AU8426591A - Liver fluke diagnostic system - Google Patents
Liver fluke diagnostic systemInfo
- Publication number
- AU8426591A AU8426591A AU84265/91A AU8426591A AU8426591A AU 8426591 A AU8426591 A AU 8426591A AU 84265/91 A AU84265/91 A AU 84265/91A AU 8426591 A AU8426591 A AU 8426591A AU 8426591 A AU8426591 A AU 8426591A
- Authority
- AU
- Australia
- Prior art keywords
- antibody
- binding region
- peptide
- glutamic acid
- binding
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/43559—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from trematodes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
"LIVER FLUKE DIAGNOSTIC SYSTEM"
The present invention relates to liver fluke epitopes or antibody-binding regions and to a diagnostic test kit for the identification of the liver fluke parasite in livestock using the epitopes, or antibodies raised against them.
Infection of sheep and cattle by the Liver Fluke parasite (Fasciola hepatica) causes severe economic loss to the livestock industries of countries with temperate climates. In some areas of Europe up to 80 per cent of animals show evidence of previous infection at slaughter. Horses and goats can also be infected, as can humans. Current farming policy in areas where the fluke is endemic involves the regular dosing of herds with anthelminthic drugs. This is an expensive procedure and may lead to the accumulation of undesirable drug residues in the animals' tissues. Current techniques for the identification of infected animals in a herd are based on the examination of faecal matter for the large hyaline, thin-shelled, operculate eggs of the parasite. This requires personnel skilled in the identification of helminth eggs and as such is not generally applied outside research centres.
British Patent Specification No. 2169606A relates to a liver fluke antigen which is specific to the juvenile stage of Fasciola organisms and which is suitable for use as a vaccine to protect against the earlier stages of primary infection. Also disclosed are antibodies against the juvenile-specific antigen which are said to be useful in extracting juvenile-specific antigens from juvenile Fasciola.
PCT Publication No. W088/01277 discloses a reco binant DNA molecule comprising at least one nucleotide sequence which codes for all or a portion of a helminth parasite antigen. Also disclosed are a synthetic polypeptide displaying the antigenicity of all or a portion of a helminth parasite antigen and vaccine compositions derived therefrom.
However, neither of these documents discloses a diagnostic system for the detection of fluke infection or a specific antibody-binding region of a Fasciola hepatica tegument protein.
The object of the present invention is to provide a cheap, user-friendly diagnostic system to test for fluke infection. This would enable the stock raiser to administer anthelminthics only to those animals in a herd that were actually infected, leading to a considerable reduction in the costs involved in the raising of cattle for both milk and meat production. It also has the advantage that it would reduce the likelihood of the development of drug resistance in the parasites.
When animals are infected with Liver Flukes their immune system rapidly begins to make protein molecules called antibodies which bind specifically to the outer surface (tegument) of the parasite. The amino acid sequence of part of a tegument protein has been determined and it has been found to contain a region which has the same seven amino acids arranged in a tandem repeating array. These amino acids can act as an epitope (antibody binding region) and bind Liver Fluke specific antibodies. This sequence has been synthesised and incorporated in an immunoassay which can distinguish between sera from infected and uninfected cattle and may thus be used to test for infection by the Liver Fluke.
According to the present invention there is provided an antibody-binding region of a Fasciola hepatica tegument protein comprising a peptide having the amino-acid sequence:-
Asparagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Glutamic acid.
Preferably the antibody-binding region comprises a tandem repeat of the peptide as defined above. A tandem repeat may be defined as multiple copies of the same sequence lying in series. Most preferably the antibody-binding region comprises the peptide tande ly repeated five times. The antibody-binding region may comprise the peptide as defined above, or a tandem repeat thereof, flanked at either end thereof by other amino-acid sequences. Alternatively, the antibody-binding region may be linked to another conventional carrier molecule.
It will be appreciated that non-essential amino acids of the above peptide sequence may be varied without significantly affecting the immunological or biological activity of the antibody-binding region. Therefore, within the scope of this invention are included antibody-binding regions comprising analogs of the above peptide sequence in which non-essential a ino-acids are replaced, the antibody-binding activity being retained. The antibody-binding region of the invention may also comprise a tandem repeat of such analogs.
The invention also provides a DNA sequence encoding the above defined antibody-binding region or which on expression codes for a peptide having similar immunological or biological activity to the antibody-binding region defined above.
The invention also provides antibodies directed against an antibody-binding region of a Fasciola hepatica tegument protein comprising a peptide having the amino-acid sequence:-
Asparagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Glutamic acid.
The antibodies may be monoclonal or polyclonal.
In a further aspect tiie invention provides a diagnostic test kit for the identification of Fasciola hepatica in livestock comprising an antibody-binding region or an antibody as defined above or a combination of both.
The diagnostic test kit may be that for any conventional diagnostic test.
The invention relates to antibody-binding regions, DNA sequences and antibodies in isolated or biologically pure form.
A cDNA library of Fasciola hepatica was produced as described in Huynh et. al, in the phage vector λgtll (Sigma Chemical Company, Fancy Road, Poole, BH17 7NA, England). Screening this library with antisera from domestic farm animals experimentally infected with Fasciola hepatica was carried out by transferring lysogenic plaques to nitrocellulose filters and screening as described by Sa brook et. l (-) • This enabled the identification of recombinant plaques containing inserts coding for Fasciola hepatica proteins which are immunogenic in the mammalian host.
One of these inserts is linked to DNA encoding the -Qalactosidase gene. When expressed, it produced a fusion protein (FPI). FP1 was excised from polyacrylamide gels (PAGE) and an emulsion was prepared with Complete Freunds Adjuvant. Mice were inoculated (0.5 ml intra-peritoneally) and after two weeks boosted with Incomplete Freunds Adjuvant and FPI. After a further two weeks sera were collected and tested for immunoreactivity. In the juvenile fluke (6 weeks post excystment) there is strong binding to both the tegumental syncytium and to the tegumental cells. In the mature fluke (more than 12 weeks post excystment) the binding to the tegument is much reduced but there is strong binding to the walls of the oviduct. This indicates that the insert contains DNA encoding at least a portion of a tegumental protein.
Agarose gel electrophoresis showed the insert to be about 1000 bp (base pairs) with an internal EcoRl site which on cleavage yields two fragments of 800 bp and 200 bp. SDS PAGE analysis of the induced insert expressed as a β-galactosidase fusion protein shows a difference of about 30 kDa (kilo Daltons) between E. coli ft-qalactosidase and the fusion protein suggesting that most of the insert is translated.
The fusion protein was excised from λgtll by digestion with the restriction enzyme EcoRl, separated by PAGE and DNA fragments were
isolated from the gels and prepared for ligation into the Ml3 cloning vector (Sigma Chemical Company, Fancy Road, Poole, BH17 7NA, England) using T4 poly erase (Sambrook et_ aj_ (2)). The DNA thus expressed in the M13 cloning vector was used for dideoxynucleotide sequencing.
Figure 1 shows part of the sequence of the 200 bp fragment. A notable feature of this part of the sequence is the presence of a tande ly repeated heptamer. Peptides corresponding to two of the possible reading frames of the heptamer were synthesised (Houghten, 1985) as either 3 x (NKEGEPE) or 3 x (RTRKENQ). The other reading frame contained a "stop" codon. In order to determine the correct reading frame an ELISA (enzyme linked immunosorbent assay) was performed using homogenised fluke antigen in the solid phase (Trudgett, Anderson & Hanna, 1988). Sera from infected cattle were titrated in the presence and absence of each of the peptides (used at 1 g/ml). The presence of 3 x (NKEGEPE) resulted in a 50% reduction in the binding of the anti-fluke antibody, thus indicating that this peptide contained a major B-cell epitope from F. hepatica. This result was confirmed by coating ELISA plates with 3 x (NKEGEPE) and then titrating sera from infected and control animals. Cattle sera from two weeks post-infection had titres (reciprocal of dilution giving significant binding) of the order of 200-4000 by this technique.
The sequence also contains a potential site for cleavage by membrane bound proteases (Arg-Lys), which allows the parasite to shed the antibody-binding site, thus evading the host's immune response. For this reason it is considered that the peptide of the invention would not be suitable for vaccination purposes.
The FPI fusion protein has been shown to be associated with the tegument and tegumental cells of juvenile flukes. In the adult it is observed in association with the female reproductive system. In this respect it is similar to the epitopes of the Tl antigen which have been detected at these sites using monoclonal antibodies (Hanna, Trudgett and Anderson, 1988). The molecular ratio (Mr) of the isogenic protein as determined by immunoblotting is not significantly different from that of the Tl antigen (Hanna and Trudgett, 1983). It is thus concluded that the FPI insert contains part of the gene for the Tl
antigen. This protein provides the major i munogenic stimulus whilst the fluke is migrating through the host tissue towards the bile ducts. (Hanna, 1980). It is believed to play a major role in the evasion of the host immune system as it is continuously shed from the tegument during this period. The tandemly repeated heptameric B cell epitope may be involved in this phenomenon as may the site for proteolytic cleavage in proximity to this region of the molecule.
Serial serum samples taken from experimentally infected cattle and sheep have indicated that a rise in specific antibody levels against the antibody-binding region of the present invention can be detected from one week post infection.
Abbreviations A Alanine T Threonine
V Valine C Cysteine
L Leucine Y Tyrosine
I Isoleucine N Asparagine
P Pro!ine Q Glutamine F Phenylalanine D Aspartic acid
W Tryptophan E Glutamic acid
M Methionine K Lysine
G Glycine R Arginine
S Serine H Histidine
References
Duffus, W.P.H. and Franks, D. (1981). The interaction in vitro between bovine immunoglobulin and juvenile Fasciola hepatica. Parasitology 82:1.
Hanna, R.E.B. (1980) Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. Experimental Parasitology 50:103.
Hanna, R.E.B. and Trudgett, A. (1983) Fasciola hepatica: development of monoclonal antibodies and their use to characterize a glycocalyx antigen in migrating flukes. Parasite Immunology 5:409.
Hanna, R.E.B., Trudgett, A. and Anderson, A. (1988) Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding. Journal of Hel inthology 62:15
Houghten R.A. (1985) General method for the rapid synthesis of large numbers of peptides: specificity of an antigen-antibody interaction at the level of individual amino-acids. Proc. Natl. Acad. Sci. USA 82:5131-5135
Huynh, T. et. __ _ in DNA cloning : a practical approach. ,1985) ed. D.M. Glover. IRL Press, Oxford.
Trudgett A, Anderson A. and Hanna R.E.B. (1988) Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays. Research in Veterinary Science 44:262
Sambrook, Fritsch and Maniatis (eds) (1) in Molecular Cloning. 2nd Edition. Cold Spring Harbor Laboratory Press, pages r 16-1220.
Sambrook, Fritsch and Maniatis (eds) (2) in Molecular Cloning. 2nd Edition. Cold Spring Harbor Laboratory Press, page 1323.
Claims (10)
1. An antibody-binding region of a Fasciola hepatica tegument protein comprising a peptide having the amino-acid sequence:-
Aspararagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Gluta ic acid,
or an analog thereof also having antibody-binding activity.
2. An antibody-binding region as claimed in claim 1 comprising a tandem repeat of the said peptide or analog thereof.
3. An antibody-binding region as claimed in claim 2 wherein the peptide or analog thereof is tandemly repeated five times.
4. An antibody-binding region as claimed in any preceding claim which is flanked at either end thereof by other amino-acid sequences.
5. A DNA sequence encoding an antibody-binding region as claimed in any preceding claim.
6. An antibody directed against an antibody-binding region of a Fasciola hepatica tegument protein comprising a peptide having the amino-acid sequence:-
Asparagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Glutamic acid,
or an analog thereof also having antibody-binding activity.
7. An antibody directed against an antibody-binding region as claimed in any of claims 1 to 4.
8. An antibody as claimed in claim 6 or 7 which is a polyclonal antibody.
9. An antibody as claimed in claim 6 or 7 which is a monoclonal antibody.
10. A diagnostic test kit for the identif cation of Fasciola hepatica in livestock comprising a reagent selected from an antibody-binding region as claimed in any of claims 1 to 4, an antibody as claimed in any of claims 6 to 9 or a combination of both.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB909018238A GB9018238D0 (en) | 1990-08-20 | 1990-08-20 | Liver fluke diagnostic system |
GB9018238 | 1990-08-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
AU8426591A true AU8426591A (en) | 1992-03-17 |
Family
ID=10680939
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU84265/91A Withdrawn AU8426591A (en) | 1990-08-20 | 1991-08-16 | Liver fluke diagnostic system |
Country Status (4)
Country | Link |
---|---|
AU (1) | AU8426591A (en) |
CA (1) | CA2089157A1 (en) |
GB (2) | GB9018238D0 (en) |
WO (1) | WO1992003735A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105277720A (en) * | 2015-10-08 | 2016-01-27 | 深圳华康生物医学工程有限公司 | ELISA kit for detection of liver fluke IgG4 antibody and preparation method thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09502988A (en) * | 1993-09-28 | 1997-03-25 | ザ・ユニバーシテイ・オブ・メルボルン | Infection protective antigen against parasites |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988001277A1 (en) * | 1986-08-18 | 1988-02-25 | The Australian National University | Helminth parasite vaccine |
JPH04507235A (en) * | 1989-01-31 | 1992-12-17 | ダラテック ピーテーワイ リミテッド | Vaccines for prophylactic treatment against liver fluke infections in ruminants |
-
1990
- 1990-08-20 GB GB909018238A patent/GB9018238D0/en active Pending
-
1991
- 1991-08-16 GB GB9117831A patent/GB2249097A/en not_active Withdrawn
- 1991-08-16 WO PCT/GB1991/001398 patent/WO1992003735A1/en not_active Application Discontinuation
- 1991-08-16 AU AU84265/91A patent/AU8426591A/en not_active Withdrawn
- 1991-08-16 CA CA 2089157 patent/CA2089157A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105277720A (en) * | 2015-10-08 | 2016-01-27 | 深圳华康生物医学工程有限公司 | ELISA kit for detection of liver fluke IgG4 antibody and preparation method thereof |
Also Published As
Publication number | Publication date |
---|---|
CA2089157A1 (en) | 1992-02-21 |
GB2249097A (en) | 1992-04-29 |
GB9117831D0 (en) | 1991-10-09 |
WO1992003735A1 (en) | 1992-03-05 |
GB9018238D0 (en) | 1990-10-03 |
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