DE19704024A1 - Proteins, DNA sequences coding for them, on the other hand specific antibodies and their use to find nematicidal active substances - Google Patents

Abstract

The present invention relates to proteins, as well as sections of said proteins, which react with active substances against parasitizing nematodes from the group of the cyclic or open-chain depsipetides consisting of amino acids and hydroxycarboxylic acids. The disclosure also comprises DNA sequences coding these proteins or sections of said proteins as well as the use of said proteins or their sections for identifying substances acting against parasitizing nematodes.

Description

The present invention relates to proteins, as well as portions of these proteins that with substances acting against parasitic nematodes from the group of cycli or open-chain depsipeptides consisting of amino acids and hydroxycarbon acids exist, react, furthermore DNA sequences for these proteins or for Coding sections of these proteins and the use of these proteins or their sections for the identification of substances with action against parasitizing Nematodes.

A number of nematicidal substances have long been used as broad-spectrum Anthelmintics used in animal breeding (e.g. benzimidazole, levamisole, Morantel or avermectinoids). Based on the substance classes mentioned conducted numerous studies to elucidate the mechanism of action and to iden tification of the respective binding protein / receptor (benzimidazole: β-tubulin, Levamisole / Morantel: nicotinic acetylcholine receptor; Avermectinoids: glutamate mediated chloride channel). By increasing resistance to this broad spectrum anthelmintics, it is desirable to have new anthelmintically active substances to develop.

A cyclic depsipeptide PF 1022A and its activity against endoparasites is known (Y. Kodama et al., Sci. Reports of Meiji Seika Kaisha 31, 1992, pp. 1-8; EP-OS 382 173 and EP-OS 503 538). Other cyclic depsipeptides (Cycloocta depsipeptide: WO 93/19053; EP-OS 634 408; WO 94/19334; WO 95/07272; EP-OS 626 375; EP-OS 626 376; EP-OS 664 297; Cyclohexadepsipeptides: WO 93/25543; WO 95/27498; EP-OS 658 551) and open-chain depsipeptides (EP-OS 657 171; EP-OS 657 172; EP-OS 657 173) and their endoparasiticides Effects are known.

A series of studies to elucidate the mechanism of action of PF 1022A in various parasitic nematodes, e.g. angio strongylus cantonensis, Haemonchus contortus, Ascaris suum (see also M. Terada et al., Jpn. J. Parasitol. 42, 1993 pp. 220-226; F.E. Dutton et al., J. Antibiotics 48, 1995, pp. 820-823; E. Baldwin et al., J. Exp. Biol. 23, 1947, Pp. 277-91).  

In addition to the broad-spectrum anthelmintics already mentioned (e.g. benzimidazoles, Levamisole, morantel or avermectinoids) is via a binding protein or Receptor in nematodes of cyclic or open chain depsipeptides as Ligands consisting of amino acids and hydroxycarboxylic acids as building blocks, in particular the cyclic depsipeptide PF 1022A, so far nothing is known become.

The present invention relates to proteins, as well as portions of these proteins that with substances acting against parasitic nematodes from the group of cycli or open-chain depsipeptides consisting of amino acids and hydroxycarbon acids exist, react. The present invention further relates to DNA sequences that code for these proteins or for portions of these proteins.

The present invention further relates to the use of these proteins or their sections for the identification of substances with action against parasitizing Nematodes from the group of cyclic or open-chain depsipeptides that consist of amino acids and hydroxycarboxylic acids.

The present invention further relates to methods for identifying the Proteins and their parts, as well as the production of the proteins or their Sections of recombinant technology.

Identification and characterization of the cDNA sequences required for the inventions encoding proteins according to the invention is carried out by constructing a statistical, representative cDNA library using polyadenylated RNA fractions isolated from parasitic nematodes and a subsequent one Immunoscreening of the cDNA library mentioned.

Identification and characterization of the cDNA sequences according to the invention take place with the help of antibodies against a conjugate consisting of a cyclic or open-chain depsipeptide hapten and a suitable carrier Protein.

So far, they are only monoclonal antibodies against various cyclodepsi peptide synthetases, for example the Enniatin synthetase (A. Billich et al., Biol. Chem. Hoppe-Seyler 368 (5), 1987, pp. 521-529; A. Lawen et al., J. Biol. Chem. 265, (19), 1990, pp. 11355-11360) or cyclosporin synthetase (A. Lawen et  al., J. Biol. Chem. 266, (24), 1991, pp. 15567-15570), but not specific Antibodies against conjugates consisting of depsipeptide hapten and a suitable one neten carrier protein.

Preferred for identification and characterization of the invention cDNA sequences are conjugated as PF 1022A or its derivatives Depsipeptide hapten used on carrier proteins.

The proteins of the invention are expressed by expression of the cDNA in suitable expression systems, for example in bacteria, in particular especially in E. coli, as a prokaryotic expression system or in an euka ryotic expression system, in particular the baculovirus system.

Active substances which are found with the aid of the proteins according to the invention, are of great interest to medicine and veterinary medicine.

Further aspects of the invention and its preferred embodiments are described in following explained in more detail or defined in the claims.

Since it is the open chain and cyclic depsipeptides, for the specific Antibodies are designed to be relatively small and simple molecules is not able to act alone after application in a test animal are to trigger an appropriate immune response there, must preparatory measures are taken (E. Harlow, D. Lane in; Antibodies, a Laboratory Manual, Cold Springer Harbor Lab., Cold Spring Harbor, USA, 1988).

Such compounds are called, because of their size (molecules with a molecular weight of less than 10,000 daltons are normal weak immunogens; Molecules with a molecular weight less than 2,000 daltons alone can hardly trigger an immune response) and simple Structuring are unable to induce an immune response than Haptens or incomplete antigens and thus represents the complete antigens (= Immunogens), which act both as an antigen and an immune response to induce. Such hapten molecules can be attached to mostly soluble, high molecular weight compounds (carrier molecules) are conjugated, making them in  their properties become comparable to complete antigens, d. H. they own now the ability to trigger an immune response.

Some of the antibodies produced in the course of the immunization reaction are then able to react with specific epitopes on the hapten molecule regardless of whether the hapten molecule is alone or still coupled to the carrier molecule.

When immunizing with hapten-carrier conjugates, in addition to the hapten specific antibodies also induce and form antibodies against the carrier or the link between carrier and hapten, or only against the comm plex from Hapten (link) carrier are directed. The specific anti-hapten Antibodies are usually directed against that part of the molecule that furthest away from the carrier.

Under the frequently used name Hapten in the Rah men of the invention, the open chain or used for immunization cyclic depsipeptides, preferably the cyclodepsipeptide PF 1022A or its Derivatives understood.

In the context of this invention, those which act as haptens are therefore open chain and cyclic depsipeptides, in particular PF 1022A or its derivatives, before the immunization of experimental animals, preferably rodents, such as. B. mice, Rats, rabbits, guinea pigs, to a high molecular weight carrier coupled, which is suitable, said depsipeptides a complete effectiveness to lend.

Suitable carrier molecules within the scope of this invention are the first Line to understand macromolecular compounds that are freely accessible reactive Have groups for the coupling reaction with the hapten and in the By coupling to the hapten, it is capable of producing an immunogenic potency confer or increase their existing immunogenicity.

Particularly preferred carriers are macromolecular compounds that are free accessible reactive amino groups included.  

Lysine-rich proteins with a are very particularly preferred as carrier molecules Molecular weight between 10,000 and 1,500,000, as z. B. "Bovine Serum Albumin "(BSA: molecular weight 66,200)," Human Serum Albumin "(HSA; Molecular Weight: 58,000) or "Keyhole Limpet Protein" (KLH; molecular weight: 1,000,000). These are commercially available.

Of course, other macromolecular compounds as carrier Molecules are used, such as. B. "Porcine Thyreoglobulin", B2 Microglobu lin, haemocyanin, immunoglobulins, toxins (cholera, tetanus, diptheria toxin u. a.), polysaccharides, lipopolysaccharides, natural or synthetic polyadenyl and polyuridylic acids, polyalanyl and polylysine polypeptides or cell membrane components, such as treated with formalin or glutaraldehyde Erythrocyte cell membranes.

The, for example, is also suitable for use as a carrier molecule purified IgG fraction against mouse IgG (H + L) from rabbits according to that at H. Kawamura and J.A. Berzofsky (J. Immunol. 58 (1986), p. 136) Method.

The coupling of haptens to carriers takes place according to established methods Techniques (P. Tijssen in: "Practice and Theory of Enzyme Immunoassays", Burdon, R.H. & Knippenberg, P.H., ed., Elsevier, Oxford, UK 1985).

The conjugation of the hapten to the carrier molecule can either be direct or but preferably take place via a bridge link, which if necessary is next attached to the hapten molecule.

The coupling of the open-chain and cyclic depsipeptides as haptens, in particular the PF 1022A or its derivatives, on the carrier molecule in such a way that the relevant structural elements of the depsipeptides are free remain accessible and thus able to respond to a specific immune response trigger, d. H. to induce the formation of specific antibodies.

Depsipeptide ligands from open-chain or are used as preferred haptens cyclic depsipeptides with 6 to 30 ring atoms, consisting of amino acids and hydroxycarboxylic acids as building blocks.  

Depsipeptide ligands from cycli serve as very particularly preferred haptens depsipeptides with 24 ring atoms, consisting of amino acids and Hydroxycarboxylic acids as building blocks.

Cyclic depsipeptides with 24 ring atoms include compounds of the general formula (I)

in which
R ₁ for optionally substituted benzyl, where hydrogen, C _1-4 -alkyl, in particular methyl, hydroxy, halogen, in particular fluorine, C _1-4 -alkoxy, in particular methoxy or tert-butyloxy, nitro, amino may be mentioned as substituents , Dialkylamino, in particular dimethylamino or diethylamino, N-morpholinyl, N-pyrrolidinyl or N-piperidinyl,
R _{2 is} hydrogen, C _1-4 -alkyl, in particular methyl, hydroxy, halogen, in particular fluorine, C _1-4 -alkoxy, in particular methoxy or tert-butyloxy, nitro, amino, dialkylamino, in particular dimethylamino or diethylamino, N-morpholinyl, N-pyrrolidinyl or N-piperidinyl,
in which

• a) in the event that R _{1 is} benzyl,
  R _{2 represents} hydrogen-hydroxy, C _1-4 -alkoxy, in particular methoxy, halogen, in particular fluorine, alkenyloxy, in particular allyloxy,
• b) in the event that R _{1 is} methyl,
  R _{2 represents} hydrogen hydroxy, C _1-6 alkoxy, in particular meth oxy, nitro, amino, dialkylamino, in particular dimethyl amino, N-morpholinyl.

The compounds of the general formula (I) can be used in their optically active, stereoisomeric forms or as racemic mixtures. Preferably the optically active, stereoisomeric forms of the compounds of gen my formula (I) used. The cyclic depsi are particularly preferred peptides used, which are composed of L-configured amino acids and D-configu reassembled hydroxycarboxylic acids as ring building blocks.

The compound PF 1022A of the following formula (Ia) known from EP-OS 382 173 and EP-OS 503 538, in which R _{1 is} benzyl and R _{2 is} hydrogen, may be mentioned as an example as a cyclic depsipeptide:

In addition, the cyclic depsipeptides are those from the PCT application WO 93/19053 and EP 0 634 408 A1 known compounds that suitable functional groups containing a direct covalent coupling  a carrier protein or a coupling via a bridge molecule to one Allow carrier protein.

From PCT application WO 93/19053 and EP 0 634 408 A1, the compounds of the following formula (Ib), in which R _{1 represents} (R ₃ ) substituted benzyl, may be mentioned in particular:

in which
R ₂ and R ₃ each represent nitro, amino or hydroxy.

Further depsipeptides are those from PCT application WO 94/19334 known compounds called.

The compounds of the following formula (Ic) in which R _{1 is} benzyl may be mentioned in particular from PCT application WO 94/19334:

in which
R _{2 represents} hydroxy.

Finally, the depsipeptides are those from PCT application WO 95/07272 known compounds called.

From PCT application WO 95/07272, the compounds of the following formula (Id), in which R ^{1 represents} methyl, may be mentioned in particular:

in which
R _{2 represents} nitro, amino or methoxycarbonylamino.

Cyclic depsipeptides with 24 ring atoms are particularly preferred general formula (Ib) used, which has a free primary amino group of Benzyl fragments, optionally by means of a suitable bridge member a primary amino function on the carrier protein for covalent hapten Carrier binding are bound.

As bridge members for conjugation of the hapten, especially the cyclo depsipeptids PF 1022A and its derivatives of the general formula (Ib), to which Carrier molecules are primarily compounds that are at least contain two or more reactive groups which are able to react with the freely accessible reactive groups of the carrier molecule interact to kick.

The use of is particularly preferred in the context of this invention Bridge members that contain between 3 and 10 bridge carbon atoms and that as reactive groups e.g. B. have amino, formyl, carboxyl or SH groups.

These reactive groups can be used with the reactive using known methods Groups of the hapten and carrier molecules are reacted under Formation of a hapten-carrier conjugate.

For example, it is possible to build a bridge over a reactive amino group, in particular the derivatives of the cyclodepsipeptide PF 1022A, with the help of Dialdehydes such as e.g. B. glutardialdehyde, to one of the free amino groups of Bind carrier molecule.

In a specific embodiment of the present invention, one proceeds from the deri mentioned in PCT application WO 93/19053 and EP 0 634 408 A1 vaten of the cyclodepsipeptide PF 1022A of the general formula (Ib), preferably from the nitrobenzyl derivative and sets these compounds using known ones Methods such as B. by hydrogenation, in a further reaction step very simply into the well-known aminobenzyl derivative of the cyclodepsipeptide PF 1022A around.

The actual coupling reaction then preferably takes place with the aid of the diazo clutch. The aminobenzyl derivative of the cyclodepsipeptide PF 1022A  first solubilized in a suitable solvent. As a suitable solution water and dilute acids are particularly suitable.

Following the conversion of the amino function into the corresponding slide zonium salt according to methods known from the literature (cf. Houben-Weyl, methods of Organic chemistry, volume X / 3) the carrier protein such. B. BSA or KLH admitted. After an incubation period of 0.1 to 12 hours, preferably 2 to It is neutralized for 4 hours, dialyzed and then lyophilized. The back can then if necessary after the interposition of further cleaning steps and subsequent determination of the degree of substitution (see P. Tijssen in: "Practice and Theory of Enzyme Immunoassays", Burdon, R.H. & Knippenberg, P.H., ed., Elsevier, Oxford, UK 1985) for the actual immunization reaction.

In addition to the diazo coupling mentioned as preferred, alternative Ver drive to be used for coupling the hapten to the carrier molecule such as B. the ship base formation, the active ester method or the mixed type hydride method. In the latter method, the carboxyl function of the Bridge member using acetic anhydride or the carbodiimide Derivatives 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide to the carrier molecule knotted.

For example, if the pontic has a reactive SH group, then alternatively, conjugation of the hapten to the carrier molecule by an oxi dation with free SH groups of the carrier.

The immunization of the donor animals, preferably rabbits (code no .: SA 1100-1102; SA 1003-1005, Fa. Eurogentec, Seraing, Belgium), is carried out by a up to repeated application of the to the high molecular carrier protein for example KLH or BSA, coupled depsipeptide haptens.

The preferred form of administration is injection, which is both intravenous, can be done intraperitoneally or subcutaneously.

However, intravenous injection is particularly preferred.  

In a specific embodiment of the present invention, the KLH-PF 1022A derivative and BSA-PF 1022A derivative conjugates (complexes) for Immunization of several donor animals, especially three rabbits, per Carrier-hapten complex used. The first boost immunization takes place 14 days after the first vaccination. Further boost immunizations, in particular another 10 boost immunizations, then follow every 4 weeks. From the third boost immunization, this is invented every 10 days Serum according to the invention obtained.

The success of the immunization can be determined by determining the concentration of specific antibodies with an enzyme known per se to the person skilled in the art coupled immunoassay (ELISA "Enzyme Linked Immuno Sorbent Assay") or control by Western blot analysis. Polyclonal antisera used in the Western blot analysis producing good signals are essential for "immune screening" usable from cDNA libraries.

Particularly preferred for determining the specificity of the antiserum against Depsipeptides, especially against PF 1022A derivative, is Western blot analysis.

The carrier-hapten complexes, in particular the KLH-PF 1022A- Derivative complex and the carrier protein KLH or the BSA-PF 1022A derivative Complex and the carrier protein BSA in an SDS-PAGE (SDS-polyacrylic amide gel) (see Laemmli et al., Nature 227, 1970, pp. 680-685) and the obtained proteins then blotted onto nitrocellulose. Alternatively be the carrier-hapten complexes or the carrier proteins also by means of a "mini fold S "apparatus (Schleicher & Schuell, Dassel) directly onto nitrocellulose filters blotted. Incubation of the filters with suitable serum dilutions is preferred in a ratio of 1: 1,000, very particularly preferably in a ratio of 1: 500 a significantly stronger detection reaction with the carrier Hapten complexes as with the carrier proteins KLH or BSA (use of alkaline NBT / B CIP solution; see. Manufacturing examples).

The specific polyclonal antibodies thus obtained then become in immunological processes, such as the one according to the invention "Immune screening", for the isolation and characterization of cDNA sequences used, which code for proteins, in different kinds of nematode species occur and for the binding of cyclic or open-chain depsipeptides  as ligands, consisting of amino acids and hydroxycarboxylic acids as building blocks, especially the cyclic depsipeptide PF 1022A, are responsible.

In addition, the use of the anti according to the invention is of course body in all known immunoassays, such as. B. immunoradiometric tests after labeling the tracer antibody with chloramine T or Bolton-Hunter-Rea genz, as well as other competitive and non-competitive binding assays, such as Fluorescence, enzyme (ELISA), chemiluminescence or other immunoassays, including all forms of radioimmunoassay (HA) possible. (Harlow, E .: Lane, i.e. (1988) Antibodies: A Laboratory Manual, CSH; 2nd Edn .; New York; 319 to 359; 353 to 612). It is irrelevant whether the layer antibody is on Polystyrene tubes, polystyrene beads, paramagnetic particles or activated Column materials of all types are bound covalently or non-covalently.

Within the scope of the invention, to represent a statistical, represent tative cDNA library polyadenylated RNA fractions resulting from parasitic Nematodes according to the Chirgwin et al. described guanidinium isothiocya nat / cesium chloride cushion method (see Biochemistry 18, 1979, pp. 5294 to 5299) are isolable, used for cDNA synthesis.

Within the scope of the present invention, a statistically representative cDNA library from polyadenylated RNA fractions parasitic nematodes of the order of the Strongylida, especially Haemonchus spp. or Trichostrongyius spp., used for cDNA synthesis.

The synthesis is carried out according to the known methods of "Great Length cDNA Synthesis Kit "from Clontech (Heidelberg).

The first strand synthesis is carried out using suitable primers, in particular an oligo (dT) ₂₅ (dN) primer. EcoRI adapters are ligated to the double-stranded cDNA and cDNA molecules <500 bp are fractionated using Chromaspin columns (Clontech).

After ligation of the cDNA into the EcoRI site of the vector ZAPII (Stra tagene, Heidelberg) the constructs are packaged in vitro (Gigapack Gold II, Stratagene).  

In a specific embodiment of the present invention, the Antisera obtained above against the carrier-hapten complexes, in particular but the KLH-PF 1022A derivative complex, now in "immune screening" used.

After removing the Anti-E. coli antibodies from the named Antisera by incubation with filter-bound E. coli lysate, approx. 5,000 "plaque forming units "(pfu) of the library per assay plated on LB agar and so long Incubate at + 37 ° C until plaques become visible. Then there is one Expression induction of the fusion proteins by means of an inductor saturated Nitrocellulose filter.

The inductor isopropyl-β-thiogalactopyrano is preferably used for this sid (LPTG).

After a further incubation period of 1 to 24 hours, preferably 4 to 12 Hours at temperatures between + 40 ° C and + 45 ° C, preferably at + 42 ° C, the filters with the carrier-hapten complexes, especially the KLH-PF 1022A complex, incubated.

The incubation period with the carrier-hapten complexes, especially the KLH PF 1022A complex, is 1 to 12 hours. The incubation takes place at Temperatures between +5 and + 45 ° C, but preferably at room temperature. After that, the filters are left for 4 hours at room temperature with the antiserum incubated. The antiserum is diluted from 1: 200 to 1: 2,000, preferably from 1: 500 to 1: 1,000.

Following the detection of the bound antibodies, for example with alkaline phosphatase-coupled anti-rabbit antibodies and NBT / BCIP Substrate solution, positive agar areas are separated and an eluent is carried out phages.

The phages obtained in this way are subjected to a "rescreening". The isolated pure phage clones can then be excised in vivo Plasmid clones are converted, and the plasmid DNA can be used for further DNA analysis be used.  

Both strands of the cDNA according to the invention are repeated several times over automatically Laser fluorescence sequencing is analyzed and the sequence data with a Software from Genetics Computer Group Inc. (Madison, WI, USA) examined.

In this way, according to the invention, two cloned cDNA's with the Obtained designation pHC-1 or pHC-2.

The ID No. in the sequence listing 1 and 3 shown in the invention Sequences each contain an "open reading frame" for a protein that the Active ingredient PF 1022A and other active ingredients with similar immunulogical Recognizes binding properties.

The first cloned cDNA called pHC-2 has 972 bases of the im Sequence listing ID No. 1 shown sequence.

The non-coding 5 'section of the pHC-2 cDNA according to the invention consists only from 10 bases. The size of the com complementary RNA (1.05 kb) agrees very well with the length of the cDNA.

For the pHC-2 cDNA sequence with 972 bases (see sequence listing ID No. 1) the first nucleotide sequence ATG at sequence position 11 represents the start codon For example, this initiation site of translation is of the Kozak type (see M. Kozak, J. Cell Biol. 108, 1989, p. 229). In addition, an N- terminal signal peptide with the cleavage site between 18 and 19 detectable (cf. by Heinje, Nucleic Acids Research 14, 1986, pp. 4,683 to 4,690).

From the nucleotide sequence of the pHC-2 cDNA with 972 bases according to the invention the ID No. in the sequence listing 1 sequence of the gastrointestinal nema shown death Haemonchus contortus, the corresponding amino acid sequence for derive the protein. The first protein of the invention that recognizes PF 1022A is characterized in that it has a sequence of 207 amino acids with a resulting molecular weight of 23,991 g / mol (see sequence protocol ID No. 2).

The first protein according to the invention is an integral membrane protein. It owns in addition to the signal peptide sequence, a potential transmembrane region (position 174-190).  

A glycosylation site is local to asparagine (Asn) at position 167 sizable. Glycosylations of the protein according to the invention are preferred this position 167 instead.

In addition, sequence protocol ID No.2 of the target protein according to the invention three potential antigenic determinants, in Derive the range of sections 69 to 75, 157 to 162 and 159 to 164.

These are determinants of the amino acid sequence of the protein of the invention preferred for the production of monoclonal antibodies against the protein from Bedeu tung.

The first protein according to the invention, which recognizes the depsipeptide PF 1022A, shows similarities to different proteins from different organisms (Swiss-Prot database with 43,470 protein sequences). 'Best Fit' analyzes show an identity of 28% or a similarity of 53% to the 25 kDa Glyco protein GP25L from Canis familiaris (EMBL Accession No. P27869). GP25L is part of a heterotetrameric Ca ²⁺ binding protein complex of the endoplasmic reticulum (ER). This complex is apparently of importance for the retention of ER-resident proteins (Wada et al., J. Biol. Chem. 266, 1991, pp. 19,599 to 19,610).

The Xenopus laevis cDNA clone X1262 (EMBL Accession No .: X90517) encodes also a GP25L-like protein (Joost et al., Biochem. J. 312, 1995, p. 205 to 213), which invented an identity of 56% or similarity of 73% has target protein according to the invention.

A human EST ("expressed sequence tag" cDNA fragment; EMBL accession No: T32283) also encodes a GP25L-like amino acid fragment that an identity of 71% or similarity of 83% to the invention Has protein.

The protein also shows an identity of 24% or a similarity of 52% to the 23 kDa Saccharomyces cerevisiae precursor protein P24B (EMBL Accession No: P32803). This protein is probably in the process of transporting involved some secretory proteins from the ER to the Golgi complex (Schimmöller et al. EMBO J. 14, 1995, pp. 1,329 to 1,339).  

Similarities of the GP25L precursor protein or GP25L-like proteins to the protein according to the invention are the signal sequence for the transport into the ER, the transmembrane domain, an N-glycosylation site and the like Molar mass.

By Northern blot analysis for the second cDNA clone pHC-1 corresponding mRNA was determined to be approximately 3.5 kb in size. The one about that Clone pHC-1 isolated from immune screening is 2 kb in size. The still missing 5 'region of the mRNA was isolated using the 5'-RACE methodology [M. A. Frohmann (1990) PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D.H. Gelfand, J.J. Sninsky & T.J. White eds.) Pp. 28-38, Academic Press, San Diego, USA]. The mRNA characterized in this way from several cDNA fragments consists of 3,539 bases of the sequence no. 3 sequence shown together.

The 5 'untranslated section of the mRNA (see sequence listing ID No. 3) comprises 98 bases. There may still be some bases at the 5 'end of the mRNA absence. The 3 'non-translated section (without poly (A) tail) consists of 462 Bases. The probable Kozak-type start codon is at the sequence position 99, although there are two more ATG codons upstream. These divorce but as the initiation point for translation for the following reasons: The ATG on Sequence position 22 is not of the Kozak type and that of this position deduced antino acid sequence already breaks with a TAA stop at position 55 from. The ATG at sequence position 75 is also not of the Kozak type and that amino acid sequence deduced from this position breaks with a TAA stop already at position 90. Starting from position 99 starting from position 99 is the second one Target protein according to the invention which recognizes PF 1022A, characterized in that that there is a sequence of 986 amino acids with a molecular weight of 109,740 g / mol (see Sequence Listing ID No. 4). This also shows inventions protein according to the invention has a pI of 6.13.

The second protein according to the invention is also an integral membrane protein. In addition to an amino terminal signal peptide (cleavage point between the Amino acids 18 and 19) five transmembrane domains (amino acid sections: 531 to 562, 595 to 615, 673 to 700, 725 to 744 and 750 to 774). Potential N- Glycosylation sites are each on the asparagine (Asn) in position 26, in position 499 and can be located in position 862.  

In addition, sequence protocol ID no. 4th of the second target protein according to the invention three potential antigenic determi names in the range of amino acid sections 150 to 156, 428 to 434 and 799 to Derive 804. These determinants of the amino acid sequence of the second invent Proteins according to the invention are preferred for generating monoclonal antibodies against the protein of importance.

A section of 20 threonine is particularly striking in the amino terminal area (Thr) residues interrupted by a serine (Ser) residue (amino acid position 128 to 147), which contains the carboxy-terminal part of the protein according to the invention Strikingly many proline (pro) residues (positions 915 to 926: extremely rich in proline Section). However, the target protein according to the invention is not noticeable high proline content. Proline-rich sections can be 'targets' for protein Protein interactions or as a 'spacer' between functional domains act (see e.g. Yu et al., Cell Vol. 78, 1994, pp. 933-945; Linial, Neuro report Vol. 5, 1994, pp. 2009-2015).

The cDNA section which codes for the second protein according to the invention showed when screening the EMBL database between bases 1790 to 2460, which in the Sequence listing ID No. 4 sequence shown, a 60% homology to one Genome section of the free-living nematode C. elegans (accession no .: Z54306, Wilson et al., Nature Vol. 368, 1994, pp. 32-38).

The similar C. elegans genome section is an exon of a gene whose dedu graced gene product is said to be similar to G protein-coupled receptors. Further Information about this gene or gene product does not exist because the published light sequence is the product of the C. elegans genome project (see Wilson et al., Nature Vol. 368, 1994, pp. 32-38). The 'Best Fit' analysis shows a 49% identity tity or 65% similarity of this deduced protein to the H. contortus Gene product. The C. elegans protein also has in the amino terminal region a threonine (Thr) section of 15 residues, also from a serine (Ser) rest is interrupted.

The 'best fit' analysis of the second protein according to the invention compared to different G protein-coupled receptors shows the following result: Pig precursor protein of the calcitonin receptor (SwissProt accession no .: P25 117) there is an identity of 22% or similarity of 44%; to gastrin / Rat cholecystokinin type B receptor (accession no: P30553) has one  Identity of 20% or a similarity of 45% and to Neuromedin K Rat receptor (accession no .: P16177) consists of 18% identity or 46% Similarity.

Other proteins that have threonine (Thr) -rich sections (from the SwissProt database), e.g. B. Integument Mucin from Xenopus laevis (accession no: P10667, Q05049), Fasciclin II from Drosophila melanogaster (P34083), intestinal mouse alkaline phosphatase (P24822) or the saliva protein SGS-3 Drosophila melanogaster (P13729) show - apart from the common Threonine (Thr) section - no other significant similarities to the invented protein according to the invention.

The two proteins according to the invention (cf. sequence listing ID No. 2 and 4) as new prototypes of specific proteins of parasitic nematodes, in particular in nematodes from the order of the Strongylida, such as, for example Haemonchus spp.

With the aid of the cDNA sequences according to the invention (cf. sequence protocols ID No. 1 and 3) it is possible, for example, to produce cell lines that each permanently express the proteins according to the invention. To do this, use the to known genetic engineering methods that code for these proteins cDNA sequences each inserted into a suitable vector with which one suitable cell line, which has not yet expressed the respective protein becomes. As a result, cell lines can be obtained which are the first and second respectively protein according to the invention (cf. sequence listing ID No. 2 and 4) constant expri lubricate.

For example, the proteins according to the invention can be expressed in a prokaryo table or eukaryotic expression system in a conventional manner be.

Suitable prokaryotic expression systems are all known hosts. Vector systems such as bacteria of the species Streptomyces, spp. Bacillus subtilis, Salmonella typhimurium or Serratia marcescens, especially E. coli, in which the expression of a controlled inducible promoter recombinant DNA is possible.  

Natural, hybrid or bacteriophage promoters come into consideration as promoters for expression in E. coli. It is preferred to use promoters or operators from the group lac-, lacuv5-, trp-, tac-, trc-, rac-, phoA-, P _L or P _R of phage λ, hsp, omp or synthetic promoters such as those used for example in DE-OS 34 30 683 and in EP-A 0 173 149 are proposed. It is advantageous for. B. the tac promoter operator sequence, which is now commercially available (z. B. Expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, p. 63).

Baculo is a particularly suitable eukaryotic expression system to name virus system.

Preferably, one is used to express the proteins of the invention suitable eukaryotic expression system, in particular the baculovirus system, in which post-translational modifications are made.

When expressing the proteins according to the invention, it can be useful prove to change individual codons. The targeted exchange of Bases in the coding region also make sense if the codons used in the proteins differ from the codon usage in heterologous express sion system to ensure an optimal synthesis of the protein. In addition deletions of non-translated 5 'or 3' sections are often useful, at for example if several destabilizing sequence motifs ATTTA in the 3 'range the cDNA are available. Then they should be in the preferred expression in Eukaryotes can be deleted.

Changes of this kind are deletions, additions or exchange of bases and also the subject of the present invention.

With the specific antibodies against the proteins according to the invention for example, by Western blot analyzes homologous or cross-reactive Proteins from other parasitic nematodes can be detected.

With the help of the proteins according to the invention and their homologues new ones can be obtained specific active substances against nematodes can be found.  

In particular, the protein according to the invention can be based on the potential recipe Gate properties for binding open-chain or cyclic depsipeptides as Ligands consisting of amino acids and hydroxycarboxylic acids as building blocks, in suitable test kits for screening for nematicidal activity of these compounds be used.

The active ingredients that are found with the help of the proteins according to the invention are suitable for combating pathogenic endoparasites, which occur in humans and in animal husbandry and animal breeding in useful, breeding, zoo, laboratory, experimental and hobby animals, with favorable warm-blooded toxicity . They are effective against all or individual stages of development of the pests and against resistant and normally sensitive species. By combating the pathogenic endoparasites, disease, deaths and reduced performance (e.g. in the production of meat, milk, wool, hides, eggs, etc.) are to be reduced, so that the use of the active compounds makes animal husbandry more economical and more professional is possible. Pathogenic endoparasites include nematodes. Particularly worth mentioning:
From the order of the Enoplida z. For example: Trichuris spp., Capillaria spp., Tri chomosoides spp., Trichinella spp.
From the order of the Rhabditia z. E.g. Micronema spp., Strongyloides spp. .
From the order of Strongylida z. For example: Strongylus spp., Triodontophorus spp., Oesophagodontus spp., Trichonema spp., Gyalocephalus spp., Cylindropharynx spp., Poteriostomum spp., Cyclococercus spp., Cylicostephanus spp., Oesopha gostabertia spp., .), Ancylostoma spp., Uncinaria spp., Bunostomum spp., Globocephalus spp., Syngamus spp.), Cyathostoma spp., Metastrongylus spp., Dictyocaulus spp., Muellerius spp., Protostrongylus spp., Neostrongylus ., Pneumostrongylus spp., Spicocaulus spp., Elaphostrongylus spp., Parelaphostrongylus spp., Crenosoma spp., Paracrenosoma spp., Angiostrongylus spp., Aelurostrongylus spp., Filaroides spp., Parafilaruschpp., Parafilaropp. , Ostertagia spp., Marshallagia spp., Cooperia spp., Nematodirus spp., Hyostrongylus spp., Obeliscoides spp., Amidostomum spp., Ollulanus spp.
From the order of the Oxyurida z. For example: Oxyuris spp., Enterobius spp., Passalurus spp., Syphacia spp., Aspiculuris spp., Heterakls spp.
From the order of Ascaridia z. For example: Ascaris spp., Toxascaris spp., Toxocara spp., Parascaris spp., Anisakls spp., Ascaridia spp.
From the order of the Spirurida z. E.g .: Gnathostoma spp., Physaloptera spp., Thelazia spp., Gongylonema spp., Habronema spp., Parabronema spp., Draschia spp., Dracunculus spp.
From the order of the Filariida z. For example: Stephanofilaria spp., Parafilaria spp., Setaria spp., Loa spp., Dirofilaria spp., Litomosoides spp., Brugia spp., Wuchereria spp., Onchocerca spp.
From the order of the Gigantorhynchida z. For example: Filicollis spp., Moniliformis spp., Macracanthorhynchus spp., Prosthenorchis spp.

The livestock and breeding animals include mammals such as B. cattle, horses, sheep, Pigs, goats, camels, water buffalos, donkeys, rabbits, fallow deer, reindeer, Fur animals such as B. mink, chinchilla, raccoon, birds such. B. chickens, geese, Turkeys, ducks, fresh and salt water fish such as B. trout, carp and eels.

Laboratory and experimental animals include mice, rats, guinea pigs, gold hamsters, dogs and cats.

The pets include dogs and cats.

The application can be prophylactic as well as therapeutic.

The active ingredients are used directly or in the form of suitable accessories Horse riding enterally, parenterally, dermally or nasally.

Antibodies, in particular monoclonal antibodies, which have nematicidal activity own and can be used as nematicidal active ingredients.  

In particular, the three antigenic determinants of the first invention according to protein (ID No. 2), sections 69 to 75, 157 to 162 and 159 to 164 serve for the generation of monoclonal antibodies.

Furthermore, in particular the three antigenic determinants of the invention according to protein (ID No. 4), sections 150 to 156, 428 to 434 and 799 to 804 for the production of monoclonal antibodies.

example 1 Preparation of the KLH-PF 1022A and BSA-PF 1022A derivative conjugates Example 2

200 mg of cyclo- (MeLeu-D-4-NH ₂ -PhLac-MeLeu-D-Lac-MeLeu-D-4-NH ₂ -PhLac- MeLeu-D-Lac-) are added in 4.39 ml of ice-cold 0.1 N dissolved hydrochloric acid and mixed at 0 ° C with 3.27 ml of a 0.05 M sodium nitrite solution. A solution of 200 mg BSA or KLH with 91.5 mg NaCl in 10 ml 0.5 M sodium hydroxide solution is also added dropwise at 0 ° C. The mixture is stirred for a further two hours at 0 ° C. and neutralized with concentrated hydrochloric acid. It is then dialyzed and lyophilized. The residue is then taken up in water and washed with methylene chloride. After the aqueous phase has been separated off, it is lyophilized again. The mostly powdery KLH-PF 1022A derivative and BSA-PF 1022A derivative conjugates can be used for subsequent immunization experiments without further purification.

Production of antiserum against PF 1022A

The KLH-PF 1022A derivative and BSA-PF 1022A derivative conjugates (complexes) were used to immunize rabbits (3 animals per carrier hapten Complex). The first boost immunizations occurred 14 days after the initial ammunition. A total of 10 more were every 4 weeks Boost immunizations performed. From the third boost immunization was on Approximately 20 ml of blood is drawn after 10 days for serum extraction. The animals were completely bled 24 days after the last immunization.

The specificity of the antiserum against the PF 1022A derivative was determined by Western blot analysis checked. The KLH-PF 1022A derivative complex and the carrier Protein KLH were used on a "slot blot" apparatus (Minifold S, Schleicher & Schuell, Dassel) in equal amounts (20 µg per "slot") of nitrocellulose filter bound. The filters were run at a 1: 500 dilution of the antiserum for four Incubated for hours at room temperature. Detection of bound antibodies was carried out with alkaline phosphatase-conjugated anti-rabbit antibodies (Sigma, Deisenhofen) and an alkaline nitro blue tetrazolium / 5-bromo-3-  chloro-3-indolyl phosphate substrate solution (NBT / BCIP). The KLH-PF 1022A-Kom plex reacted much more strongly with the antiserum than KLH.

Example 3 Identification and characterization of the cDNA a) Construction of the cDNA library

Extraction of total RNA from the gastrointestinal nematode Haemon chus contortus was performed according to the Chirgwin et al. described guani dinium isothiocyanate / cesium chloride cushion method (Biochemistry 18, 1979, pp. 5,294 to 5,299). The polyadenylated RNA fraction was turned on finally purified via oligo (dT) chromatography (Aviv & Leder, Proc. Natl. Acad. Sci., USA, 69, 1972, pp. 1,408 to 1,412).

To construct a statistically representative cDNA library, 5 µg of the polyadenylated RNA fraction were used for the cDNA synthesis. The synthesis followed the regulations of the "Great Lenght cDNA Synthesis Kit" from Clontech (Heidelberg). The first strand synthesis was carried out with an Oligo (dT) ₂₅ (dN) primer. EcoRI adapters were ligated to the double-stranded cDNA and cDNA molecules <500 bp were fractionated using Chromaspin columns (Clontech). After ligation of the cDNA into the EcoRI site of the vector λZAPII (Stratagene, Heidelberg), the constructs were packaged in vitro (Gigapack Gold II, Stratagene).

b) Immuno-screening of the cDNA library

The antiserum against the KLH-PF 1022A derivative complex was used for the Immune screening used. To the anti-E. coli antibodies from the anti To remove serum was diluted 1: 100 antiserum for four hours incubated at room temperature with filter-bound E. coli lysate. Approx. 5,000 "plaque forming units" (pfu) from the library were assayed LB agar plated and incubated at 37 ° C until plaques are visible were. It then became saturated with isopropyl-β-thiogalacto-pyranoside The nitrocellulose filter placed on the plate to allow expression of the fusion  to induce proteins. After an incubation period of four to 12 hours at 42 ° C the filter was removed again and with the KLH-PF 1022A- Incu conjugate (20 mg / ml) for at least one hour at room temperature beer. The filters were then diluted with antiserum (1: 500 to 1: 1,000 dilution incubation) for four hours at room temperature. Bound antibody per were anti-rabbit anti conjugated with alkaline phosphatase bodies and NBT / BCIP substrate solution detected.

Positive agar areas were cut out (20 to 30 different Plaques) and the eluted phages were subjected to a "rescreening" fen. The pure phage clones which were ultimately isolated were analyzed in vitro Excision converted to plasmid clones and the plasmid DNA for further Analyzes used.

c) DNA analysis

The clone was analyzed using automated laser fluorescence Sequencing (A.L.F.-DNA Sequencer, Pharmacia Freiburg, or Licor 4000, MWG, Ebersberg) using "Auto Read Sequencing" System (Pharmacia) or the "Thermo-Sequenase Fluorescence" sequence decorative kit (Amersham, Braunschweig). Both strands of the invention cDNA were sequenced several times. Sequence data were created using GCG Software (Genetics Computer Group Inc., Madison, WI, USA) analyzed. The protein or DNA libraries were used the EMBL (Heidelberg).

d) Glyoxal-denatured poly-A⁺-RNA (10 µg / lane) was separated in agarose gels and transferred to a Hybond-N membrane (Amersham, Braunschweig) (Chomczynski, Anal. Biochem. 201, 1992, pp. 134 bis 139). The filter was hybridized overnight at 65 ° C. in 6 × SSC, 5 × Denhardt's reagent with radioactively labeled plasmid DNA (“random priming” with 50 μCi [α- ³² P] dCTP). The membranes were then washed with high stringency in 0.2 × SSC, 0.5% SDS at 65 ° C. (Sambrock et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) .

Using the techniques described in the present application two mRNA species are cloned from nematodes and fully sequenced (see Sequence Listing ID) No. 1 and 3, Appendix).

The first cloned cDNA called pHC-2 comprises 972 bases and encodes a protein with 207 amino acids (see Sequence Listing ID No. 2).

The second mRNA characterized from several cDNA fragments (pHC-1) sets is composed of 3,539 bases and codes for a protein with 986 amino acids (see sequence listing ID No. 4).

The following sequence listing shows:
Seq. ID No. 1 shows the nucleotide sequence of the cDNA (pHC-2) according to the invention from the gastrointestinal nematode Haemonchus contortus. The cDNA shown comprises 972 bases, which for 207 amino acids of the protein according to Seq. ID) No. 2 code. Start of coding for Protein ID No. 2 is position 11.

Seq. ID No. 2 which results from the nucleotide sequence Seq. ID No. 1 derived amino acid sequence of the first protein from the gastrointestinal nematode Haemonchus contortus. In this sequence lie on the positions

• a) 1-18 NH ₂ -terminal signal peptide, cleavage site between amino acids 18 and 19,
• b) 174-190 a transmembrane region,
• c) 69-75 an antigenic determinant,
• d) 157-162 an antigenic determinant,
• e) 159-164 an antigenic determinant,
• f) 167 a site for N-glycosylation.

Seq. ID No. 3 the nucleotide sequence of the cDNA (pHC-1) according to the invention the gastrointestinal nematode Haemonchus contortus. The one out of several cDNAs characterized mRNA consists of 3,539 bases, which for 986 amino acids of the protein according to Seq. ID No. 4 code. Start coding for protein ID No. 4 is position 99.  

Seq. ID No. 4 which results from the nucleotide sequence Seq. ID No. 3 derived amino acid sequence of the second protein from the gastrointestinal nematode Haemonchus contortus. In this sequence lie on the positions

• a) 1-18 NH ₂ -terminal signal peptide, cleavage site between amino acids 18 and 19,
• b) 531-562 first transmembrane domain,
• c) 595-615 second transmembrane domain,
• d) 673-700 third transmembrane domain,
• e) 725-744 fourth transmembrane domain,
• f) 750-774 fifth transmembrane domain,
• g) 26 a site for N-glycosylations,
• h) 499 a site for N-glycosylations,
• i) 862 a site for N-glycosylations,
• j) 150-156 an antigenic determinant,
• k) 428-434 an antigenic determinant,
• l) 799-804 an antigenic determinant.

Fig. 1 shows the nucleotide sequence from Seq ID 1 and assigned the amino acids of the protein from Seq ID 2.

Fig. 2 shows the nucleotide sequence from Seq ID 3 and assigned the amino acids of the protein from Seq ID 4.

Fig. 1 Translation of the nucleic acid sequence (pHC-2 DNA)

Claims (22)

1. Proteins, as well as sections of these proteins that are used against parasitic Nematode-acting substances from the group of cyclic or open chain depsipeptides consisting of amino acids and hydroxycarboxylic acids exist, react. 2. DNA sequences for proteins or for parts of these proteins code out the substances acting against parasitic nematodes the group of cyclic or open-chain depsipeptides, which from amino acids and hydroxycarboxylic acids exist, react. 3. Proteins or their fragments that are used against parasitic nematodes acting substances from the group of cyclic or open-chain Depsi peptides consisting of amino acids and hydroxycarboxylic acids react ren, for the identification of substances with action against parasitizing Nematodes from the group of cyclic or open-chain depsipeptides, which consist of amino acids and hydroxycarboxylic acids. 4. Method of identifying the proteins and their fragments associated with against parasitic nematode substances from the group of cyclic or open chain depsipeptides consisting of amino acids and Hydroxycarboxylic acids exist, react, as well as the production of the Pro teine or its sections on recombinant technology. 5. Protein that is made up of substances acting against parasitic nematodes the group of cyclic or open-chain depsipeptides, which from amino Acids and hydroxycarboxylic acids exist, react, isolated from a para sitinary nematode species, characterized in that it is a sequence of 207 amino acids and has a molecular weight of 23,991 g / mol owns. 6. Protein according to claim 5, characterized in that it is the Amino acid sequence according to sequence listing ID Seq No. 2 has. 7. Protein according to claim 1, characterized in that it has a transmembrane region, an N-glycosylation site, three determinants and a signal peptide which the following positions and sequences of the protein in sequence listing ID No. 2 correspond to:

• a) 1-18 NH ₂ -terminal signal peptide,
• b) 174-190 a transmembrane region,
• c) 69-75 an antigenic determinant,
• d) 157-162 an antigenic determinant,
• e) 159-164 an antigenic determinant,
• f) 167 a site for N-glycosylation.

8. Protein that is made up of substances acting against parasitic nematodes the group of cyclic or open chain depsipeptides consisting of Amino acids and hydroxycarboxylic acids consist of, react, isolated a parasitic nematode species, characterized in that it is a Sequence of 986 amino acids and has a molecular weight of Has 109,740 g / mol. 9. Protein according to claim 8, characterized in that it is the amino acid sequence according to the sequence listing ID Seq. No. 4 has. 10. Protein according to claim 1, characterized in that it has five transmembrane regions, three N-glycosylation sites, three determinants and a signal peptide which the following positions and sequences of the protein in sequence listing ID No. 4 correspond to:

• a) 1-18 NH ₂ -terminal signal peptide,
• b) 531-562 first transmembrane domain
• c) 595-615 second transmembrane domain
• d) 673-700 third transmembrane domain
• e) 725-744 fourth transmembrane domain
• f) 750-774 fifth transmembrane domain
• g) 26 a site for N-glycosylation
• h) 499 a site for N-glycosylation
• i) 862 a site for N-glycosylation
• j) 150-156 an antigenic determinant
• k) 428-434 an antigenic determinant
• l) 799-804 an antigenic determinant.

11. Use of the protein according to claim 1 for distinguishing nematicidal cyclic or open chain depsipeptides, best Based on amino acids and hydroxycarboxylic acids as building blocks. 12. Test systems using the proteins according to claim 1 for Identification of nematicidal cyclic or open chain Depsipeptides consisting of amino acids and hydroxycarboxylic acids as Building blocks. 13. complexes consisting of a cyclic or open-chain depsipeptide, which consists of amino acids or hydroxycarboxylic acids and suitable Ligands to generate antibodies against these depsipeptides. 14. Polyclonal or monoclonal antibodies against cyclic or open chain Depsipeptides consisting of amino acids and hydroxycarboxylic acids as Building blocks produced using the complexes according to claim 10th 15. Use of the antibodies according to claim 11 as nematicidal active ingredients. 16. DNA sequence according to claim 2, characterized in that the entire cDNA the sequence according to sequence listing ID No. 1 has. 17. DNA sequence according to claim 2, characterized in that the entire cDNA the sequence according to sequence listing ID No. 3 has. 18. Recombinantly produced DNA, which is for the entire amino acid sequence, Amino acid partial sequences, analogs, homologs, derivatives or from them resulting combinations of the amino acid sequence of the proteins according to Claim 1, encoded and optionally in a suitable expression system is installed. 19. A method for producing antibodies according to claim 14, characterized in that

• a) a nematicidal active ingredient from the group of cyclic or open-chain depsipeptides, consisting of amino acids and hydroxycarboxylic acids as building blocks, conjugated as a hapten to a carrier molecule,
• b) a donor animal is immunized with this hapten-carrier conjugate by means of suitable application,
• c) determines the specificity of the antibodies formed,
• d) if appropriate, the concentration of the specific antibodies formed is determined,
• e) the specific antibodies obtained from sera or antibodies producing lymphocytes are separated and, if necessary, purified.

20. The method according to claim 19, characterized in that as Carrier molecule uses macromolecular compounds that are freely accessible Liche reactive groups for the coupling reaction with open chain or have cyclic depsipeptides and are capable of said ver to give bonds an immunogenic potency. 21. The method according to claim 19, characterized in that one lysine-rich protein with a molecular weight between 10,000 and 1,500,000 used. 22. A method according to claim 19, characterized in that one selects a protein from the group consisting of "Bovine Serum Albumin "(BSA)," Keyhole Limpet Protein "(KLP), Human Serum albumin (HSA), "Porcine thyroglobulin", B2 microglobulin, Haemo cyanine, immunoglobulins; Toxins; Polysaccharides; Lipopolysaccharides; natural or synthetic polyadenylic and polyuridylic acids; Polyalanyl and polylysine polypeptides; or cell membrane components used.