by Southern et al. in
J. of Mol. Bio., 98:503 and as detailed in Maniatis, Molecular
Cloning: A Laboratory Manual, 2nd ed., Chapter 9, 1989.
[0018] "Immunoreactive fragment" means a fragment of an antigen
that is recognized by an antibody raised against the entire
antigen.
[0019] "Immunoreactive analog" means a polypeptide which differs
from a naturally occurring or recombinant protein by the
substitution, deletion and/or addition of one or more amino acids
but which retains the ability to be recognized by an antibody
raised against the entire protein. A nonlimiting example is a
carrier/antigen fusion polypeptide of the whole antigen or an
immunoreactive fragment thereof, where the antigen or fragment can
be embedded within the carrier polypeptide or linked to the carrier
polypeptide at either end.
[0020] "Detecting" means determining the presence (or absence) or
quantity of a substance (e.g. an anitigen-antibody complex).
[0021] "Antibody" includes intact antibody molecule or fragments
thereof that recognize antigen (e.g. Fab or F(ab')2 fragments) and
can be of polyclonal or monoclonal type.
[0022] "Epitope" means any antigenic determinant responsible for
immunochemical binding with an antibody molecule. Epitopes usually
reside within chemically active surface groupings of protein
molecules (including amino acids and often also sugar side-chains)
and have specific three-dimensional structural characteristics and
specific charge characteristics.
[0023] "Peptide Antigen" means a peptide, dipeptide, or polypeptide
that can elicit (or react with) antibodies recognizing a particular
protein.
[0024] The search for secreted blood stage antigens for P. vivax
began by making monoclonal antibodies specific for blood stage
parasites. As described in detail in Example 1, the mAbs were made
by conventional techniques through the fusion of spleen cells
isolated from a mouse immunized with P. vivax infected red blood
cells with mouse myeloma cells to produce mAb secreting hybridomas.
Three of these mAbs were found to react with the P. vivax proteins
described herein. These proteins have been shown to be synthesized
by the parasite by several criteria. First, mAbs do not react with
uninfected erythrocytes as shown by control experiments and the
specificity of the mAb for P. vivax, described in Example 6. A
reaction to all species would be seen if the proteins were
erythrocytic. Second, as seen by IFA and IEM, the mAbs do not react
with uninfected erythrocytes which are present in the preparations.
(See FIGS. 6 and 7) Third, the mAbs have been used to
immunoprecipitate radiolabelled proteins from extracts of parasites
that have been biosynthetically labelled with .sup.35S-methionine.
These results indicate that the mAbs recognize P. vivax
proteins.
[0025] Specifically, mAb 1D11.G10 recognizes a P. vivax protein of
approximately 225,000 daltons in size as judged by SDS-PAGE (FIG.
1A, lane 1). The hybridoma which produces mAb 1D11.G10 has been
deposited with the ATCC, Bethesda, Md., on ______ and is Accession
No. ______. The protein recognized by this mAb has been designated
P. vivax ESP-1 or PvESP-1. It is found in the culture supernatant
when intact infected erythrocytes are incubated in vitro for 10-24
hours (FIG. 2A, lane labelled SUP) as well as supernatant collected
from in vitro cultures of rupturing mature schizont infected red
cells (lane labelled IRBC). These data indicate that this protein
is secreted. It is localized by immunofluorescent assay (IFA) and
immuno-electron microscopy (IEM) to the erythrocyte membrane of
infected erythrocytes. (FIGS. 6A and 7A)
[0026] mAbs 3D4.A2 and 1A3.B4 recognize a P. vivax protein of
approximately 70,000 daltons in size as judged by SDS-PAGE (FIG.
1B, lane 1). The hybridomas which produces mAbs 3D4.A2 and 1A3.B4
have been deposited with the ATCC, Bethesda, Md., on ______ and are
Accession Nos. ______ and ______, respectively. The protein
recognized by these mAb has been designated P. vivax ESP-2 or
PvESP-2. Like PvESP-1, PvESP-2 is found in the supernatants of in
vitro cultured intact trophozoite-infected erythrocytes (FIG. 2B,
lane labelled SUP), and thus is a secreted protein. The PvESP-2
protein is also found in culture supernatants collected after
schizont-infected erythrocytes have ruptured and released
merozoites (lane labelled IRBC). The protein is localized by IFA
and IEM to the caveola-vesicle complexes (CVC) of P. vivax infected
erythrocytes. (FIGS. 6B and 7B.) The CVC are membranous sac-like
vesicles attached to and contiguous with areas of flask-shaped
indentations called caveolae in the erythrocyte plasma
membrane.
[0027] Polyclonal antibodies can also be produced, for example,
using isolated PvESP-1 and/or PvESP-2 or fragments thereof. General
methodology for making such polyclonal antibodies is well-known in
the art, and can be made using protocols similar to that described
in Pink et al. (Eur. J. Immunol. 4:426-429, 1974).
[0028] The mAbs described above were used to screen a .lamda.ZAP
recombinant phage library of the P. vivax genome, although other
equivalent P. vivax libraries could have been used. The preparation
of this library is described in Example 2. After induction, mAb
1D11.G10 specifically recognized one plaque, designated PvMB3.3.1.
The 3.34 kB plasmid insert was isolated and sequenced. The
resulting sequence is SEQ ID No: 1. The nucleic acid sequence was
analyzed for open reading frames (ORF) and the deduced amino acid
sequence of the encoded protein was determined. The amino acid
sequence is SEQ ID No: 2. A schematic structure of the gene and
features of the encoded protein is presented in FIG. 3A. The gene
appears to be missing a small portion of its 5' end.
[0029] As shown in FIGS. 3A and 5, the deduced amino acid sequence
has an initial (N-terminal) sequence of hydrophobic amino acids.
This is followed by a short 139 base pair (bp) intron with typical
malaria intervening sequence splice sites. There follows a 2964 bp
ORF, ending in the TAA stop codon which is 53 bp before the end of
the cloned 3.34 kB insert DNA. A protein having this deduced
peptide sequence is hydrophilic with a low pI (3), consistent with
a large proportion of glutamate (Glu or E) residues in the deduced
amino acid sequence.
[0030] As indicated in the Figures, there are two sets of repeated
amino acid units in the sequence. One repeat unit is characterized
by the sequence D(L/M)EAGEE(A/T)G. This sequence is repeated 7
times at the N-terminal end of the protein. The second repeat is
located in the C-terminal portion of the protein, has the sequence
EEVEEVP, and is repeated 10 times. The hydrophobic amino acid
sequence could potentially be, as judged by its computer analyzed
hydrophobicity profile, a transmembrane domain, or a leader or
signal peptide sequence, or act as both. Completion of the 5' gene
sequence will shed more light on these possibilities, and is well
within the skill of the art in light of the present disclosure.
[0031] To determine the remainder of the gene sequence, the
complete intact gene can be isolated and sequenced using a large
DNA fragment, for example, in a Lambda replacement vector such as
Lambda DASH (Stratagene, LaJolla, Calif.) or equivalent library
using the insert as a probe. Methodology for this is provided by
Galinski et al. (Cell, 69:1213-1226, 1992) or other similar
methods. Alternatively, the 5' end could be isolated by the PCR
amplification or other method of amplification of the cDNA using
appropriate primers, for example, as described by Frohman et al.
(Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988).
[0032] The MB3.3.1 plasmid expresses in E. coli a large recombinant
protein recognized by the mAb 11D.G10 in Western immunoblots. The
topmost band recognized is approximately 205-210 Kd in size,
confirming that a small portion of the complete PvESP-1 gene
remains unsequenced since the native protein migrates in SDS-PAGE
at 225 Kd under identical conditions. This protein is easily
isolated from the culture using well-known techniques (Maniatis,
Molecular Cloning: A Laboratory Manual, 2nd ed., Chapter 18, 1989).
Mouse and rabbit antisera generated by immunization with the
1D11.G10 affinity purified recombinant protein recognizes both the
recombinant and native PvESP-1 indicating that the recombinant
phagmid MB3.3.1 authentically encodes PvESP-1.
[0033] Screening of the .lamda.ZAPII expression libraries with the
mAB 3D4.E2 revealed one phage plaque recognized by antibody. This
clone, PvMB2.5.1, was found to contain a plasmid having a 3.7 kB
insert. This plasmid has been deposited with the ATCC, Bethesda,
Md. on ______ having Accession No. ______. A partial restriction
map of the plasmid insert is depicted in FIG. 3B. The sequence of
this DNA is easily obtainable using, e.g., traditional nested
deletion and subcloning techniques or using nucleic acid primers
obtained by dideoxy sequencing of the insert (Maniatis, Molecular
Cloning: A Laboratory Manual, 2nd ed., Chapter 13, 1989).
[0034] The MB2.5.1 plasmid expresses a recombinant polypeptide of
approximately 60 KD, which is easily isolated from the culture
medium using standing protein isolation techniques (Maniatis,
Molecular Cloning: A Laboratory Manual, 2nd ed., Chapter 18, 1989).
The size of this protein suggests that a portion of the coding
region of this protein is not present in the insert, as the native
protein migrates under identical conditions in SDS-PAGE at
approximately 70 KD. The 60 KD polypeptide is recognized by mAb
3D4.E2 in Western blots, indicating the recognized epitope is
encoded by the insert. Additionally confirmation that plasmid MB
2.5.1 authentically encodes PvESP-2 can be done as for PvESP-1, by
immunizing with recombinantly expressed antigen and using the
antisera to determine if it recognizes native 70 kD PvESP-2.
[0035] Both PvESP-1 and PvESP-2 are successfully expressed in E.
coli and expression of these proteins in other systems, such as
viral systems, other bacterial systems, yeast systems or mammalian
cell culture, is also contemplated and is well within the skill of
the art. Using such alternate expression systems may be preferred
if glycosylation or other post-translational modification is
desired.
[0036] Purified native or recombinant peptide antigens of the
present invention can be used in immunogenic preparation to raise
additional antibodies. Such preparations will include
immunogenically effective amounts of the present antigens as well
as pharmaceutically acceptable vehicles, carriers, buffers, fillers
adjuvants and/or diluents.
[0037] Peptide antigens of the present invention can be purified by
well-known chromatographic techniques. Examples include SDS or
native PAGE gel elution, size exclusion gel filtration,
anion/cation exchange, antibody affinity, protein binding to
immobilized glutathione or MBP fusion partner binding to
immobilized amylase, metal binding with a polyhistadine linker
peptide after expression in a suitable plasmid vector and cell
host, or combinations thereof.
[0038] As is evident to one of ordinary skill, it is only certain
portions or epitopes of the proteins which are recognized by Mabs.
The portions of the protein(s) containing the relevant epitopes can
be identified. Fragments of the gene can be subcloned into the
appropriate reading frame of a plasmid expression vector and used
to transfect E. coli (or other host system) and expressed by
induction. Defined gene fragments can be generated using
restriction enzymes with cutting sites within the gene.
Alternatively, appropriate oligonucleotide primers can be used in a
PCR-based amplification reaction to engineer the DNA fragment to be
subcloned into the plasmid expression vector. Once the expression
vector is constructed, the recombinant immunoreactive fragments (or
analogs, e.g. as a fusion polypeptide) are expressed. The produced
fragments are then reacted with antibodies as above in Western
immunoblots, ELISA tests, or other immunochemical assay methods to
determine which portion or portions of the protein specifically
interact with the antibodies. These methods work well for defining
relatively large or small regions of a protein to locate the
corresponding epitope and is effective in identifying
conformation-dependent (discontinuous) epitopes, or linear
epitopes. An alternative method for identifying antigenic
determinants is the use of overlapping synthetic peptides of 8-15
amino acids that correspond to the deduced amino acid sequence of
the gene. Reactivity of these peptides can be determined using
ELISA-based assays, such as the methodology of Geysen et al.
(Journal of Immunological Methods, 102:259-274, 1987) or using
commercial-based peptide synthesis kits, i.e., Pepscan or
Inimotope. (Cambridge Research Biochemicals, Valley stream, N.Y.
and Chiron, Emeryville, Calif., respectively) This method is
especially effective in determination of linear epitopes.
[0039] The detection of parasite antigens present in a biological
fluid (e.g. plasma), such as PvESP-1 and PvESP-2, can constitute a
method for the diagnosis of acute or chronic P. vivax malaria
infections. To be useful, such an antigen should contain epitopes
unique to the P. vivax species to allow specific diagnosis and
differential diagnosis from other malarial infections, and should
preferably be conserved within all or most isolates of that species
(more than one antigens can be used to generate antibodies if
necessary to accommodate strain variations). Either monoclonal
antibodies or polyclonal antibodies could be used in the assay,
with monoclonals preferred. The epitopes recognized by the
monoclonal antibodies 1D11.G10 (anti-PvESP-1) and 3D4.E2
(anti-PvESP-2) are present in all or most P. vivax so far tested
(25/26 for 1D11.G10 and 26/26 for 3D4.E2). However, these antigens
are not present in P. falciparum (FIG. 4A, lane 3), P. malariae
(lane 2), P. coatneyi (lane 4), P. knowlesi (lane 5), or P. berghei
(lane 1). 1D11.G10 does cross-react with P. cynomolgi (lane 6), a
simian malaria parasite very closely related to P. vivax (lane 7),
but never found to occur as a naturally acquired human malaria
infection. The mAB 3D4.E2 in particular only recognizes P. vivax
and thus far does so 100% of the time. Of course, any strain
differences that may be encountered may be accounted for in an
assay by provision of additional appropriate antibodies, or by
provision of antibodies directed to inter-strain conserved
epitopes, which can be conveniently raised against recombinant
versions of PvESP-1 and PvESP-2 as well as immunoreactive fragments
and analogs thereof.
[0040] The detected antigens are relatively stable in vivo; that
is, they are not rapidly degraded and/or removed from circulation.
PvESP-1 and PvESP-2 can be detected by Western immunoblot in the
plasma of squirrel (Saimiri) monkeys experimentally infected with
P. vivax (FIG. 4A, lane 3) and in the plasma of humans from endemic
areas that are infected with P. vivax (FIGS. 4C, lanes 8-11 and 4D,
lanes 5-7). The antigens are not detected in plasma of individuals
infected only with P. falciparum (FIGS. 4C, lanes 3-7 and 4D, lanes
6 and 7), the major human malaria parasite that must be
differentiated from P. vivax. The squirrel monkey model closely
approximates what would occur in naturally infected humans, but
under more controlled conditions than that of work conducted in the
field within endemic areas. In Saimiri monkey infections, the
antigen can be detected with the present antibodies when there are
1000 parasites/.mu.l blood. In humans, early acute infections are
detected. Again, as is evident to one of ordinary skill, the
isolation of the genes means that high-titer, high-affinity (e.g.
of the order of 10.sup.10 liters/mol) antibodies can be produced
using standard methodology. These antibodies will be used to
increase the sensitivity and specificity of the assay.
[0041] Other serological assay formats based on antigen capture and
a reporter signal have produced similar results as described above
using mABs 3D4.1E2 and 1D11.G10. Based on these successes, it is
anticipated that these mAbs or others to be produced using the
recombinant proteins or immunogenic fragments thereof can be
adapted for use in immunoassay systems (using either labelled Abs
or labelled antigens) well-known in the diagnostic testing art.
[0042] All well-known methods of labelling antibodies are
contemplated, including without limitation enzymatic conjugates,
direct labelling with dye, radioisotopes, fluorescence, or
particulate labels, such as liposome, latex, polystyrene, and
colloid metals or nonmetals. Multiple antibody assay systems, such
as antigen capture sandwich assays, are also within the scope of
this invention. Further, competitive immunoassays involving
labelled protein or assays using the labelled protein to detect
serum antibodies are also contemplated forms of the diagnostic
assays of the present invention. Beyond diagnostic assays which
occur in solution, assays which involve immobilized antibody or
protein are also considered within the scope of the invention.
(See, for example, Miles et al., Lancet 2:492, 1968; Berry et al.,
J. Virol. Met. 34:91-100, 1991; Engvall et al., G. Immunochemistry,
8:871, 1971, Tom, Liposomes and Immunology, Elsevier/North Holland,
New York, N.Y., 1980; Gribnau et al., J. of Chromatogr. 376:175-89,
1986 and all references cited therein).
[0043] Examples of the types of labels which can be used in the
present invention include, but are not limited to, enzymes,
radioisotopes, fluorescent compounds, chemiluminescent compounds,
bioluminescent compounds, particulates, and metal chelates. Those
of ordinary skill in the art will know of other suitable labels for
binding to the monoclonal or polyclonal antibody (or to an antigen)
or will be able to ascertain the same by the use of routine
experimentation. Furthermore, the binding of these labels to the
monoclonal or polyclonal antibody (or antigen) can be accomplished
using standard techniques commonly known to those of ordinary skill
in the art.
[0044] One of the ways in which an assay reagent (generally, a
monoclonal antibody, polyclonal antibody or antigen) of the present
invention can be detectably labeled is by linking the monoclonal
antibody, polyclonal antibody, or antigen to an enzyme. This
enzyme, in turn, when later exposed to its substrate, will react
with the substrate in such a manner as to produce a chemical moiety
which can be detected as, for example, by spectrophotometric or
fluorometric means.
[0045] Examples of enzymes which can be used to detectably label
the reagents of the present invention include malate dehydrogenase,
staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase
and acetylcholine esterase.
[0046] The presence of the detectably labeled reagent of the
present invention can also be detected by labeling the reagent with
a radioactive isotope which can then be determined by such means as
the use of a gamma counter or a scintillation counter. Isotopes
which are particularly useful for the purpose of the present
invention are .sup.3H, .sup.125I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.36Cl, .sup.57Co, .sup.58Co, .sup.59Fe and
.sup.75Se.
[0047] It is also possible to detect the binding of the detectably
labeled reagent of the present invention by labeling the monoclonal
or polyclonal antibody with a fluorescent compound. When the
fluoroescently labeled reagent is exposed to light of the proper
wave length, its presence can then be detected due to the
fluorescence of the dye. Among the most commonly used fluorescent
labelling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0048] The reagents according to the invention also can be
detectably labeled using fluorescent emitting metals such as
.sup.152Eu, or others of the lanthanide series. These metals can be
attached to the reagent molecule using such metal chelating groups
as diethylenetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA) and salts thereof.
[0049] The reagents of the present invention also can be detectably
labeled by coupling it to a chemiluminescent compound. The presence
of the chemiluminescent-tagged reagent is then determined by
detecting the presence of luminescence that arises during the
course of the chemical reaction. Examples of particularly useful
chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0050] Likewise, a bioluminescent compound may be used to label the
reagent of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent reagent is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0051] Another technique which may also result in greater
sensitivity when used in conjunction with the present invention
consists of coupling the monoclonal or polyclonal antibody of the
present invention to low molecular weight haptens. The haptens can
then be specifically detected by means of a second reaction. For
example, it is common to use such haptens as biotin (reacting with
avidin) or dinitrophenol, pyridoxal and fluorescamine (reacting
with specific antihapten antibodies) in this manner.
[0052] Any biological sample containing the detectable yet unknown
amount of P. vivax specific blood-stage antigen can be used to
assay. Normally, the sample is preferably a liquid, such as, for
example, urine, saliva, cerebrospinal fluid, blood, serum and the
like, or a solid or semi-solid, such as, for example, tissue, feces
and the like.
[0053] It appears that 1D11.G10 may recognize a repeated epitope
since it has been successfully used in a two-site antigen capture
immunoassay using the same mAb for capture and an alkaline
phosphatase labelled mAb or mAb conjugated to liposomes
encapsulating a marker dye for the reporter antibody (Example 6).
Eleven of 15 plasma samples from P. vivax infected individuals were
positive by alkaline phosphatase conjugated antibody. Thirteen of
15 samples were positive by liposome conjugated antibody. None of
18 P. falciparum infected plasma samples were positive. Therefore,
antibodies to PvESP-1 and PvESP-2 appear quite effective when used
in a diagnostic assay of P. vivax infection and further, such
assays appear to specifically identify P. vivax infection. It is
anticipated that assays based on mAb specific for particular
epitopes and selected for their high titer and/or affinity will
serve to increase the specificity and sensitivity of the assay.
[0054] In general, increases in sensitivity are a development
consideration and are achieved by optimization of all reagents,
including the concentrations conjugated to reporter systems,
adsorbed to solid phase surfaces, specificity of the Abs, and
affinity of the Abs. These steps are routinely done and evaluated
during assay development and are well within the skill of those
working in the art.
[0055] As is evident to one of ordinary skill, the diagnostic assay
of the present invention includes kit forms of such an assay. This
kit would include anti-PvESP-1 and/or anti-PvESP-2 monoclonal or
polyclonal antibodies (raised against whole PvESP or immunoreactive
fragments or analogs thereof) which can be optionally immobilized,
as well as any necessary reagents and equipment to prepare the
biological sample for and to conduct analysis, e.g. preservatives,
reaction media such as nontoxic buffers, microtiter plates,
micropipettes, etc. The reagent (Abs and/or antigens) can be
lyophilized or cryopreserved. As described above, depending on the
assay format, the antibodies can be labelled, or the kit can
further comprise labelled PvESP-1 or PvESP-2 protein or fragments
or analogs thereof containing the relevant epitopes.
[0056] The types of immunoassays which can be incorporated in kit
form are many. Typical examples of some of the immunoassays which
can utilize the antibodies of the invention are radioimmunoassays
(RIA) and immunometric, or sandwich, immunoassays.
[0057] "Immunometric assay" or "sandwich immunoassay", includes
simultaneous sandwich, forward sandwich and reverse sandwich
immunoassays. These terms are well understood by those skilled in
the art. Those of skill will also appreciate that the monoclonal
antibodies, polyclonal antibodies and/or antigens of the present
invention will be useful in other variations and forms of
immunoassays which are presently known or which may be developed in
the future. These are intended to be included within the scope of
the present invention.
[0058] In a forward sandwich immunoassay, a sample is first
incubated with a solid phase immunoadsorbent containing monoclonal
or polyclonal antibody(ies) against the antigen. Incubation is
continued for a period of time sufficient to allow the antigen in
the sample to bind to the immobilized antibody in the solid phase.
After the first incubation, the solid phase immunoadsorbent is
separated from the incubation mixture and washed to remove excess
antigen and other interfering substances, such as non-specific
binding proteins, which also may be present in the sample. Solid
phase immunoadsorbent containing antigen bound to the immobilized
antibody is subsequently incubated for a second time with soluble
labeled antibody or antibodies. After the second incubation,
another wash is performed to remove unbound labeled antibody(ies)
from the solid phase immunoadsorbent and removing non-specifically
bound labeled antibody(ies). Labeled antibody(ies) bound to the
solid phase immunoadsorbent is then detected and the amount of
labeled antibody detected serves as a direct measure of the amount
of antigen present in the original sample.
[0059] Alternatively, labeled antibody which is not associated with
the immunoadsorbent complex can also be detected, in which case the
measure is in inverse proportion to the amount of antigen present
in the sample. Forward sandwich assays are described, for example,
in U.S. Pat. Nos. 3,867,517; 4,012,294 and 4,376,110.
[0060] In carrying out forward immunometric assays, the process may
comprise, in more detail: (a) first forming a mixture of the sample
with the solid phase bound antibody(ies) and incubating the mixture
for a time and under conditions sufficient to allow antigen in the
sample to bind to the solid phase bound antibody(ies), (b) adding
to the mixture after said incubation of step (a) the detectably
labeled antibody or antibodies and incubating the new resulting
mixture for a time and under conditions sufficient to allow the
labeled antibody to bind to the antigen-antibody complex on the
solid phase immunoadsorbent; (c) separating the solid phase
immunoadsorbent from the mixture after the incubation in step (b);
and (d) detecting either the labeled antibody or antibodies bound
to the antigen-antibody complex on the solid phase immunoadsorbent
or detecting the antibody not associated therewith.
[0061] In a reverse sandwich assay, the sample is initially
incubated with labeled antibody(ies), after which the solid phase
immunoadsorbent containing multiple immobilized antibodies is added
thereto, and a second incubation is carried out. The initial
washing step of a forward sandwich assay is not required, although
a wash is performed after the second incubation. Reverse sandwich
assays have been described, for example, in U.S. Pat. Nos.
4,098,876 and 4,376,110.
[0062] In carrying out reverse immunometric assays, the process may
comprise, in more detail; (a) first forming a mixture of the sample
with the soluble detectably labeled antibody for a time and under
conditions sufficient to allow antigen in the sample to bind to the
labeled antibody; (b) adding to the mixture after the incubation of
step (a) the solid phase bound antibodies and incubating the new
resulting mixture for a time and under conditions sufficient to
allow antigen bound to the labeled antibody to bind to the solid
phase antibodies; (c) separating the solid phase immunoadsorbent
from the incubating mixture after the incubation in step (b); and
(d) detecting either the labeled antibody bound to the solid phase
immunoadsorbent or detecting the labeled antibody not associated
therewith.
[0063] In a simultaneous sandwich assay, the sample, the
immunoadsorbent having multiple immobilized antibodies thereon and
labeled soluble antibody or antibodies are incubated simultaneously
in one incubation step. The simultaneous assay requires only a
single incubation and does not include washing steps. The use of a
simultaneous assay is by far the preferred one. This type of assay
brings about ease of handling, homogeneity, reproducibility, and
linearity of the assays and high precision. The sample containing
antigen, solid phase immunoadsorbent with immobilized antibodies
and labeled soluble antibody or antibodies is incubated under
conditions and for a period of time sufficient to allow antigen to
bind to the immobilized antibodies and to the soluble
antibody(ies). In general, it is desirable to provide incubation
conditions sufficient to bind as much antigen as possible, since
this maximizes the binding of labeled antibody to the solid phase,
thereby increasing the signal. Typical conditions of time and
temperature are two hours at 45.degree. C., or twelve hours at
37.degree. C. Antigen typically binds to labeled antibody more
rapidly than to immobilized antibody, since the former is in
solution whereas the latter is bound to the solid phase support.
Because of this, labeled antibody may be employed in a lower
concentration than immobilized antibody, and it is also preferable
to employ a high specific activity for labeled antibody. For
example, labeled antibody might be employed at a concentration of
about 1-50 ng per assay, whereas immobilized antibody might have a
concentration of 10-500 ng per assay per antibody. The labeled
antibody might have a specific activity with, for instance, one
radioiodine per molecule, or as high as two or more radioiodines
per molecule of antibody.
[0064] Of course, the specific concentrations of labeled and
immobilized antibodies, the temperature and time of incubation as
well as other assay conditions can be varied, depending on various
factors including the concentration of antigen in the sample, the
nature of the sample and the like. Those skilled in the art will be
able to determine operative and optimal assay conditions for each
determination by employing routine experimentation.
[0065] After the single incubation period, the solid phase
immunoadsorbent is removed from the incubation mixture. This can be
accomplished by any of the known separation techniques, such as
sedimentation and centrifugation. A washing step is not required
prior to detection of bound labeled antibody. Detection can be
performed by a scintillation counter, for example, if the label is
a radioactive gamma-emitter, or by a fluorometer, for example, if
the label is a fluorescent material. In the case of an enzyme
label, the detection can be done by calorimetric methods employing
a substrate for the enzyme.
[0066] In carrying out the simultaneous immunometric assay on a
sample containing a multivalent antigen, the process may comprise,
in more detail:
[0067] (a) simultaneously forming a mixture comprising the sample,
together with the solid phase bound antibody and the soluble
labeled antibody or antibodies;
[0068] (b) incubating the mixture formed in step (a) for a time and
under conditions sufficient to allow antigen in the sample to bind
to both immobilized and labeled antibodies;
[0069] (c) separating the solid phase immunoadsorbent from the
incubation mixture after the incubation; and
[0070] (d) detecting either labeled antibody bound to the solid
phase immunoadsorbent or detecting labeled antibody not associated
therewith.
[0071] Other such steps as washing, stirring, shaking filtering and
the like may of course be added to the assays, as is the custom or
necessity for any particular situation.
[0072] In the preferred mode for preforming the assays it is
important that certain "blockers" be present in the incubation
medium (usually added with the labeled soluble antibody). The
"blockers" are added to assure that non-specific proteins,
protease, or human antibodies to mouse immunoglobulins present in
the experimental sample do not cross-link or destroy the monoclonal
or polyclonal antibodies on the solid phase support, or the
radiolabeled indicator antibody, to yield false positive or false
negative results. The selection of "blockers" therefore adds
substantially to the specificity of the assays described in the
present invention.
[0073] It has been found that a number of nonrelevant (i.e.,
nonspecific) monoclonal or polyclonal antibodies of the same class
or subclass (isotype) as those used in the assays (e.g., IgG1, IgG
2a2, IgM, etc.) can be used as "blockers". The concentration of the
"blockers" (normally 1-100, .mu.g/.mu.l) is important, in order to
maintain the proper sensitivity yet inhibit any unwanted
interference by mutually occurring cross reactive proteins in human
serum. In addition, the buffer system containing the "blockers"
needs to be optimized. Preferred buffers are those based on weak
organic acids, such as imidazole, HEPPS, MOPS, TES, ADA, ACES,
HEPES, PIPES, TRIS, and the like at physiological pH ranges.
Somewhat less preferred buffers are inorganic buffers such as
phosphate, borate or carbonate. Finally, known protease inhibitors
should be added (normally at 0.01-10 microns/ml) to the buffer
which contains the "blockers".
[0074] There are many solid phase immunoadsorbents which have been
employed and which can be used in the present invention. Well-known
immunoadsorbents include nitrocellulose, glass, polystyrene,
polypropylene, dextran, nylon and other materials; tubes, beads,
and microtiter plates formed from or coated with such materials,
and the like. The immobilized antibodies can be either covalently
or physically bound to the solid phase immunoadsorbent, by
techniques such as covalent bonding via an amide or ester linkage,
or by absorption. Those skilled in the art will know many other
suitable solid phase immunoadsorbents and methods for immobilizing
antibodies thereon, or will be able to ascertain such, using no
more than routine experimentation.
[0075] Details of the operation and practice of the present
invention are set forth in the specific examples which follow.
However, these examples are not to be interpreted as limiting the
scope of the present invention.
EXAMPLE 1
Method of Making the Monoclonal Antibodies Specific for PvESP-1 and
PvESP-2
[0076] Balb/c mice were immunized intraperitoneally with
5.times.10.sup.8 purified P. vivax (of the Belem strain infected
red blood cells (IRBC) in complete Freud's adjuvant. Immunization
was repeated at 2 and 7 weeks using incomplete Freund's adjuvant
and finally at 14 weeks without adjuvant. 3 days later, spleen
cells from the immunized mouse were fused with myeloma cell line
NY-FOX (Hyclone, Utah; Taggart, Science, 219:1228-1230, 1983)
according to the basic method of Galfre et al. (Nature,
266:550-552, 1977). Cells were plated directly into microtiter
wells and cultured (Rener et al., Proc. Natl. Acad. Sci. USA,
77:6797-6799, 1980) such that 1 to 2 weeks later, 1 or more hybrid
colonies were observed in all wells. Culture supernatants were
collected and screened by immunofluorescence assay using smears of
P. vivax infected blood that also contained normal red blood cells.
Those cells producing antibodies which selectively reacted with
IRBCs were expanded and cryopreserved. Secondary screening was
performed by SDS-PAGE with hybridoma culture supernatants from
expanded cultures that had been obtained by centrifugation. Those
mAbs which reacted with P. vivax blood stage extracts and culture
supernatants (prepared essentially as described in Galinski et al.,
Cell, 69:1213-1226, 1992) were selected for further study. Three
such mAbs are designated 1D11.G10, 3.D4.A2, and 1A3.B4.
EXAMPLE 2
Screening of P. vivax .lamda.ZAPII Expression Library with the
mAbs
[0077] P. vivax genomic DNA was isolated and digested with mung
bean nuclease (U.S. Biochemical) following the procedures of
Vernick et al. (Nucl. Acids Res., 16:6883-6896, 1988) and as
modified by Galinski et al. (supra) Specifically, the DNA was
digested with 42.5-45% formamide. The digested DNA was ligated into
the .lamda.ZAPII vector (Stratagene, LaJolla, Calif.) and the
resulting phage were used to infect E. coli. Expression was induced
by growth on IPTG (isopropylthio-.beta.-D-galactoside) containing
nitrocellulose plates overlaying the agar plates, and the resulting
plaques were screened with the mAbs using standard immunodetection
methods (see, for example, Maniatis, Molecular Cloning: A
Laboratory Manual, 2nd ed., Chapter 12, 1989).
[0078] After screening approximately 3.times.10.sup.5 recombinant
plaques, mAb 1D11.G10 specifically recognized one recombinant phage
plaque. This phage clone, PvMB3.3.1 was purified, and in vivo
excised with the aid of helper phage R408 (Stratagene, LaJolla,
Calif.) to yield the clone as a pBluescript plasmid retaining the
recombinant DNA as a 3.34 kb insert (Short et al., Nucleic Acids
Res., 16:7583, 1988).
[0079] After screening approximately 4.times.10.sup.5 plaques, mAb
3D4.E2 also revealed one phage plaque recognized by the antibody.
This clone, PvMB2.5.1 was plaque purified and in vivo excised, as
above, to yield a pBluescript plasmid containing the 3.7 kb DNA
insert.
EXAMPLE 3
Expression of the Cloned Proteins in E. coli
[0080] The isolated pBluescript plasmids were transformed into E.
coli and expression was induced by growth in the presence of IPTG
using standard methodology (Maniatis, Molecular Cloning: A
Laboratory Manual, 2nd ed., Chapter 1, 1989). Proteins produced by
the cultures were isolated, separated on a gel, blotted and probed
using standard techniques (Maniatis, Molecular Cloning: A
Laboratory Manual, 2nd ed., Chapter 18, 1989). Probing the blot of
PvMB3.3.1 with 1D11.G10 revealed multiple bands, the largest of
which was 205-210 kD. Probing the blot of PvMB2.5.1 revealed a 60
kD band. These results indicate that the cloned inserts encode the
epitopes recognized by these mAbs.
EXAMPLE 4
Sequencing of the Pv3.3.1 Insert
[0081] The insert was directly sequenced from the pBluescript
excision plasmid. Nested deletions of 100-300 bp intervals were
created using exonuclease III and mung bean nuclease (U.S.
Biochemical, Cleveland, Ohio) and standard methodology. DNA
sequences were generated using the dideoxy termination method
sequencing methodology (Sanger et al., Proc. Natl. Acad. Sci. USA,
74:5463-5467, 1977). The entire PvESP-1 gene was sequenced on both
DNA strands, and is SEQ ID No: 1. The deduced protein sequence (SEQ
ID No: 2) was analyzed using Pustell and MacVector software
programs (IBI). GenBank (release 70) and the Swiss Protein Data
Bank (release 20) were screened for DNA and protein sequence
homologies using the GCG Sequence Analysis Software Package,
Version 7.0 (Genetics Computer Group, Inc.).
EXAMPLE 5
Cross-Reactivity Test with Other Plasmodium Species
[0082] FIG. 4A was produced as follows. P. vivax
trophozoite-infected erythrocytes (2.times.10.sup.4), 25 .mu.l of
supernatants from P. vivax trophozoite and rupturing
schizont-infected red blood cell cultures, and 20 .mu.l of a 1:10
dilution of P. vivax infected Saimiri monkey plasma were mixed with
sample buffer and electrophoresed on an SDS-PAGE gel. The gel was
electrophoretically transferred to 0.2 .mu.m nitrocellulose (NC) by
Western blot (Towbin, H. et al., Proc. Natl. Acad. Sci. USA,
76:4350, 1979). The NC was blocked with 3% non-fat dry milk and
probed with mAb 1D11.G10 at 2 mg/ml in TBS. The blot was washed
with TBS/0.05% tween 20 and reprobed with alkaline phosphatase
conjugated anti-mouse IgG (Promega, Madison, Wis.) and developed
with P-nitroblue tetrazolium chloride/5-bromo-4-chloro-3 indolyl
phosphate (U.S. Biochemicals, Cleveland, Ohio).
[0083] FIG. 4B. was produced as follows. P. vivax IRBC were
acquired from infected Saimiri monkey, P. cynomolgi (M strain) IRBC
was from infected Rhesus monkeys, P. knowlesi IRBC were Saimiri
monkey, P. coatneyi IRBC were from Rhesus monkeys, P. falciparum
from human rbc in in vitro culture, P. malariae from infected Aotus
monkeys, and P. berghei from infected rats. SDS-PAGE and
nitrocellulose transfer were done as above with 1.times.10.sup.5
parasites/lane dissolved in SDS-PAGE sample buffer. Indirect
immunofluorescence assay was performed by making smears of IRBC on
slides and reacting 1D11.G10 or 3D4.E2 with smears and using FITC
conjugated goat anti-mouse IgG as secondary antibody with the same
results as Western blot.
[0084] These results show that there is no cross-reactivity with
other malarial species.
EXAMPLE 6
Diagnostic Assay using Alkaline Phosphatase and Liposome Conjuated
mAb
[0085] Unlabelled mAb is absorbed to nitrocellulose sheets (5 .mu.m
average pore size) at 5 mg Ab/ml in PBS. The sheet is washed and
blocked with 3% non-fat dry milk in TBS. The sheet is layered on an
ELISA apparatus (Pierce) and a 96-well plexiglass top (like a slot
blot apparatus) is secured in place over the nitrocellulose sheet.
Diluted plasma (1:10-100 .mu.l) samples are applied to the wells
and drawn through the nitrocellulose by vacuum. The wells are
washed by vacuum and mAb 1D11.G10 conjugated to alkaline
phosphatase is applied to the wells. Alkaline phosphatase
conjugation was accomplished by the glutaraldehyde method of
Avrameas (Immunochemistry, 6:43, 1967). The alkaline phosphatase
conjugated mAb is pulled through the nitrocellulose by vacuum, the
wells are washed, and then the developer substrates NBT-BCIP are
added to and pulled through the wells. Positive reactions are
assessed by the appearance of a purple-violet to blue-black
precipitate forming in the wells at the surface of the
nitrocellulose. Eleven of the 15 plasma samples from P. vivax
individuals were positive using the alkaline phosphatase conjugated
mAb1D11.G10. All samples were assessed for infection with P. vivax,
P. falciparum, or both, by Giemsa-stained thick films of blood
samples. False positives thus, would show a positive reaction, but
would be negative for P. vivax parasites in thick films. No such
reactions were seen.
[0086] The liposome-based test was similar to the alkaline
phosphatase-based test. As in the alkaline phosphatase assay, the
secondary (reporter) mAb was conjugated to liposomes that contained
a bright red to maroon dye. Thus, the appearance of red on the
nitrocellulose was the reporter system and an enzymatic development
step is not needed as in the alkaline phosphatase system. Thirteen
of 15 infected samples were positive using the liposome conjugated
1D11.G10. This assay can also be adapted to a strip test where a
mAb or polyclonal Ab is absorbed to a NC strip that overlays an
absorbent pad. Then, test plasma, antibody conjugated liposomes,
and washing solutions are wicked upwards by diffusion and a
positive test is indicated by a red-to-magenta line across the NC
strip assay.
EXAMPLE 7
Competitive Diagnostic Test for Malaria which Indicates Specific
Infection with P. vivax
[0087] In a colorimetric immunoassay for PvESP-1 and/or PvESP-2,
large, unilamellar phospholipid vesicles approximately 0.2
micrometers in diameter are loaded with high concentrations of
Sulforhodamine B or a similar dye. The PvESP-1 and/or PvESP-2 is
coupled to phosphatidylethanolamine or another component of the
lipid vesicle, and incorporated into the lipid formulation, thus
conferring immunological specificity. Methods of formation of the
vesicles, loading the vesicles, and coupling the protein to the
phosphatidylethanolamine are disclosed in O'Connell et al. (Clin.
Chem, 31:1424-1426). The liposomes are then used as tracers in
simple competitive-binding immunoassays with antibody-coated tubes.
The results are read spectrophotometrically. Specific immunoassay
methods are described in O'Connell et al., supra, as well as
O'Connell, MG and DI, December, 1985, pp. 31-36. As this is a
competitive assay, the less signal seen, the more PvESP-1 and/or
PvESP-2 will be present in the sample. It is anticipated that this
assay will be selective for P. vivax infection, given the
selectivity of the antibodies 1D11.G10, 3D4.A2, and 1A3.B4 as shown
in Example 5.
Sequence CWU 1
1
4 1 3337 DNA Plasmodium vivax 1 gaattccggt aaagtaacaa ctatggtttc
gtatctatat ataaccttac taattttatc 60 ttttgctttt cttttaattc
atgcttcaac agtaagataa aaataatcta taaaaactgc 120 tatatataca
tatatattca taagtggcat ttgtgaattg cgatcattta aatttacgta 180
aaaacaatat tgaaaaaaat tttttttttt tttttttttt tgttctacag aacgatttag
240 aattggaaaa tgcttctgat gatgttgtag aggtggagga tccttcaaac
gacggtttag 300 aattagaaga ggaaaatttt gatgagaatt caggtgatga
tgaaactctt ttagatgcta 360 cccccgaaga tgactttgcc ttaacagatt
tgccaattga agacgatgag gaagtcaacg 420 aaacgttaga tggaggtgaa
tcattaggag aggtttccac tgaagatatg gaaacagaag 480 atggctcaac
agatgatacg gaaacagaag aaggactacc tggtgatatg gaaggagaag 540
aagaagctgg cgatatggaa gcaggggaag aagctggtga tttggaagca ggggaagaaa
600 ctggcgattt ggaagcaggg gaagaaactg gcgatttgga agcaggggaa
gaagctggtg 660 atttggaagc aggggaagaa actggcgatt tggaagcagg
ggaagaaact ggagatgcgg 720 aaactgaaga aggagcaact ggagatgcgg
aaactgaaaa tggagcaact gtgtatgtag 780 acacagaaga tagttcagct
gatggagcag aaaaagtaca tgttcctgct caagaaaatg 840 tacaacctgc
cgatagtaat gatgccctct ttggaagtat tttggataaa gatataattt 900
ttgatcatat taaagatttc gagccactat tcgaacaaat tgtggcgggt actgctaaac
960 atgttacggg acaagaattg ccaatgaaac ctgtaccatt accagtggca
gaagagcccg 1020 cgcaagtacc agcggaagaa ttagatgcca ctccagagga
tgacttcgca ttagatgtta 1080 cagaatctcc cgaggaagta gaattagtat
tagatgaaga ggcaactgaa gaagaatcaa 1140 cggaagtggg accaacggaa
gaaggaccaa ccgaagaatt agatgccact ccagaggatg 1200 gatttcgcat
tagacgaaac tgcagaagga gaaacagaag aaacgtagag ggagaagaaa 1260
cagaagaagc tgcagaagga gaagtatcag aagaaactcc agaaggagaa gaagagttag
1320 aggcaactcc agaggatgat ttcgcattag atggaactac attagaagaa
accgaagaaa 1380 ctgcagaagg agaagaaacc gtagagggag aagaaaccgt
agagggagaa gaaaccgtag 1440 agggagaaga agctgcagaa ggagaagaag
agttagaggc aactccagag gatgacttcc 1500 aattagaaga accatcagga
gaaggagaag gggaaggaga aggagaaggg gaaggagaag 1560 gagaagcgtt
agtagcagtg ccagtagtgg ccgaaccggt agaagtagtg actcctgctc 1620
agcctgtcaa accaatggtc gctccaacgg cagatgaaac tttattcgtt gatatcttag
1680 ataacgattt aacgtatgca gacattacat cctttgagcc attatttaaa
caaatcctca 1740 aggatcctga tgcaggagag gctgtaacag taccatcaaa
ggaagcacct gtacaagtac 1800 cagtggcagt agggcccgcg caagaagtgc
caacggaaga attgatgcaa ctccaagagg 1860 acgatttcga attagaagga
actgcagaag ctccagagga aggagaatta gtattagaag 1920 gagaaggaga
accaacggaa gaagagccaa gagaaggaga gccaacagaa ggagaagtgc 1980
cagaagaaga attagaggca actccagagg acgatttcga attagaagaa ccaacaggag
2040 aagaagtaga agaaaccgta gagggcgaag aaactgcaga aggagaagaa
gtggaagagg 2100 tacctgcaga agtagaagaa gtggaagagg tacctgcaga
agtagaagaa gtggaagagg 2160 taccagaaga agtagaagag gtacccgcag
aagtagaaga agtggaagag gtaccagaag 2220 aagtggaaga ggtaccagaa
gaagtggaag aggtaccaga agaagtggaa gaggtaccag 2280 aagaagtgga
agaagtggaa gaagtagaag aagtagaggt accagcggta gtagaagtag 2340
aagtaccagc ggtagtagaa gaagaggtgc cagaagaagt agaagaagaa gaagaagagg
2400 aagaaccagt agaggaagaa gatgtattac aattagtaat accatcggaa
gaagatatac 2460 aattagacaa accaaagaaa gacgaattag gctctggaat
tttatctatc atcgacatgc 2520 actaccaaga cgttccaaag gaatttatgg
aagaagaaga agaaactgca gtgtatccat 2580 tgaaaccaga agattttgca
aaggaagatt cacaatctac agaatggctc acattcattc 2640 aaggcctaga
aggcgactgg gaacgattag aagtgagctt aaataaggct agagaaagat 2700
ggatggaaca aagaaataaa gaatgggctg gctggcttcg cttaattgaa aataaatggt
2760 cagaatatag tcaaatttca acaaaaggaa aggacccagc tggtttgaga
aaacgagagt 2820 ggagcgacga gaaatggaaa aaatggttta aagcagaagt
caaatcccaa attgattcac 2880 acttgaaaaa atggatgaac gacactcatt
ccaatttatt taaaattctt gtgaaagata 2940 tgtcacaatt tgaaaacaag
aaaaccaaag aatggttaat gaatcactgg aaaaagaacg 3000 aacggggtta
tggttctgaa tcatttgaag ttatgaccac atcaaaatta ttaaatgtgg 3060
ctaagagtcg agaatggtac cgtgccaatc ctaatataaa tagagaaaga agagaactca
3120 tgaaatggtt tctcctaaaa gaaaacgaat atttaggaca aagaatggaa
aaaatggact 3180 cattggaaaa aagttaaatt ttttgtgttc aattcaatgt
gtacaacatt ttctggaaaa 3240 cgcctaacca aggaagaatg gaatcaattt
gttaatgaaa taaaagtttg aattatagaa 3300 aaaagaacag attattctct
tataaaataa ataattc 3337 2 1018 PRT Plasmodium vivax 2 Asn Ser Gly
Lys Val Thr Thr Met Val Ser Tyr Leu Tyr Ile Thr Leu 1 5 10 15 Leu
Ile Leu Ser Phe Ala Phe Leu Leu Ile His Ala Ser Thr Asn Asp 20 25
30 Leu Glu Leu Glu Asn Ala Ser Asp Asp Val Val Glu Val Glu Asp Pro
35 40 45 Ser Asn Asp Gly Leu Glu Leu Glu Glu Glu Asn Phe Asp Glu
Asn Ser 50 55 60 Gly Asp Asp Glu Thr Leu Leu Asp Ala Thr Pro Glu
Asp Asp Phe Ala 65 70 75 80 Leu Thr Asp Leu Pro Ile Glu Asp Asp Glu
Glu Val Asn Glu Thr Leu 85 90 95 Asp Gly Gly Glu Ser Leu Gly Glu
Val Ser Thr Glu Asp Met Glu Thr 100 105 110 Glu Asp Gly Ser Thr Asp
Asp Thr Glu Thr Glu Glu Gly Leu Pro Gly 115 120 125 Asp Met Glu Gly
Glu Glu Glu Ala Gly Asp Met Glu Ala Gly Glu Glu 130 135 140 Ala Gly
Asp Leu Glu Ala Gly Glu Glu Thr Gly Asp Leu Glu Ala Gly 145 150 155
160 Glu Glu Thr Gly Asp Leu Glu Ala Gly Glu Glu Ala Gly Asp Leu Glu
165 170 175 Ala Gly Glu Glu Thr Gly Asp Leu Glu Ala Gly Glu Glu Thr
Gly Asp 180 185 190 Ala Glu Thr Glu Glu Gly Ala Thr Gly Asp Ala Glu
Thr Glu Asn Gly 195 200 205 Ala Thr Val Tyr Val Asp Thr Glu Asp Ser
Ser Ala Asp Gly Ala Glu 210 215 220 Lys Val His Val Pro Ala Gln Glu
Asn Val Gln Pro Ala Asp Ser Asn 225 230 235 240 Asp Ala Leu Phe Gly
Ser Ile Leu Asp Lys Asp Ile Ile Phe Asp His 245 250 255 Ile Lys Asp
Phe Glu Pro Leu Phe Glu Gln Ile Val Ala Gly Thr Ala 260 265 270 Lys
His Val Thr Gly Gln Glu Leu Pro Met Lys Pro Val Pro Leu Pro 275 280
285 Val Ala Glu Glu Pro Ala Gln Val Pro Ala Glu Glu Leu Asp Ala Thr
290 295 300 Pro Glu Asp Asp Phe Ala Leu Asp Val Thr Glu Ser Pro Glu
Glu Val 305 310 315 320 Glu Leu Val Leu Asp Glu Glu Ala Thr Glu Glu
Glu Ser Thr Glu Val 325 330 335 Gly Pro Thr Glu Glu Gly Pro Thr Glu
Glu Leu Asp Ala Thr Pro Glu 340 345 350 Asp Gly Phe Arg Ile Arg Arg
Asn Cys Arg Arg Arg Asn Arg Arg Asn 355 360 365 Val Glu Gly Glu Glu
Thr Glu Glu Ala Ala Glu Gly Glu Val Ser Glu 370 375 380 Glu Thr Pro
Glu Gly Glu Glu Glu Leu Glu Ala Thr Pro Glu Asp Asp 385 390 395 400
Phe Ala Leu Asp Gly Thr Thr Leu Glu Glu Thr Glu Glu Thr Ala Glu 405
410 415 Gly Glu Glu Thr Val Glu Gly Glu Glu Thr Val Glu Gly Glu Glu
Thr 420 425 430 Val Glu Gly Glu Glu Ala Ala Glu Gly Glu Glu Glu Leu
Glu Ala Thr 435 440 445 Pro Glu Asp Asp Phe Gln Leu Glu Glu Pro Ser
Gly Glu Gly Glu Gly 450 455 460 Glu Gly Glu Gly Glu Gly Glu Gly Glu
Gly Glu Ala Leu Val Ala Val 465 470 475 480 Pro Val Val Ala Glu Pro
Val Glu Val Val Thr Pro Ala Gln Pro Val 485 490 495 Lys Pro Met Val
Ala Pro Thr Ala Asp Glu Thr Leu Phe Val Asp Ile 500 505 510 Leu Asp
Asn Asp Leu Thr Tyr Ala Asp Ile Thr Ser Phe Glu Pro Leu 515 520 525
Phe Lys Gln Ile Leu Lys Asp Pro Asp Ala Gly Glu Ala Val Thr Val 530
535 540 Pro Ser Lys Glu Ala Pro Val Gln Val Pro Val Ala Val Gly Pro
Ala 545 550 555 560 Gln Glu Val Pro Thr Glu Glu Leu Met Gln Leu Gln
Glu Asp Asp Phe 565 570 575 Glu Leu Glu Gly Thr Ala Glu Ala Pro Glu
Glu Gly Glu Leu Val Leu 580 585 590 Glu Gly Glu Gly Glu Pro Thr Glu
Glu Glu Pro Arg Glu Gly Glu Pro 595 600 605 Thr Glu Gly Glu Val Pro
Glu Glu Glu Leu Glu Ala Thr Pro Glu Asp 610 615 620 Asp Phe Glu Leu
Glu Glu Pro Thr Gly Glu Glu Val Glu Glu Thr Val 625 630 635 640 Glu
Gly Glu Glu Thr Ala Glu Gly Glu Glu Val Glu Glu Val Pro Ala 645 650
655 Glu Val Glu Glu Val Glu Glu Val Pro Ala Glu Val Glu Glu Val Glu
660 665 670 Glu Val Pro Glu Glu Val Glu Glu Val Pro Ala Glu Val Glu
Glu Val 675 680 685 Glu Glu Val Pro Glu Glu Val Glu Glu Val Pro Glu
Glu Val Glu Glu 690 695 700 Val Pro Glu Glu Val Glu Glu Val Pro Glu
Glu Val Glu Glu Val Glu 705 710 715 720 Glu Val Glu Glu Val Glu Val
Pro Ala Val Val Glu Val Glu Val Pro 725 730 735 Ala Val Val Glu Glu
Glu Val Pro Glu Glu Val Glu Glu Glu Glu Glu 740 745 750 Glu Glu Glu
Pro Val Glu Glu Glu Asp Val Leu Gln Leu Val Ile Pro 755 760 765 Ser
Glu Glu Asp Ile Gln Leu Asp Lys Pro Lys Lys Asp Glu Leu Gly 770 775
780 Ser Gly Ile Leu Ser Ile Ile Asp Met His Tyr Gln Asp Val Pro Lys
785 790 795 800 Glu Phe Met Glu Glu Glu Glu Glu Thr Ala Val Tyr Pro
Leu Lys Pro 805 810 815 Glu Asp Phe Ala Lys Glu Asp Ser Gln Ser Thr
Glu Trp Leu Thr Phe 820 825 830 Ile Gln Gly Leu Glu Gly Asp Trp Glu
Arg Leu Glu Val Ser Leu Asn 835 840 845 Lys Ala Arg Glu Arg Trp Met
Glu Gln Arg Asn Lys Glu Trp Ala Gly 850 855 860 Trp Leu Arg Leu Ile
Glu Asn Lys Trp Ser Glu Tyr Ser Gln Ile Ser 865 870 875 880 Thr Lys
Gly Lys Asp Pro Ala Gly Leu Arg Lys Arg Glu Trp Ser Asp 885 890 895
Glu Lys Trp Lys Lys Trp Phe Lys Ala Glu Val Lys Ser Gln Ile Asp 900
905 910 Ser His Leu Lys Lys Trp Met Asn Asp Thr His Ser Asn Leu Phe
Lys 915 920 925 Ile Leu Val Lys Asp Met Ser Gln Phe Glu Asn Lys Lys
Thr Lys Glu 930 935 940 Trp Leu Met Asn His Trp Lys Lys Asn Glu Arg
Gly Tyr Gly Ser Glu 945 950 955 960 Ser Phe Glu Val Met Thr Thr Ser
Lys Leu Leu Asn Val Ala Lys Ser 965 970 975 Arg Glu Trp Tyr Arg Ala
Asn Pro Asn Ile Asn Arg Glu Arg Arg Glu 980 985 990 Leu Met Lys Trp
Phe Leu Leu Lys Glu Asn Glu Tyr Leu Gly Gln Arg 995 1000 1005 Met
Glu Lys Met Asp Ser Leu Glu Lys Ser 1010 1015 3 9 PRT Plasmodium
vivax MISC_FEATURE (1)..(9) where Xaa1 can be either Leu or Met
MISC_FEATURE (1)..(9) where Xaa2 can be either Ala or Thr 3 Asp Xaa
Glu Ala Gly Glu Glu Xaa Gly 1 5 4 7 PRT Plasmodium vivax 4 Glu Glu
Val Glu Glu Val Pro 1 5
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