Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-05-20
2003-03-18
Navarro, Mark (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S389200, C530S397000, C530S399000, C530S303000, C435S007900, C424S198100
Reexamination Certificate
active
06534634
ABSTRACT:
This application is a national phase of PCT/EP97/05075, filed Sep. 17, 1997, which is based on DE 196 41 378.8, filed Sep. 27, 1996.
The present invention relates to diagnosis aids and processes for detecting pregnancy in ruminants based on the relaxin-like factor detectable in ruminants. Antibodies against the relaxin-like factor of ruminants as well as against fragments and/or active derivatives of the same with the same immunogenicity are also provided.
At present, several immunological processes exist for determining the pregnancy of numerous mammals. Thus it is already known for example that pregnancies in women can be reliably detected by the detection of specific antibodies against the human chorionic gonadotrophin in the urine of the person to be examined, hemagglutination inhibition test, latex agglutionation inhibition tests or radioimmunological detection of &bgr;-HCG being used in particular. In addition it is possible to detect pregnancy in several mammals by determining the yellow body hormone relaxin formed above all in the uterus and placenta during the pregnancy, which very early on during the pregnancy reaches a certain level which by contrast is never exceeded in the normal non-pregnant cycle. It was even shown that a specific increase in the relaxin in the peripheral serum takes place during a very early stage, namely even before the nidation of the blastocytes and even before a clear positive HCG test (D. R. Stewart et al., Relaxin in the peri-implantation period, J.Clin. Endocrinol. Metab., 70: 1771-1773 (1990)). Consequently, it has been proposed to provide alternative pregnancy tests on the basis of detecting relaxin. For example, processes are described in U.S. Pat. No. 5,108,897 for determining the pregnancy of bitches, with which the relaxin values detectable in certain body fluids or tissues of the animals in the case of pregnancy are measured.
Alongside relaxin, another peptide hormone, called relaxin-like factor (abbreviation: RLF) has recently been identified (E. Bullesbach et al., A novel Leydig cell cDNA-derived protein is a relaxin-like factor, J. Biol. Chem., 270: 16011-16015 (1995)). While the function of regulating the physiology of female animals of most mammal types is essentially attributed to relaxin, RLF seems to be expressed exclusively in the Leydig cells of the male gonads in most types according to current knowledge, as substantial amounts of specific mRNA were detected in this tissue (W. Pusch et al., Molecular cloning and expression of the relaxin-like factor from the mouse testis, Endocrinology, 137 (1996, printing); I. M. Adham et al., Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells, J. Biol. Chem., 268: 26668-26672 (1993)). By using the reverse transcription polymerase chain reaction (RT-PCR) and succeeding Northern hybridisation, specific signals for RLF-mRNA were however also detected in ovaries and trophoblasts of humans (L. Tashima et al., The human Leydig insulin-like (Ley-I-L) gene is expressed in the corpus luteum and trophoblast, J. Clin. Endocrinol. Metab., 80: 707-710 (1995)).
However, ruminants occupy a special place amongst mammals insofar as, although they show clear signs of a relaxin-dependent physiology in the case of pregnancy, no expression of a relaxin gene has so far been detected (S. Hartung et al., The search for ruminant relaxin, in: A. H. McLennan, G. Tregear and G. D. Bryant-Greenwood (Publ.) “Progress in Relaxin Research”, Singapore, World Scientific Publishing, pp. 439-456 (1995)). A deletion in the relaxin gene has in fact been established in sheep (P. J. Roche et al., A single copy relaxin-like gene sequence is present in sheep, Mol. Cell. Endocrinol., 91: 21-28 (1993)).
As the yellow body hormone relaxin has thus up until now been detected only in non-ruminant species such as humans, horses, cats, pigs, rats, mice, guinea pigs and hamsters, and the pregnancy-dependent gonadotrophin is detectable exclusively in primates and horses, there is a considerable need for diagnostic possibilities for determining pregnancy in other mammal types, such as in particular in ruminants.
The object of the present invention is consequently to provide diagnosis aids and processes as well as suitable substances for same with which a pregnancy can be reliably detected in ruminants and particularly in cattle.
To achieve the object, the diagnosis aid for detecting pregnancy in ruminants, characterized in that it comprises antibodies against the relaxin-like factor of ruminants or fragments of the same with the same immunospecificity, the process for detecting the pregnancy of ruminants, characterized in that body fluids or tissue are removed from the animal to be examined, in which the relaxin-like factor is detectable during the pregnancy of the animal and the presence of the relaxin-like factor is detected in the body fluid or the tissue of the animal. The object of the invention is also to provide antibodies directed against the relaxin-like factor of ruminants as such or against fragments and/or active derivatives of the same with the same immunogenicity; the use of the same for detecting pregnancy in ruminants; and further versions of the diagnosis aid, processes of the using the same and antibodies useful therein are also further described herein.
Within the framework of the research efforts which finally led to the present invention, a relaxin-like factor has now also been detected in female ruminants and particularly in cows, which is expressed in very large quantities in the yellow body (corpus luteum). The expression patterns of the RLF gene of cattle are very similar to those of the human relaxin genes with an increase in the late luteal phase and with continuing expression into the pregnancy, whereby the applicability of the means and processes proposed according to the invention for determining the pregnancy of ruminants and in particular cattle is emphasized.
The RLF protein of pigs is known to have a primary structure which is similar to an A-, B-, C- (connecting) peptide of the primary structure of insulin. All three peptide regions are contained in the RLF precursor protein which is cleaved post-translationally into its components. The A- and B- peptides form a factor present as a heterodimer, while the C-peptide is presumably discarded. The primary sequence of the cattle RLF shown in
FIG. 1
is similar to that of pigs in its primary structure and presumably leads equally to a heterodimeric factor. The RLF-protein contains additionally a signal sequence at the N terminus which is used for the cotranslational introduction of the molecule into the secretory system of the cells and is subsequently cleaved off. As this structure also exists in the RLF-precursor in cattle, it is to be assumed that this protein is also secreted into the blood path and can accordingly be detected.
Within the framework of the invention, the entire coding region of the RLF-gene of the cattle has been cloned and sequenced with the help of the RT-PCR (see Example 1). Furthermore, with the help of the Northern RNA hybridisation as well as the RT-PCR, a large quantity of tissue of the female and male cattle was examined with regard to the expression of the gene (see Example 2). The results show that the RLF of the cattle is expressed exclusively in the Leydig cells of the bull and in the yellow bodies of the cow. The amino acid sequence shown in
FIG. 1
(SEQ ID NO: 2) of the relaxin-like factor of ruminants according to the invention offers the possibility of providing, with the help of established processes, polyclonal and monoclonal antibodies or fragments of the same with the same immunospecificity for use in diagnosis aids and immunological detecting processes. Such antibodies can be prepared on the basis of the complete RLF as well as on the basis of fragments and/or active derivatives of the same with the same immunogenicity.
The term “antibody” used within the framework of the present description relates to a protein which consists of one or more polypeptides which are essentially coded by
IHF Institut fur Hormon- und Fortpflanzungsforschung GmbH
Navarro Mark
Portner Ginny Allen
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