Growth hormone potentiating molecules

Antigenic molecules cause antibodies to be raised against at least some of the 91 to 102 region of a natural growth hormone when parenterally administered in vivo. The molecules can include portions of the 91 to 102 region, particularly the 94 to 98 region. The antibodies are believed to bind to growth hormone on administration and enhance its effect.

The present invention relates to biologically active molecules, 
particularly peptides. More particularly, the invention relates to peptide 
fragments of growth hormone (including bovine growth hormone (bGH), 
porcine growth hormone (pGH), chicken growth hormone (cGH), ovine growth 
hormone (oGH) and their mutant derivatives), which are rendered antigenic 
and which can enhance or promote growth hormone activity. Human growth 
hormone (hGH), rat growth hormone (rGH), mouse growth hormone (mGH), horse 
growth hormone (eGH) and salmon growth hormone (sGH) are other growth 
hormones of interest. 
Polypeptide hormones are important for both medical and veterinary 
application. One such hormone, growth hormone, is found in vertebrates and 
is important for promoting somatic growth. Growth hormones from different 
species share both structural and functional characteristics. Growth 
hormones consist of amino acid sequences generally of about 191 residues 
in length. It is known that growth hormone can stimulate somatic growth, 
promote wool growth in sheep, affect body composition, improve food 
efficiency and promote lactation in appropriate species. Different aspects 
of the structural and functional characteristics of growth hormones have 
been described (Nicoll et al, Endocrine Rev. 7(2) 169-203 (1986); Isaksson 
et al, Ann. Rev. Physiol. 47 483-499 (1985); Wallis, "Chemistry and 
Biochemistry of Amino Acids, Peptides and Proteins" (Ed. B. Weinstein), 
Vol. 5, pp 213-320 (Dekker, New York (1978)). 
Antibodies to hormones have been shown to be capable of (i) enhancing 
hormone activity, (ii) have no effect on hormone activity or (iii) inhibit 
hormone activity (Thompson, Proc. Soc. Expl. Biol. NY 35 640-644 (1937); 
Rolands, J. Endocrinol. 1 177-183 (1939); Goodfriend et al, J. Clin. 
Endocrinol. Metab. 30 565-572 (1970); Schechter et al, Proc. Natl. Acad. 
Sci. USA 76(6) 2720-2724 (1979); Schechter et al, Nature 278 835-838 
(1979); Cole et al, Biol. Reprod. 12 516-521 (1975); Aston et al, J. 
Endocrinol. 110 381-388 (1986); Aston et al, Molec. Immunol. 24 143-150 
(1987); Ferguson, Nature 174 411 (1954)). More specifically, Aston et al 
1986, 1987 loc. cit. have shown that certain antibodies to growth hormones 
can enhance the biological activity of the hormone in vivo. It was 
concluded in these studies that enhancement of hormone by antibodies is 
characteristic of particular specificities, a property also described 
previously (Cole et al 1975 loc. cit.; Goodfriend et al 1970 loc. cit.). 
However, in none of these studies is a regime available to indicate how to 
generate reproducibly an enhancing antiserum by using a small peptide. 
EP-A-0137234 discloses that the large 7 kiloDalton fragment of growth 
hormone can produce antibodies that enhance growth hormone activity; 
however, this fragment may not always be suited for this purpose due to 
its size and its limited ability to produce a consistent enhancing 
antiserum. Currently, manufacture of such a large polypeptide may be 
problematic by peptide synthetic routes. 
Enhancement of hormonal activity by the administration of a peptide 
fragment of GH has been disclosed in WO-A-8404915. In this particular 
disclosure it was shown that a short peptide derived from the amino 
terminal portion of the GH molecule potentiated hypoglycaemic activity; 
however this peptide was not administered in an immunogenic fashion. Both 
GH and insulin induce hypoglycaemia when administered to animals. The 
insulin-potentiating activity of peptide fragments of GH have also been 
described elsewhere (Pullin et al, Int. J. Peptide Protein Res. 18 318-323 
(1981); Ng. et al, Diabetes 29 782-787 (1980)). 
More recent contributions to the art have included the identification of 
various sequence fragments of natural growth hormones which, when made 
appropriately antigenic (for example by coupling them to a suitable 
carrier) have the capability of enhancing or potentiating the activity of 
endogenous or exogenous growth hormone. For example: WO-A-8807547 
identifies the 35 to 53 region of natural growth hormones; WO-A-8901166 
identifies the 112-159 region in general and the smaller regions 120-140 
and 134-154. more specifically; and EP-A-0303488 identifies the 1-18, 
55-72, 97-110 and 122-138 regions generally but focuses attention on the 
122-138, 119-131, 130-143, 123-137 and 133-146 regions. More recently 
still, Aston et al (Molecular Immunology 28 (1/2) 41-50 (1991)) confirmed 
the significant enhancement of hormonal activity by antibodies raised 
against the 120-140 and 134-154 regions, as well as other regions within 
the overall 112-159 region. 
Significantly, though, Aston et al 1991 report a marked absence of growth 
hormone enhancing activity in antigenic peptides derived from the 
sequences immediately upstream (ie, towards the amino terminus) of the 
112-159 sequence. Specifically, no enhancement at all was observed for the 
80-100 sequence, and only a slight enhancement was seen for the 95-115 
sequence which probably reflects the partial overlap with the 112-159 
sequence. 
It would still be desirable to identify further regions or sequences of 
growth hormones which give rise to antigenic peptides for enhancing growth 
hormone activity. This is the goal to which the present invention is 
addressed. 
The invention is based on the surprising discovery that, in spite of the 
teaching of Aston et al 1991 discussed above, there is a region of growth 
hormone, not far upstream of the 112-159 region, which can form the basis 
of antigenic peptides which give rise to enhancement of growth hormone 
activity. 
According to a first aspect of the invention, there is provided an 
antigenic molecule which causes antibodies to be raised against at least 
some of the 91 to 102 region of a natural growth hormone. This may be 
achieved by providing a molecule having antigenic equivalence to at least 
part of the 91 to 102 region. 
According to a second aspect of the invention, there is provided a molecule 
(other than a natural growth hormone) at least part of which is 
antigenically equivalent to an oligopeptide selected from residues 91 to 
102 of a natural growth hormone. 
The 91 to 102 regions of various natural growth hormones have the following 
amino acid sequences: 
______________________________________ 
(bovine) bGH QFLSRVFTNSLV (SEQ ID NO: 1) 
(human) hGH QFLRSVFANSLV (SEQ ID NO: 2) 
(porcine) pGH QFLSRVFTNSLV (SEQ ID NO: 1) 
(ovine) oGH QFLSRVFTNSLV (SEQ ID NO: 1) 
(equine) eGH QLLSRVFTNSLV (SEQ ID NO: 3) 
(mouse) mGH QFLRSVFANSLV (SEQ ID NO: 2) 
(chicken) cGH QYLSKVFTNNLV (SEQ ID NO: 4) 
(rat) rGH QFLSRIFTNSLV (SEQ ID NO: 5) 
(salmon) sGH QTL-----NSLL (SEQ ID NO: 6) 
______________________________________ 
Bovine, ovine, porcine and chicken growth hormones are preferred. Ovine 
growth hormone is very similar to bGH. 
In the above, and throughout this specification, the amino acid residues 
are designated by the usual IU single letter nomenclature. The single 
letter designations may be correlated with the classical three letter 
designations of amino acid residues as follows: 
______________________________________ 
A = Ala G = Gly M = Met S = Ser 
C = Cys H = His N = Asn T = Thr 
D = Asp I = Ile P = Pro V = Val 
E = Glu K = Lys Q = Gln W = Trp 
F = Phe L = Leu R = Arg Y = Tyr 
______________________________________ 
The oligopeptide will be of at least the minimum size necessary to confer 
epitope specificity: usually it will be of at least six or seven residues, 
but may be of any suitable length up to, for example, 20 amino acid 
residues. The best oligopeptides may be expected to correspond to 
topographical surface features of a natural growth hormone molecule, that 
is to say those features having some three-dimensional feature protruding 
from or extending into the ambient surface level of the hormone. Preferred 
oligopeptides correspond to regions 94-98. 
Probably the most simple way of ensuring that at least part of the molecule 
is antigenically equivalent to the oligopeptide is for that part of the 
molecule to comprise a sequence of amino acid residues which is identical 
to or conformationally similar to the oligopeptide. However, any other way 
of producing antigenic equivalence may be used: an example is to use an 
anti-idiotype antibody or other (even non-proteinaceous) analogue. 
The invention therefore encompasses antigenic molecules including short 
peptides (preferably of less than 10 amino acid residues, but generally of 
at least 4 or 5 amino acid residues, for example 6 to 8 residues) sharing 
structural homology with growth hormone and which when administered to an 
animal can enhance hormone activity. Potentiation of hormone activity (in 
particular growth hormone activity), may occur through direct or indirect 
effects on the hormone in question. Non-antigenic peptides are useful as 
chemical intermediates in the preparation of antigenic peptides and are 
also within the scope of the invention. 
The invention therefore encompasses a peptide other than a natural growth 
hormone comprising the amino acid sequence (SEQ ID NO: 1): 
EQU QFLSRVFTNSLV 
or an active fragment thereof and/or conservative mutant thereof. This 
sequence is taken from oGH, residues 91 to 102, and is identical to pGH 
and bGH. Peptides comprising amino acid sequences corresponding to this 
region from other growth hormones are also within the scope of the 
invention. 
Prior to administration the peptide will generally be rendered antigenic 
and capable of stimulating the production of antibodies which, when in an 
appropriate formulation, potentiate the effect of growth hormone. 
As stated above, an active subfragment of the specified sequence may be 
used. Active subfragments may consist of or include pentapeptides, 
including (in the case of oGH) one or more of: 
______________________________________ 
QFLSR (SEQ ID NO: 7) 
FLSRV (SEQ ID NO: 6) 
LSRVF (SEQ ID NO: 9) 
SRVFT (SEQ ID NO: 10) 
RVFTN (SEQ ID NO: 11) 
VFTNS (SEQ ID NO: 12) 
FTNSL (SEQ ID NO: 13) 
TNSLV. (SEQ ID NO: 14) 
______________________________________ 
Active subfragments may also consist of or include hexapeptides, including 
one or more of: 
______________________________________ 
QFLSRV (SEQ ID NO: 15) 
FLSRVF (SEQ ID NO: 16) 
LSRVFT (SEQ ID NO: 17) 
SRVFTN (SEQ ID NO: 18) 
RVFTNS (SEQ ID NO: 19) 
VFTNSL (SEQ ID NO: 20) 
FTNSLV. (SEQ ID NO: 21) 
______________________________________ 
Active subfragments may alternatively consist of or include heptapeptides, 
including one or more of: 
______________________________________ 
QFLSRVF (SEQ ID NO: 22) 
FLSRVFT (SEQ ID NO: 23) 
LSRVFTN (SEQ ID NO: 24) 
SRVFTNS (SEQ ID NO: 25) 
RVFTNSL (SEQ ID NO: 26) 
VFTNSLV. (SEQ ID NO: 27) 
______________________________________ 
Further, active subfragments may consist of or include octapeptides, 
including: 
______________________________________ 
QFLSRVFT (SEQ ID NO: 28) 
FLSRVFTN (SEQ ID NO: 29) 
LSRVFTNS (SEQ ID NO: 30) 
SRVFTNSL (SEQ ID NO: 31) 
RVFTNSLV. (SEQ ID NO: 32) 
______________________________________ 
Preferred fragments include those containing some, for example at least 
four residues of, or all of the pentapeptide motif SRVFT (SEQ ID NO:10). 
Most preferred fragments contain from residues 94 to 100, which appears to 
be the minimum functional unit for optimal results. In ovine growth 
hormone, this motif comprises SRVFTNS (SEQ ID NO:25). 
Each of the above sequences can be varied to take into account variations 
in the sequence of corresponding regions of growth hormones from other 
species, particularly those previously referred to. Sequences 
corresponding to the preferred oGH 94-100 sequence (SRVFTNS) (SEQ ID 
NO:25) are as follows: 
______________________________________ 
(bovine) bGH 94-100 SRVFTNS (SEQ ID NO: 25) 
(human) hGH 94-100 RSVFANS (SEQ ID NO: 33) 
(porcine) 
pGH 94-100 SRVFTNS (SEQ ID NO: 25) 
(equine) eGH 94-100 SRVFTNS (SEQ ID NO: 25) 
(mouse) mGH 94-100 RSVFANS (SEQ ID NO: 33) 
(chicken) 
cGH 94-100 SKVFTNN (SEQ ID NO: 34) 
(rat) rGH 94-100 SRIFTNS (SEQ ID NO: 35) 
______________________________________ 
It should be noted that combinations of more than one of the above 
sequences may be used. 
Although the use of natural sequences in an antigenic molecule can 
certainly lead to generation of appropriate antibodies, it is also the 
case that a non-natural sequence may give rise to an antibody which binds 
to a given natural sequence. It has been found, as part of the studies 
involved in the present invention, that an antibody which binds the 
preferred sequence SRVFTNS can bind certain modifications of that sequence 
almost as strongly and, in some cases, even more strongly than the natural 
sequence. It is therefore well within the scope of the invention to use 
such modified sequences in epitopes of antigenic molecules. 
Generally, the natural 94-100 sequence and its acceptable variations may be 
set out as follows (SEQ ID NO:36): 
##STR1## 
In the above general formula, the natural sequence is shown as the top 
line, substitute amino acids are shown vertically aligned with the 
corresponding natural amino acid. 
Replacement of S.sub.94 with C, D or E appears to give better binding than 
the wild type sequence in the model system studied. Such replacements are 
therefore preferred over the wild type. D and E are preferable to C. 
Similarly, V.sub.96 is preferably replaced by I as better binding results. 
Although, F.sub.97 may be replaced by W, Y or (less preferably) C, it is 
preferred to keep the natural residue at this position. N.sub.99 is the 
residue of choice at position 99, but H may be substituted. S.sub.100 may 
be replaced by G or, preferably, A, but the natural residue is preferred. 
Bearing the above comments in mind, a subset of the natural 94-100 sequence 
and its preferred variations may be set out as follows (SEQ ID NO:37). 
##STR2## 
of course, more than one substitution may be made. 
According to a further aspect of the invention, a method of promoting the 
activity of growth hormone (or a substance having growth hormone activity) 
comprises administering to a vertebrate an effective amount of an 
antigenic peptide or other molecule as described above. The invention thus 
encompasses the use of a peptide or other molecule as described above in 
the preparation of an agent for use in the promotion of the activity of 
growth hormone or a substance having growth hormone activity. 
Peptides and other molecules in accordance with the invention may be 
rendered antigenic, or presented, in a variety of ways. For preference, an 
antigenic region (such as a peptide fragment or sub-fragment) in a 
molecule in accordance with the invention will contain the amino acid 
sequence of choice linked to a carrier peptide or protein. It is generally 
preferred to have a plurality, for example 5 to 10, copies of a peptide 
sequence (for example one or more of the above sequences) linked to the 
carrier. The carrier can for convenience be a generally large protein, 
which is inert in material respects, and which is derived from a different 
species or genus from that associated with the natural growth hormone. 
Examples of carriers include albumins such as human serum albumin, bovine 
serum albumin and ovalbumin (although not so many peptides will probably 
be able to be carried in this last case). Alternatively, keyhole limpet 
haemocyanin can be used. The carrier will generally preferably come from a 
different species from that on which the fragment is based. 
It is not essential that peptide sequences as described above be linked to 
albumins: they may be linked to other macromolecules, such as 
.beta.-galactosidase, especially of bacterial origin. 
The invention encompasses molecules being peptides or having peptide 
regions which share substantial (eg greater than 30%, 50% or even 70%) 
sequence homology with the above peptides. Similarly, conservative amino 
acid substitutions may not decrease the immunogenicity or antigenicity of 
peptides. Thus antigenically similar homologues will elicit antibody which 
binds to GHs in the same region as the above peptides define. It is well 
known that the use of homologues can be a means of circumventing `self` 
tolerance. Thus the use of the corresponding sequences from other species 
may be advantageous in this invention. Examples of homologous sequences to 
the preferred bovine, porcine and ovine growth hormones are those derived 
from the corresponding sequence regions of equine GH, mouse GH, chicken 
GH, trout or salmon GH or rat GH. 
It is alternatively possible for molecules in accordance with the invention 
which are or which comprise peptides to be or to include polymers of 
sequences as described above. Appropriate sequences can be polymerised 
either by cross-linking of two cysteine residues to form disulphide bonds 
or by using external chemical coupling agents (such as carbodiimide, 
glutaraldehyde or other dialdehydes or di- (or poly-) functional 
carboxylic acids). As a further alternative, recombinant DNA techniques 
could be used to produce a peptide polymer. 
It should be noted that the chemical coupling (which could for example take 
place through the agency of lysine residues) and disulphide bond formation 
are not limited to when the coupling residues are at the end of the 
sequence: internal residues could also be appropriate. Coupling residues, 
for example cysteine residues, may be added as desired. 
It may be found that it is not necessary to couple any of the sequences 
described above with external peptides. 
They may be antigenic on their own. In such a case, it may be advisable to 
select particular adjuvants such as DEAE DEXTRAN adjuvant and MERCK 7426 
adjuvant. 
According to a further aspect of the present invention, there is provided a 
pharmaceutical or veterinary composition comprising an antigenic molecule 
as described above in conjunction with a pharmaceutically or veterinarily 
acceptable carrier. The composition may contain an adjuvant, for example, 
DEAE DEXTRAN adjuvant and MERCK 7426 adjuvant, a muramyl peptide compound 
such as muramyl dipeptide, saponin and aluminium hydrogel. Alternatively 
or in addition Freund's complete adjuvant could be used. As noted above, 
certain adjuvants are more likely to be preferred in particular 
circumstances. 
Compositions in accordance with the invention will normally be sterile, as 
they will be intended for implantation or injection. Intravenous injection 
is not preferred: subcutaneous injection is the route of choice, although 
possibly intramuscular and/or intraperitoneal injection could be used. 
The preferred location of the subcutaneous injection would be at the back 
of the neck in the case of an animal, as that is a place where 
commercially useful meat is less likely to be damaged by tissue breakage 
or bruising. 
The carrier will generally be an isotonic buffer plus is saline, such as 
PBS or physiological saline. 
Dosages will be generally as prescribed in accordance with the directions 
of the physician or veterinary surgeon, but 5 to 500 .mu.g per dose, 
particularly 50 to 100 .mu.g of peptide or other molecule may be found to 
be suitable for bovine or porcine applications. 
A substance having or promoting growth hormone activity can be administered 
shortly after (or may be in some circumstances prior to or at the same 
time as) peptides or other molecules in accordance with the present 
invention. The activity of the growth hormone is then enhanced. This can 
lead to enhanced growth where growth is relevant; improved body 
compositions (for example, in pigs there may be less fat and more muscle 
on the back); better wool growth in sheep; improved growth efficiency 
(that is to say, better growth for a given unit amount fed); and 
enhancement of lactation in cows and sheep. This latter application is not 
only important for providing milk or human consumption, but it may also 
enable sheep to rear their young more effectively. 
Examples of substances promoting growth hormone activity other than growth 
hormones themselves and other than molecules in accordance with this 
invention include antibodies to growth hormone inhibitors and antibodies 
to other growth hormone antagonists, such as antibodies against 
somatostatin or leutinising hormone releasing hormone (LHRH). The 
production of an antibody to somatostatin, for example, would increase 
circulating GH levels and may thus potentiate the effect of molecules in 
accordance with the present invention. Another substance which may be 
regarded as promoting growth hormone activity is growth hormone releasing 
hormone (GRF), which may also be administered. 
In another aspect, the invention provides antibodies raised against, or 
otherwise capable of specifically binding to, molecules of the first 
aspect. Such antibodies may be parenterally administered to animals, 
generally in an appropriate formulation to produce a growth hormone 
potentiating effect. Preferred formulation and administration details may 
be as described above, with changes as appropriate.

EXAMPLE 1 
Epitope Scan of oGH Using MAb (OA15) 
Ovine GH (oGH) was prepared as described previously by Aston et al (Mol. 
Immunol. 24 143-150 (1987)); porcine GH (pGH) was obtained from the USDA 
(batch 20705) pituitary hormone programme. Generation and characterisation 
of MAbs to oGH, including that designated OA15, and in vivo bio-assay of 
oGH and oGH-MAb complexes have also been described previously by Aston et 
al (1987) loc. cit. Amounts of MAb OA15 are expressed in ABT.sub.50 
values: this is the amount of MAb required to bind 50% of .sup.125 I-oGH 
by solution phase titration assay. 
An adaptation of the multiple pin peptide synthesis method of Geysen et al 
(Proc. Natl. Acad. Sci. USA 81 3998-4002 (1984)) was used for epitope 
analysis, as follows. Briefly, octamers representing the entire sequence 
of rbGH were assembled with a one residue overlap onto activated 
polyethylene pins (ie 1-8, 2-9, 3-10, . . . 184-191). Peptides were 
synthesised on duplicate pins and therefore the 184 sets of octapeptides 
were accommodated on 4 blocks of 96 pins. The peptides were custom 
synthesized by Cambridge Research Biochemicals (Northwich, Cheshire, 
England). Mapping of the epitope recognised by MAb OA15 was performed as 
described below. 
Pins were blocked by incubation for 1 hour at ambient temperature in a 
solution containing 2% (w/v) BSA, 0.1% (v/v) TWEEN 20 detergent in 10 mM 
phosphate buffered saline (PBS) pH 7.4. Following incubation, excess 
blocking solution was shaken from the pins and they were placed in a 
solution of first antibody (ie mouse MAb OA15 hybridoma S supernatant 0.6 
mg/ml protein diluted 1:2000 in blocking buffer containing 0.1% (w/v) Na 
azide. 175 .mu.l/well of first antibody was added and incubation was 
continued overnight at 4.degree. C. Blocks of pins were then washed in a 
bath of 10 mM PBS (4.times.10 min washes) and incubated with appropriate 
second antibody-horse radish peroxidase (HRP) conjugate 175 .mu.l/well of 
a 1:1000 dilution in a buffer containing 1% (v/v) normal sheep serum, 0.1% 
(w/v) sodium caseinate, 0.1% (v/v) TWEEN 20 detergent, in PBS pH 7.4. Pin 
blocks were washed (4.times.10 min) in 0.01M PBS as before and bound 
antibody was detected by immersion of pins in 150 .mu.l/well of substrate 
buffer (0.1M Na.sub.2 HPO.sub.4 :0.1M citric acid pH 4) containing 0.05% 
(w/v) 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid (ABTS), and 
0.01% hydrogen peroxide. Colour development was monitored over a period of 
30 min-2 hr and absorbances were determined at 405 nm. All antibody 
incubation and washing steps were performed on a flat-bed shaking table at 
100 rpm. 
After each epitope scan, antibody was removed from the pins by sonication 
for 10 min at 65.degree. C. in a buffer containing 1% (w/v) SDS, 0.1% 
(v/v) 2-mercaptoethanol in 10 mM PBS pH 7.4. Pin blocks were then washed 
in distilled water at an initial temperature of 65.degree. C. for 30min. 
Finally blocks were immersed in methanol at 60.degree. C. for 15 sec, left 
to air dry and stored in the presence of silica gel dessicant at 4.degree. 
C. This procedure for regeneration of peptides is also exactly in 
accordance with the manufacturer's instructions. In the applicants' 
studies pin blocks have been used 25 times with no observable decrease in 
antibody binding or increase in background absorbance. For MAb OA15, three 
epitope scans were performed at 1:2000. to confirm the efficient removal 
of antibody from pins control experiments using second antibody or HRP 
substrate buffer alone were performed periodically. These experiments gave 
values equivalent to the background obtained with non-immune antisera. 
FIG. 1 shows the results of the epitope scan using MAb OA15. As is clearly 
evident, OA15 binds to a continuous antigenic determinant on the bGH 
molecule which is located between residues 91 and 102. In FIG. 2(a) the 
sequence of bGH fragment 91-102 is indicated. In FIG. 2(b), the sequences 
of the five overlapping pin-bound octapeptides derived from this region 
are shown, together with the relative reactivities of the peptides towards 
OA15. Also, shown boxed in FIG. 2(b) is a putative core recognition 
sequence which is shared amongst four of the five immunoreactive peptides. 
In FIG. 2(c) the location of this epitope is depicted diagrammatically 
within the three-dimensional structure of the molecule. This site 
comprises six residues from the C-terminal portion of helix 2 and the 
adjoining six residues from the region of random coil linking helices 2 
and 3. 
EXAMPLE 2 
Enhancement of oGH Activity by OA15 
FIG. 3 shows the dose-dependent enhancing effect of OA15 on oGH mediated 
.sup.35 So.sub.4.sup.2- incorporation into mouse costal cartilage (Holder 
et al, J. Endocrinol 85 35-47 (1980), Holder et al, J. Endocrinol 107 
R9-R12 (1985)) when OA15 pre-bound to oGH is administered, compared to the 
results obtained with oGH (50 .mu.g/day) alone. At the maximum dose of 
OA15 used (5000 ABT.sub.50 units; approximately 100 .mu.g pure MAb protein 
(Aston et al (1987) loc. cit.)) there was a 165% increase in 
35so.sub.4.sup.2- uptake compared to the value obtained with oGH alone 
(15077.+-.602 versus 5687.+-.214 dpm .sup.35 SO.sub.4.sup.2- /mg costal 
cartilage). If the basal value for .sup.35 SO.sub.4.sup.2- uptake 
(2140.+-.137 dpm .sup.35 SO.sub.4.sup.2- /mg costal cartilage) is 
subtracted then this becomes a 3.5-fold increase with administration of 
OA15-oGH complex compared to administration of oGH alone. 
EXAMPLE 3 
Enhancement of oGH Activity by OA15 
pGH (40 .mu.g/day) was substituted for oGH in Example 3 and administered to 
dwarf mice; similar results were obtained, as is shown in FIG. 4. At the 
maximum dose of OA15 (5000 ABT.sub.50 units) there was a 212% increase in 
.sup.35 SO.sub.4.sup.2- uptake compared to the value obtained with pGH 
alone (7022.+-.580 vs. 2250.+-.187 dpm .sup.35 SO.sub.4.sup.2- per mg 
costal cartilage). If the basal value for .sup.35 SO.sub.4.sup.2- uptake 
(460.+-.60 dpm .sup.35 SO.sub.4.sup.2- per mg costal cartilage) is 
subtracted then this becomes a 3.7-fold increase with administration of 
OA15-pGH complex compared to administration of pGH alone. 
EXAMPLE 4 
OA15 Binds oGH Remotely from the Receptor Binding Site 
The contact area for OA15 on growth hormone is removed from those areas on 
growth hormone which are believed to be involved in receptor binding. 
In a radio-receptor assay, pregnant rabbit liver membranes were prepared as 
described by Posner et al (Endocrinol. 95 521-531 (1974)): stock 
suspensions of membranes (approximately 20 mg/ml membrane protein) were 
diluted to 1 mg/ml in 20 mM CaCl.sub.2 --20 mM Tris Cl (pH 7.4): to 100 
.mu.l of this preparation (100 .mu.g membrane protein) was added 100 .mu.l 
.sup.125 I-oGH (20-30,000 cpm per tube) and 100 .mu.l of various dilutions 
of OA15 MAb (.box-solid.) or unlabeled oGH (.circle-solid.): tracer, 
unlabelled hormone and MAb dilutions were made in RRA buffer (20 mM Tris 
Cl (pH 7.4)--1% BSA--1% normal rabbit serum). Following overnight 
incubation at room temperature, 500 .mu.l of 0.9% (w/v) NaCl was added, 
tubes centrifuged 1700 g for 30 min, supernatants decanted and bound 
radioactivity determined by .gamma.-counting; oGH was iodinated to a 
specific activity of 50-100 .mu.Ci/.mu.g by the iodogen coated tube method 
(Fraker and Speck, Biochem. Biophys. Res. Comm. 80 849-857 (1978)). 
The data presented in FIG. 5 show that OA15 failed to inhibit the binding 
of .sup.125 I-oGH to pregnant rabbit liver membrane microsomes. 
EXAMPLE 5 
Enhancement of oGH Activity by Polyclonal Antibodies 
This example seeks to find whether the enhancing activity of OA15 shown in 
Examples 2 and 3 could be replicated by antisera prepared against the 
peptide which represents the epitope defined by MAb OA15. Sheep (n=5) were 
immunised with the 12-mer (Q F L S R V F T N S L V) (SEQ ID NO: 1) 
cross-linked to keyhole limpet haemocyanin (KLH) using glutaraldehyde, as 
follows. 
The 12-mer peptide representing residues oGH 91-102-sequence Q F L S R V F 
T N S L V (SEQ ID NO: 1) was synthesised using solid phase F-moc chemistry 
on an Applied Biosystems 431A automated peptide synthesiser. Details of 
peptide synthesis, purification and characterisation are as described by 
Beattie and Flint (Biochem. J. 283 473-478 (1992)). Peptide was conjugated 
to keyhole limpet haemocyanin by addition of 50 .mu.l 25% glutaraldehyde 
to 20 ml of 10 mM Na phosphate (pH 6.8) containing 0.5 mg/ml peptide and 
0.5 mg/ml KLH. After stirring for 2-3 h at room temperature, the mixture 
was aliquoted, snap-frozen in liquid N.sub.2 and stored at -20.degree. C. 
prior to use. For immunisation, peptide-KLH conjugate was emulsified with 
an equal volume of Freunds' complete adjuvant and 1 ml (250 .mu.g peptide 
equivalent) was injected into each hind leg of five sheep. Subsequent 
challenges were with 250 .mu.g peptide in Freunds incomplete at 21 day 
intervals. Blood was taken prior to immunisation (A; .DELTA.) and 10 days 
after each peptide immunisation (B; .smallcircle., C and D; .quadrature.). 
Antibody production was monitored by the ability of serial dilutions of 
antisera to bind .sup.125 I-bGH in a liquid phase RIA. Each tube contained 
50 .mu.l antiserum, 50 .mu.l .sup.125 I-bGH (prepared using iodogen ref. 
28; 20,000 cpm/tube) and 100 .mu.l of assay buffer. Tubes were incubated 
overnight at 4.degree. C. and bound .sup.125 I-bGH was separated from free 
using PEG 6000. Each sheep antiserum is identified individually. There was 
no increase in titre after bleed C and these data have been omitted; serum 
from bleed C was used in subsequent experiments (see below). Results are 
means of triplicate determinations and background counts were not 
subtracted. 
FIGS. 6.1 to 6.5 show that each of the five sheep produced an antiserum 
capable of recognising .sup.125 I-bGH in a liquid phase radioimmunoassay 
(RIA), and that titres increased with successive immunisations and bleeds 
(A, B, D) 
Serum was prepared from blood taken from sheep H148 and HISS at bleed C. 
The immunoglobulin (Ig) fraction of the antisera was precipitated using 
Na.sub.2 SO.sub.4 (Aston et al (J. Endocrinol. 110 381-88 (1986)); the 
precipitate was redissolved in a small quantity of PBS and dialysed 
extensively against PBS. The resulting antibody preparation was 
reconstructed to 1/2 the original serum volume and mixed with an equal 
volume of either bGH or pGH (USDA, Beltsville, Md., USA) such that the 
final solution contained 40 .mu.g GH/0.1 ml. At this point antibody was 
present at physiological levels with regard to the concentration found in 
sheep serum. Dwarf mice (n=6/treatment group) were injected with 0.1 ml 
(sc) of GH/antibody complex or GH alone once/day for 2 days. On the third 
day mice received .sup.35 SO.sub.4.sup.2- (0.5 .mu.Ci/g body wt ip), and 
were killed 20h later and .sup.35 SO.sub.4.sup.2- uptake into costal 
cartilage measured. These methods have been described in more detail by 
Holder et al (J. Endocrinol. 85 35-47 (1980)). All results are expressed 
as means .+-.SEM. Differences between groups receiving GH plus antibody 
and the appropriate GH alone were assessed using students-t test (*p&lt;0.05; 
***p&lt;0.001). Values in parentheses represent increases over the 
appropriate GH alone controls. 
In FIG. 7 it is demonstrated that the antisera from sheep H148 and H155 
(see FIGS. 6.2 and 6.3) are able to enhance the biological actions of both 
bGH and pGH, thus mimicking the properties of MAb OA15. The degree to 
which these antisera can enhance bGH and pGH (ie percent increases over 
the appropriate GH alone control) are given in parentheses (FIG. 7); it is 
apparent that the ability of these antisera to enhance bGH was greater 
than their ability to enhance pGH. This may be attributed to varying 
potencies of the GH preparations used. Preimmunisation sera from these 
animals failed to enhance GH action (data not shown). Enhancing activity 
of the other three antipectide antisera was not tested but it is predicted 
that they would also enhance GH action. 
EXAMPLE 6 
Window Size and Replacement Net Analyses 
In order to gain further insight into the structure of the OA15 epitope, 
"window size" and "replacement net" analyses on the OA15 epitope were 
conducted. For the window size analysis, completely overlapping sets of 
5-8 mers from the dodecapeptide bGH 91-102 (Q F L S R V F T N S L V) (SEQ 
ID NO:1) were assembled covalently onto activated polyethylene pins and 
probed for OA15 binding in an identical manner to that described in 
Example 1. Sizing analysis of the epitope revealed that a 7-mer peptide 
was required for substantial binding to remain. Peptides 4-6 residues in 
length showed either no, or much reduced binding activity. Within the 
series of 7-mers, the heptapeptide S R V F T N S (SEQ ID NO:25) had 
binding activity comparable with the most strongly reactive octapeptides 
seen in this and in previous scans (such as FIG. 1). This heptapeptide may 
therefore be defined as the minimum functional epitope for OA15 
(asterisked in FIG. 8). The contribution of each residue within the 
epitope to OA15 binding was examined by a replacement net analysis, where 
each amino acid of the epitope is systematically replaced with each of the 
other nineteen naturally occurring L-amino acids (FIG. 9). For each 
replacement, reactivity against the native heptapeptide is asterisked; 
reactivity against substituted peptides is indicated over the 
corresponding amino acid replacing the native residue; single-letter codes 
are used for amino acids, which have been arranged alphabetically. As 
indicated, for R95 and T98 little replacement is permitted and in the 
context of this epitope, these may be viewed as critical residues for OA15 
binding. The remaining residues S94, V96, F97, N99 and S100 appear 
replaceable to a greater or lesser degree. For example V96 is replaceable 
only by isoleucine (but not to any great degree by leucine). This is a 
conservative substitution illustrating the importance of an aliphatic, 
hydrophobic side chain in this part of the epitope. The act that I but not 
L will replace V at this position indicates a further level of structural 
specificity in the requirement for a .beta.-methyl substituted side chain. 
Similarly, F97 is best replaced by the two other aromatic side 
chains--tryptophan (W) and tyrosine (Y) although in this instance limited 
replaceability is seen by other amino acid side chains. The two serine 
residues in the epitope--S94 and S100 show different patterns of 
replaceability. S94 is replaceable by cysteine (C), aspartic acid (D) and 
glutamic acid (E). In fact heptapeptides containing D or E show 
substantially higher is binding to OA15 than the parent sequence. For 
S100, replaceability is allowed, to a lesser extent, by alanine (A) and 
glycine (G) and for a few other residues. Finally N99 is only replaceable 
to any extent by histidine. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 37 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "91-102 REGION OF BOVINE 
PORCINE OR OVINE GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlnPheLeuSerArgValPheThrAsnSerLeuVal 
1510 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "91-102 REGION OF HUMAN 
OR MOUSE GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GlnPheLeuArgSerValPheAlaAsnSerLeuVal 
1510 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "91-102 REGION OF EQUINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GlnLeuLeuSerArgValPheThrAsnSerLeuVal 
1510 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "91-102 REGION OF CHICKEN 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GlnTyrLeuSerLysValPheThrAsnAsnLeuVal 
1510 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..12 
(D) OTHER INFORMATION: /note= "91-102 REGION OF RAT 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlnPheLeuSerArgIlePheThrAsnSerLeuVal 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "91-102 REGION OF SALMON 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GlnThrLeuAsnSerLeuLeu 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "91-95 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GlnPheLeuSerArg 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "92-96 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
PheLeuSerArgVal 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "93-97 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
LeuSerArgValPhe 
15 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "94-98 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
SerArgValPheThr 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "95-99 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
ArgValPheThrAsn 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "96-100 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ValPheThrAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "97-101 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
PheThrAsnSerLeu 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "98-102 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ThrAsnSerLeuVal 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "91-96 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GlnPheLeuSerArgVal 
15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "92-97 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
PheLeuSerArgValPhe 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "93-98 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
LeuSerArgValPheThr 
15 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "94-99 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
SerArgValPheThrAsn 
15 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "95-100 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ArgValPheThrAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "96-101 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ValPheThrAsnSerLeu 
15 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "97-102 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
PheThrAsnSerLeuVal 
15 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "91-97 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GlnPheLeuSerArgValPhe 
15 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "92-98 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
PheLeuSerArgValPheThr 
15 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "93-99 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
LeuSerArgValPheThrAsn 
15 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "94-100 REGION OF OVINE, 
BOVINE, EQUINE OR PORCINE GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
SerArgValPheThrAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "95-101 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
ArgValPheThrAsnSerLeu 
15 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "96-102 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
ValPheThrAsnSerLeuVal 
15 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..8 
(D) OTHER INFORMATION: /note= "91-98 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
GlnPheLeuSerArgValPheThr 
15 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..8 
(D) OTHER INFORMATION: /note= "92-99 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
PheLeuSerArgValPheThrAsn 
15 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..8 
(D) OTHER INFORMATION: /note= "93-100 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
LeuSerArgValPheThrAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..8 
(D) OTHER INFORMATION: /note= "94-101 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
SerArgValPheThrAsnSerLeu 
15 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..8 
(D) OTHER INFORMATION: /note= "95-102 REGION OF OVINE 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
ArgValPheThrAsnSerLeuVal 
15 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "94-100 REGION OF HUMAN OR 
MOUSE GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ArgSerValPheAlaAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "94-100 REGION OF CHICKEN 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
SerLysValPheThrAsnAsn 
15 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "94-100 REGION OF RAT 
GROWTH HORMONE" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
SerArgIlePheThrAsnSer 
15 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "MODIFIED 94-100 REGION OF 
GROWTH HORMONE: Xaa position 1 is Ser, Cys, Asp, or Glu 
Xaa position 3 is Val or Ile 
Xaa position 4 is Phe, Trp, Tyr, Cys 
Xaa position 6 is Asn or His 
Xaa position 7 is Ser, Ala, or Gly" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
XaaArgXaaXaaThrXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "MODIFIED REGION 94-100 OF 
GROWTH HORMONE: Xaa position 1 is Ser, Cys, Asp, or Glu 
Xaa position 3 is Val or Ile" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
XaaArgXaaPheThrAsnSer 
15 
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