GB2249097A - Antigen and antibodies for a liver fluke diagnostic system - Google Patents

Antigen and antibodies for a liver fluke diagnostic system Download PDF

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Publication number
GB2249097A
GB2249097A GB9117831A GB9117831A GB2249097A GB 2249097 A GB2249097 A GB 2249097A GB 9117831 A GB9117831 A GB 9117831A GB 9117831 A GB9117831 A GB 9117831A GB 2249097 A GB2249097 A GB 2249097A
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Prior art keywords
antibody
binding region
glutamic acid
peptide
binding
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GB9117831D0 (en
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Alan George Trudgett
Alan Thomas Mcnair
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National Research Development Corp UK
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National Research Development Corp UK
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    • 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

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  • 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)

Abstract

Current techniques for the diagnosis of liver fluke infection in animals involve examination of faecal matter for parasite eggs and as such are difficult to use outside a research environment. The invention provides an antibody-binding region of a liver fluke tegument protein comprising a peptide with an amino-acid sequence: Asparagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Glutamic acid as well as antibodies raised against it. Either the antibody-binding sequence or antibodies raised against it can be used in a test system for the diagnosis of liver fluke infection.

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 inflected, 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 recombinant 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 tandemly 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 amino-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 the 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 Xgtll (Sigma Chemical Company, Fancy Road, Poole, BH17 7NA, England). Screening this library with antisera from domestic farm animals experimentally infected with Fasciola heqatica was carried out by transferring lysogenic plaques to nitrocellulose filters and screening as described by Sambrook et al (1). 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 ss-galactosidase 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 FP1. 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 EcoR1 site which on cleavage yields two fragments of 800 bp and 200 bp. SDS PAGE analysis of the induced insert expressed as a ss-galactosidase fusion protein shows a difference of about 30 kDa (kilo Daltons) between E. coli ss-galactosidase and the fusion protein suggesting that most of the insert is translated.
The fusion protein was excised from Xgtll by digestion with the restriction enzyme Eco1, separated by PAGE and DNA fragments were isolated from the gels and prepared for ligation into the M13 cloning vector (Sigma Chemical Company, Fancy Road, Poole, BH17 7NA, England) using T4 polymerase (Sambrook et al (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 tandemly 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 & BR< Hanna, 1988). Sera from infected cattle were titrated in the presence and absence of each of the peptides (used at 1 ug/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 FP1 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 T1 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 T1 antigen (Hanna and Trudgett, 1983). It is thus concluded that the FP1 insert contains part of the gene for the T1 antigen. This protein provides the major immunogenic 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 Proline 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 Helminthology 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 al 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 1216-1220.
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 henatica tegument protein comprising a peptide having the amino-acid sequence: Aspararagine-Lysine-Glutamic acid-Glycine-Glutamic acid-Proline-Glutamic 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 identification 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.
GB9117831A 1990-08-20 1991-08-16 Antigen and antibodies for a liver fluke diagnostic system Withdrawn GB2249097A (en)

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GB9117831A Withdrawn GB2249097A (en) 1990-08-20 1991-08-16 Antigen and antibodies for a liver fluke diagnostic system

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CA (1) CA2089157A1 (en)
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WO (1) WO1992003735A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2172492A1 (en) * 1993-09-28 1995-04-06 Elza Nicole Theresia Meeusen Protective antigens against parasites
CN105277720B (en) * 2015-10-08 2017-03-22 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

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001277A1 (en) * 1986-08-18 1988-02-25 The Australian National University Helminth parasite vaccine
CA2045663A1 (en) * 1989-01-31 1990-08-01 Sonia Louise Crameri Vaccine for the preventative treatment of infection of liver fluke in ruminants

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Experimental Parasitology, Vol.50, 1980, pages 103-114 *
Journal of Clinical Microbiology, Vol.12, No.5, 1980, pages 695-699 *
Journal of Helminthology, Vol.60, No.3, 1986, pages 173-178 *
Parasite Immunology, Vol.5, 1983, pages 409-420 *
Research In Veterinary Science, Vol.33, 1982, pages 328-332 *

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WO1992003735A1 (en) 1992-03-05
AU8426591A (en) 1992-03-17
GB9117831D0 (en) 1991-10-09
CA2089157A1 (en) 1992-02-21
GB9018238D0 (en) 1990-10-03

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