Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-21
2001-03-27
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007900, C435S007920, C435S007930, C435S007940, C435S007950, C435S947000, C436S518000, C530S388100, C530S388800
Reexamination Certificate
active
06207395
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of diagnostic assays for detecting infection of an animal by the protozoan parasite
Entamoeba histolytica.
2. Background
Entamoeba histolytica
affects an estimated 480 million people annually; about 10 percent of these people develop colitis, liver abscesses, or other symptoms. Recently, a non-pathogenic species,
E. dispar,
has been described (Diamond and Clark (1993)
J. Euk. Microbiol.
40: 340-344).
E. dispar
is morphologically identical to the pathogenic species
E. histolytica.
Diagnosis of
E. histolytica
infection is often difficult. Amoebic dysentery caused by
E. histolytica
is easily confused with monocytic erythrophagocytosis and erythrophagocytosis caused by
Entamoeba coli
(Long and Christie (1995)
Clin. Lab. Med.
15: 307-331). Early diagnostic assays included microscopy and culture. One more recently developed diagnostic method involves detection of Entamoeba-specific IgG, IgM and IgA antibodies in serum (Healy (1986)
Rev. Infect. Dis.
8: 239-246; Arvind et al. (1988)
Serodiagn. Inmunother. Infect. Dis.
2: 79-84). However, seropositivity can persist for years, thus resulting in a high background due to healthy subjects giving positive results (Krupp (1970)
Am. J. Trop. Med. Hyg.
19: 57-62; Lobel et al. (1970)
Ann. Rev. Microbiol.
32: 379-347).
Another diagnostic method involves detection of a lectin found on the surface of
E. histolytica
and
E. dispar
trophozoites. Infection of a cell by Entamoeba involves binding of this lectin to Gal/Ga1NAc residues on the surface of the target cell (Petri et al. (1989)
J. Biol. Clem.
264: 3007-3012). The lectin, which has a molecular mass of 260 kDa, is composed of two subunits of 170 kDa and 35 kDa. Diagnostic assays that use monoclonal antibodies raised against purified native 170 kDa antigen were found to have problems with false positives (Ravdin et al. (1990)
J. Infect. Dis.
162: 768-772). Monoclonal antibodies against a recombinantly produced form of the 170 kDa subunit and the use of the antibodies for detecting the 170 kDa antigen are discussed in U.S. Pat. No. 5,272,058 (see also, Mann et al. (1993)
Infect. Immun.
61: 1772-1778; Petri et al. (1990)
Infect. Immun.
58: 1802-1806). Other immunoassays for diagnosing
E. histolytica
infection are discussed in, for example, Root et al. (1978)
Arch. Invest. Med.
(Mex) 9: Supplement 1: 203.
Two commercially available immunoassays for
E. histolytica
detection were recently compared to culture and PCR methods (Haque et al. (1996) 96
th
ASM General Meeting, New Orleans La.). The TechLab “Entamoeba Test” uses a monoclonal antibody to detect a the 170 kDa subunit of the Gal/Ga1NAc lectin that is present in both pathogenic
E. histolytica
and non-pathogenic
E. dispar
. The Alexon “ProSpecT
Entamoeba histolytica
Microplate Assay” also detects both pathogenic and non-pathogenic Entamoeba species, through use of rabbit polyclonal antisera. Comparison of these two tests to PCR and/or culture methods found that the Alexon test had a sensitivity of only 55% and a correlation of 66%, while the TechLab test had a sensitivity of 100% and a correlation of 84% (Id.). Both tests, however, are unable to distinguish between pathogenic and non-pathogenic strains.
Pathogenic
E. histolytica
trophozoites display a 29 kDa cysteine-rich surface antigen (Torian et al. (1990) Proc. Nat'l Acad. Sci. USA 87: 6358-6362). Monoclonal antibodies raised against this antigen were tested for ability to detect
E. histolytica
infection, but not all clinical isolates were detected (Id.). Thus, a need exists for sensitive and reliable assays for detecting
E. histolytica
infection in a clinical setting. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of diagnosing infection of a mammal by an Entamoeba species, in particular
E. histolytica
and
E. dispar.
The methods involve contacting a capture reagent which binds to a 29 kDa antigen of
Entamoeba histolytica
or
Entamoeba dispar
with a stool sample obtained from the mammal. The capture reagent forms a complex with the 29 kDa antigen if the 29 kDa antigen is present in the test sample. The presence or absence of the 29 kDa antigen bound to the capture reagent is then detected; the presence of the 29 kDa antigen is indicative of Eutamoeba infection of the mammal.
The invention also provides devices and kits for diagnosing infection of a mammal by an Entamoeba species, in particular
E. histolytica
and
E. dispar
. The kits typically include, inter alia, a solid support upon which is immobilized a capture reagent which binds to a 29 kDa antigen of
Entamoeba histolytica,
and a detection reagent which binds to the 29 kDa antigen.
Also provided by the invention are recombinant monoclonal and polyclonal antibodies that bind to the 29 kDa antigen.
REFERENCES:
patent: 4200690 (1980-04-01), Root et al.
patent: 5130417 (1992-07-01), Stanley, Jr. et al.
patent: 5272058 (1993-12-01), Petri, Jr. et al.
patent: 5459042 (1995-10-01), Flores de Castaneda
Haque et al. “Rapid Diagnosis of Entamoeba Infection by Using EntamoebaEntamoeba histolyticaStool Antigen Detection Kits” Journal of Clinical Microbiology, vol. 33, No. 10 (Oct. 1995), pp. 2558-2561. QR46.J87.*
Flores et al. “Serologic Reactivity to Purified Recombinant and Native 29-Kilodalton Peripheral Membrane Protein of PathogenicEntamoeba histolytica” Journal of Clinical Microbiology, vol. 31, No. 6 (Jun. 1993), pp. 1403-1407. QR46.J87.*
Diamond and Clark (1993)J. Euk. Microbiol.40:340-344.
Long and Christie (1995)Clin. Lab. Med.15:307-331.
Healy (1986)Rev. Infect. Dis.8:239-246.
Arvind et al. (1988)Serodiagn. Immunother. Infect. Dis.2:79-81.
Krupp (1970)Am. J. Trop. Med. Hyg.19:57-62.
Lobel et al. (1978)Ann. Rev. Microbiol.32:329-347.
Petri et al. (1989)J. Biol. Chem.264:3007-3012.
Ravdin et al. (1990)J. Infect. Dis.162:768-772.
Mann et al. (1993)Infect Immun.61:1772-1778.
Petri et al. (1990)Infect. Immun.58:1802-1806.
Root et al. (1978)Arch. Invest. Med.(Mex) 9:Supplement 1:203.
Haque et al. (1996) 96thASM General Meeting, New Orleans,LA: TechLab Entamoeba Test and Alexon ProSpecTEntamoeba histolyticaMicroplate Assay.
Torian et al. (1990)Proc. Nat'l. Acad. Sci. USA87:6358-6362.
Bruchhaus and Tannich (1993)Trop. Med. Parasitol44:116-118.
Reed et al. (1992)Infect. Immun.60:542-549.
Tachibana et al. (1991)J. Clin. Microbiol.29:2234-2239.
Soong et al. (1995)Infect. Immun.63:472-477.
Buechler Joe
Gray Jeff
Valkirs Gunars E.
Biosite Diagnostics, Inc.
Stucker Jeffrey
Townsend and Townsend / and Crew LLP
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