Monoclonal antibodies which differentiate between native and modified porcine somatotropins

Monoclonal antibodies are provided which differentiate between native and modified sequence proteins. Also provided are methods for using monoclonal antibodies to determine the relative amount of native and modified sequence proteins in a sample.

This invention relates generally to monoclonal antibodies and particularly 
to monoclonal antibodies which differentiate between native and modified 
sequence proteins and methods for using such monoclonal antibodies to 
determine the relative amounts of native and modified sequence proteins in 
a sample. 
BACKGROUND OF THE INVENTION 
1. Recombinant Proteins 
Methods for producing recombinant proteins are well known in the art; 
heterologous DNA segments that encode for a particular protein are 
inserted into host organisms using recombinant DNA technology. By growing 
the transformant organisms under conditions which induce the expression of 
proteins, heterologous proteins such as insulin, somatotropins, 
interleukins, interferons, somatomedins, and the like can be produced. For 
example, U.S. Pat. Nos. 4,604,359 and 4,332,717 disclose methods for 
producing human recombinant somatotropin; U.S. Pat. No. 4,431,739 
discloses a method for producing recombinant somatotropins; E.P. Patent 
Application 0 104 920 discloses a method for producing recombinant porcine 
somatotropin; U.S. Pat. No. 4,443,539 discloses a method for producing 
recombinant bovine somatotropin; Schoner, Biotechnology, 3(2):151-54, 
discloses a method for producing recombinant somatotropin, and Buell, 
Nucleic Acid Res., 13, 1923-38 (1985) discloses a method for producing 
recombinant somatomedin C. 
Typically, the recombinant protein produced has an amino acid sequence 
which is the same as the amino acid sequence of the native protein. 
Often, however, recombinant proteins produced using recombinant DNA 
techniques have an amino acid sequence which is not the same as the amino 
acid sequence of the native protein--a modified sequence protein. 
It may be desirable to modify the amino acid sequence in a recombinant 
protein for several reasons. For example, a recombinant protein with a 
modified amino acid sequence may have physical or chemical properties 
which make it easier to recover the protein from the fermentation broth, 
refold and purify the protein during the recovery process, or formulate 
and administer the protein for the intended purpose. In addition, the 
modified sequence recombinant protein may have greater bioactivity than 
the native protein and cause less adverse side effects when administered 
for its intended purpose. 
Also, when administering a modified sequence recombinant protein to an 
animal for its intended use, it is often difficult to distinguish between 
the native protein endogenous to the animal and the modified sequence 
protein administered to the animal. For example, when modified sequence 
somatotropin is administered to an animal to promote growth, the animal's 
serum can be assayed for total somatotropin levels using radioimmunoassay 
(RIA) or other well known techniques. However, it is difficult to 
differentiate between native and modified sequence somatotropin levels and 
determine if the modified sequence somatotropin is causing an increase in 
growth or if the increase in growth is caused by some factor which has 
increased native somatotropin levels. 
Similarly, when somatotropin or any other endogenous protein is being 
delivered to an animal with a delivery device, it is difficult to 
determine if the modified sequence protein is being delivered to the 
animal in the required amounts or if all or part of the the protein can be 
attributed to endogenous protein. 
Methods are, therefore, needed for differentiating between native and 
modified sequence proteins and for determining the relative amounts of 
native and modified sequence proteins, particularly modified sequence 
recombinant proteins, in a sample. 
2. Protein Immunology 
A macromolecular protein immunogen has several antigenic determinants. 
Immunizing an animal with a macromolecular protein results in the 
formation of different antibodies with different specificities for each 
antigenic determinant; the number of different antibodies depends on the 
number of antigenic determinants on the macromolecular protein and their 
inherent immunogenicity. 
The immunogenicity of a particular antigenic determinant is dependent upon 
several factors including the amino acid sequence, conformation, segmental 
mobility, or hydropathicity of the antigenic determinant. 
Antibodies, particularly monoclonal antibodies, formed in response to 
protein immunogens contain antigen combining sites that are highly 
specific for individual antigenic determinants on the protein. Thus, an 
antibody specific for a particular antigenic determinant of a protein will 
not react with that protein if the antigenic determinant has been deleted, 
modified or otherwise altered to change its immunogenicity. 
When recombinant or synthetic proteins have an amino acid sequence which is 
not the same as the amino acid sequence of the native protein, the altered 
amino acids may affect the immunogenicity of an antigenic determinant 
containing the altered amino acid sequence. 
3. Description of References 
Pestka, U.S. Pat. No. 4,623,621, discloses the use of antibodies to 
distinguish monomeric from oligomeric forms of peptides and proteins. The 
assay employs a single monoclonal antibody in two different assay steps. 
Sharp et al, U.S. Pat. No. 4,487,829, discloses the production and use of 
monoclonal antibodies against adenoviruses.

SUMMARY OF THE INVENTION 
It is, therefore, an object of the present invention to provide monoclonal 
antibodies which differentiate between native and modified sequence 
proteins. 
It is another object of the present invention to provide monoclonal 
antibodies which differentiate between native and modified sequence 
somatotropins. 
It is another object of the present invention to provide monoclonal 
antibodies which differentiate between native somatotropins and modified 
sequence somatotropins having deleted sequences. 
It is another object of the present invention to provide monoclonal 
antibodies which differentiate between native somatotropins and modified 
sequence somatotropins having N-terminal deleted sequences. 
It is a further object of the present invention to provide a method for 
determining the relative amount of native and modified sequence proteins 
in a sample. 
It is another object of the present invention to provide a method for 
determining the relative amount of native and modified sequence 
somatotropins in a sample. 
It is another object of the present invention to provide a method for 
determining the relative amount of native somatotropins and modified 
sequence somatotropins having deleted sequences in a sample. 
It is another object of the present invention to provide a method for 
determining the relative amount of native somatotropins and modified 
sequence somatotropins having N-terminal deleted sequences in a sample. 
These and other objects are achieved using monoclonal antibodies which 
differentiate between native and modified sequence proteins, preferably 
somatotropins. The monoclonal antibodies are native antibodies having an 
antigen combining site specific for a native antigenic determinant in the 
native protein. As such, the monoclonal antibodies will not react with 
modified sequence proteins which have had the native antigenic determinant 
deleted, modified or otherwise altered. 
The monoclonal antibodies are formed by any acceptable method, preferably 
using the HAT selection technique well known to skilled artisans. 
In the most preferred embodiment, monoclonal antibodies are provided which 
differentiate between native somatotropins and modified sequence 
somatotropins having N-terminal deleted sequences. The monoclonal 
antibodies are native antibodies having an antigen combining site specific 
for a native antigenic determinant located in about the first 1-40 amino 
acids on the N-terminal end of the native somatotropin. Monoclonal 
antibodies specific for native antigenic determinants located in the 
N-terminal 27 amino acids of porcine somatotropin have been produced; the 
hybridoma producing the antibodies has been deposited with The American 
Type Culture Collection (ATCC), Rockville, Md. and assigned Accession No. 
HB10308. Monoclonal antibodies produced by ATCC HB10308 will distinguish 
between native and N-terminal deleted sequence porcine somatotropins such 
as delta-7 porcine somatotropin. 
Other objects, advantages, and novel features of the present invention will 
become apparent from the following detailed description of the invention. 
DETAILED DESCRIPTION OF THE INVENTION 
1. Definition of Terms 
The term "modified sequence protein" is defined herein to mean a protein 
having bioactivity of a native protein but having an amino acid sequence 
different from the amino acid sequence of the native protein and includes 
synthetic proteins produced by chemical synthesis, transgenic animals, 
recombinant microorganisms and other suitable means. Modified sequence 
proteins may have an abbreviated amino acid sequence or an amino acid 
sequence similar to the native protein and their analogs and muteins 
having substituted, deleted, elongated, replaced, or otherwise modified 
sequences. In particular, modified sequence protein as used herein refers 
to a recombinant protein having the same amino acid sequence as the native 
protein but having amino acids deleted from the amino and/or carboxy 
terminal end. 
The term "modified sequence somatotropin" is defined herein to mean a 
somatotropin having bioactivity of a native somatotropin but having an 
amino acid sequence different from the amino acid sequence of the native 
somatotropin and includes synthetic somatotropins produced by chemical 
synthesis and recombinant somatotropins produced by recombinant organisms. 
Modified sequence somatotropins may have an abbreviated amino acid 
sequence or an amino acid sequence similar to the native somatotropin and 
their analogs and muteins having substituted, deleted, elongated, 
replaced, or otherwise modified sequences. In particular, modified 
sequence somatotropin as used herein refers to a recombinant somatotropin 
having the same amino acid sequence as the native somatotropin but having 
amino acids deleted from the amino and/or carboxy terminal end. Examples 
of such proteins include but are not limited to delta-7 recombinant 
porcine somatotropin, delta-9 recombinant bovine somatotropin, (native 
somatotropins having 7 and 9 residues deleted from the amino terminal end, 
respectively), and the like. 
The term "native antigenic determinant" is primarily defined herein to mean 
an amino acid sequence in the native protein which has been substituted, 
deleted, elongated, replaced, or otherwise modified to produce a modified 
sequence protein and a reasonable number of native amino acids surrounding 
the altered sequence which may be required to produce an antigenic 
determinant having the immunogenicity of the native protein. The term also 
includes an amino acid sequence in the native protein which has had its 
conformation and antigenicity changed by a modification in the protein in 
a location not in the sequence itself. 
The term "native antibody" is defined herein to mean an antibody which has 
an antigenic combining site specific for the native antigenic determinant. 
The native antibody will, therefore, be specific for the native protein 
while not combining with the modified sequence protein. 
2. The Invention 
According to the present invention, monoclonal antibodies are provided 
which differentiate between native and modified sequence proteins. The 
monoclonal antibodies are native antibodies having an antigen combining 
site specific for the native antigenic determinant in the native protein; 
the monoclonal antibodies will not react with modified sequence proteins 
which have had the native antigenic determinant deleted, modified or 
otherwise altered. 
The monoclonal antibodies and methods of the present invention can be used 
to differentiate between and determine the amount of any native and 
modified sequence protein which has a native antigenic determinant. These 
include but are not limited to somatotropins, prolactins, placental 
lactogens, somatomedins, somatostatins, insulins, interleukins, and the 
like. 
The monoclonal antibodies of the present invention are formed by using the 
amino acid sequence of the native antigenic determinant as an immunogen. 
The immunogen is injected into an animal and immune spleen cells are 
isolated and fused with myelomas to produce hybridomas. A hybridoma which 
makes monoclonal antibody specific for the native antigenic determinant is 
isolated and used to make native antibodies which will combine with the 
native protein but will not combine with the modified sequence protein 
since the modified sequence has had the native antigenic determinant 
deleted, modified or otherwise altered. 
Methods for making hybridomas which produce monoclonal antibodies from 
immune spleen cells and myelomas are well known in the art, particularly 
methods using the HAT (hypoxanthine, aminopterin, thymidine) selection 
technique. 
The native antigenic determinant used as an immunogen is prepared by any 
suitable means, typically by synthesizing an amino acid sequence 
corresponding to the native protein amino acid sequence containing the 
amino acid sequence which has been altered to produce the modified 
sequence protein or by excising a section of a protein which has the 
immunogenicity of the native antigenic determinant. It may be necessary to 
include a number of amino acids on either side of the native antigenic 
determinant to produce an immunogen which will elicit an antibody response 
from the immune system of the injected animal or to maintain the desired 
characteristics of the native antigenic determinant. These additional 
amino acids may be part of the native amino acid sequence or may be 
necessary to elicit an immune response, particularly if the altered 
sequence is a hapten or if the additional amino acids are necessary to 
insure the conformation of the native antigenic determinant. It is 
sometimes necessary to insure the secondary structure of an antigenic 
determinant to elicit the proper antibody response. 
In the preferred embodiment, monoclonal antibodies are provided which 
differentiate between native and modified sequence somatotropins. The 
monoclonal antibodies are native antibodies having an antigen combining 
site specific for the native antigenic determinant in the native 
somatotropin; the monoclonal antibodies will not react with modified 
sequence somatotropins which have had the native antigenic determinant 
deleted, modified or otherwise altered. The somatotropins useful in the 
present invention can be from any species but are preferably bovine, 
porcine, avian, ovine, piscine or human somatotropin, most preferably 
porcine or bovine somatotropin. 
In the most preferred embodiment, monoclonal antibodies are provided which 
differentiate between native somatotropins and modified sequence 
somatotropins having N-terminal deleted sequences. The monoclonal 
antibodies are native antibodies having an antigen combining site specific 
for a native antigenic determinant located in about the first 1-40 amino 
acids of the N-terminal end of the native somatotropin; the monoclonal 
antibodies will not react with modified sequence somatotropins which have 
had the N-terminal native antigenic determinant deleted, modified or 
otherwise altered. 
Several somatotropins having an N-terminal native antigenic determinant 
modification are known in the art. European Patent Application Publication 
No. 0 103 395 describes the construction of a transformant strain of E. 
coli containing a first plasmid which codes for delta-9 (Ser) bovine 
somatotropin (somatotropin less its 9 N-terminal amino acids and having an 
additional serine residue at the N-terminus) under the control of the 
lambda P.sub.L promoter-operator which has a Shine-Dalgarno region derived 
from bacteriophage mu. The transformant also contains a second plasmid, 
pcI857, which codes for the production of the pcI857 temperature-sensitive 
repressor protein. The repressor protein can be inactivated by raising the 
temperature to about 42.degree. C., thereby inducing expression of delta-9 
(Ser) bovine somatotropin. A transformant strain of this type, E. coli 
HB101 (P.sub.L -mu-delta-9 (Ser) bovine somatotropin and pcI857) has been 
deposited, with The American Type Culture Collection (ATCC), Rockville, 
Md. and assigned Accession No. 53030. 
Construction of a similar transformant strain which codes for the 
production of delta-7 porcine somatotropin (porcine somatotropin less its 
first 7 N-terminal amino acids) is described in European Patent 
Application Publication No. 0 104 920. A transformant strain of this type, 
E. coli HB101 (P.sub.L -mu-delta-7 porcine somatotropin and pcI857) has 
been deposited with ATCC and assigned Accession No. 53031. 
Strains 53030 and 53031 are prolific producers of delta-9 (Ser) bovine 
somatotropin and delta-7 porcine somatotropin, respectively. Other methods 
for many similar proteins are known in the art. 
Monoclonal antibodies specific for native antigenic determinants located in 
the N-terminal amino acids of porcine somatotropin have been produced; the 
hybridoma producing the antibodies has been deposited with The American 
Type Culture Collection (ATCC), Rockville, Md. and assigned Accession No. 
HB10308. Monoclonal antibodies produced by ATCC HB10308 will distinguish 
between native and N-terminal deleted sequence porcine somatotropins such 
as delta-7 porcine somatotropin. 
According to the present invention, a method is provided for determining 
the relative amount of native and modified sequence proteins in a sample. 
The method comprises three steps: (1) determining the total amount of the 
native and modified sequence protein in the sample, (2) determining the 
amount of the native protein in the sample by reacting a native antibody 
having an antigen combining site specific for the native antigenic 
determinant in the native protein and determining the amount of native 
antibody-native protein complex formed by the reaction, and (3) 
calculating the amount of the modified sequence protein in the sample by 
subtracting the amount of the native protein in the sample from the total 
amount of the native and modified sequence protein in the sample. 
The total amount of the native and modified sequence protein in the sample 
can be determined by means known to skilled artisans. Typical methods 
include but are not limited to radioimmunoassays, enzyme-linked 
immunosorbent assays, and the like. 
The antibody-antigen reaction between the native antibody and the native 
protein is conveniently accomplished by mixing a sample containing the 
native protein with a complexing amount of the native antibody. The amount 
of native antibody reacted with the sample should be more than sufficient 
to form a complex with all the native protein in the sample (antibody 
should be present in excess). The amount will vary depending on the sample 
size, amount of native protein in the sample, and the like. 
The amount of native antibody-native protein complex formed by the reaction 
can be determined by conventional means such as immumoprecipitation of the 
complex, color formation in enzyme-linked immunosorbent assays, and the 
like. 
In the preferred embodiment, a method is provided for determining the 
relative amount of native and modified sequence somatotropins in a sample. 
The method comprises steps 1-3 as described above using samples containing 
native and modified sequence somatotropin and a native antibody having an 
antigen combining site specific for the native antigenic determinant in 
the native somatotropin. 
More preferably, a method is provided for determining the relative amount 
of native somatotropin and modified sequence somatotropin having 
N-terminal deleted sequences in a sample. The method comprises steps 1-3 
as described above using (1) samples containing native somatotropin and 
modified sequence somatotropin having N-terminal deleted sequences and (2) 
a native antibody having an antigen combining site specific for a native 
antigenic determinant located in about the first 1-40 amino acids of the 
N-terminal end of the native somatotropin. The native antibody is 
preferably a monoclonal antibody which will not react with modified 
sequence somatotropins which have had the N-terminal native antigenic 
determinant deleted, modified or otherwise altered. 
Most preferably, the native antibody is a monoclonal antibody specific for 
native antigenic determinants located in the N-terminal amino acids of 
porcine somatotropin; the hybridoma producing the antibody has been 
deposited with The 
Type Culture Collection (ATCC), Rockville, Md. and assigned Accession No. 
HB10308. 
Also according to the present invention, a method is provided for 
determining the amount of modified sequence protein in a sample. The 
method comprises (1) reacting a native antibody having an antigen 
combining site specific for a native antigenic determinant in the native 
protein to form a native antibody-native protein complex, (2) separating 
the native antibody-native protein complex from the sample, and (3) 
determining the amount of the modified sequence protein in the sample. 
The antibody-antigen reaction between the native antibody and the native 
protein is conveniently accomplished by mixing a sample containing the 
native protein with a complexing amount of the native antibody. The amount 
of native antibody reacted with the sample should be more than sufficient 
to form a complex with all the native protein in the sample. The amount 
will vary depending on the sample size, amount of native protein in the 
sample, and the like. 
The native antibody-native protein complex can be removed from the sample 
by conventional means such as column chromatography, immunoprecipitation, 
high pressure liquid chromatography, and the like. 
The amount of modified sequence protein in the sample can be determined by 
means known to skilled artisans. Typical methods include but are not 
limited to radioimmunoassays, enzyme-linked immunosorbent assays, and the 
like. 
In the preferred embodiment, a method is provided for determining the 
amount of modified sequence somatotropin in a sample. The method comprises 
steps 1-3 as described above using samples containing native and modified 
sequence somatotropin and a native antibody having an antigen combining 
site specific for the native antigenic determinant in the native 
somatotropin. 
Most preferably, a method is provided for determining the amount of 
modified sequence somatotropin having N-terminal deleted sequences in a 
sample. The method comprises steps 1-3 as described above using (1) 
samples containing native somatotropin and modified sequence somatotropin 
having N-terminal deleted sequences and (2) a native antibody having an 
antigen combining site specific for a native antigenic determinant located 
in about the first 1-40 amino acids of the N-terminal end of the native 
somatotropin. The native antibody is preferably a monoclonal antibody 
which will not react with modified sequence somatotropins which have had 
the N-terminal native antigenic determinant deleted, modified or otherwise 
altered. 
Most preferably, the native antibody is a monoclonal antibody specific for 
native antigenic determinants located in the N-terminal 27 amino acids of 
porcine somatotropin; the hybridoma producing the antibody has been 
deposited with The American Type Culture Collection (ATCC), Rockville, Md. 
and assigned Accession No. HB10308. 
The invention having been generally described, the following examples are 
given as particular embodiments of the invention and to demonstrate the 
practice and advantages thereof. It is understood that the examples are 
given by way of illustration and are not intended to limit the 
specification or the claims to follow in any manner. In particular, 
recombinant proteins used in the experiments were prepared from 
transformed E. Coli strains which produce delta-7 porcine somatotropin. 
The somatotropin was isolated from E. Coli host strain HB101 transformed 
with a first plasmid (pL-mu-delta-7 porcine somatotropin) coding for 
delta-7 porcine somatotropin and a second plasmid (pCI 857) coding for the 
temperature sensitive lambda phage repression protein. Many other strains 
of organisms produce many types of recombinant proteins which will 
function in the present invention. 
EXAMPLE 1 
An amino-terminus fragment consisting of the first 27 amino acids (N27) of 
native somatotropin, shown in FIG. 1, was synthesized for use as an 
immunogen using techniques well known in the art. This particular fragment 
size was chosen because it contains both the 7 amino acids which have been 
deleted from the amino-end of delta-7 recombinant porcine somatotropin and 
a complete alpha helix. Thus, this fragment was likely to structurally 
resemble the corresponding segment in the native porcine somatotropin, not 
only in the primary structure, but also in secondary and tertiary 
structure. In addition, a peptide of 27 amino acids would be immunogenic 
whereas a 7 amino acid sequence would be a hapten and require conjugation 
to a carrier to elicit an immune response. 
EXAMPLE 2 
Biotinylation of porcine somatotropin: Porcine somatotropin was added to 
carbonate buffer (pH 9.6) to a final concentration of 10 mg/ml. A 50 mg/ml 
solution of biotin ester was made by adding biotin-N-hydroxysuccinimide 
ester (Bethesda Research Laboratories) to dimethylformamide which had been 
stored over molecular sieves. The two solutions were combined in a 
reaction vial and stirred at room temperature for 1.5 hours. The reaction 
was stopped by the addition of 1M NH.sub.4 Cl to a final concentration of 
0.1M. The biotinylated porcine somatotropin was dialyzed against PBS (pH 
7.6) 3 times over 2 days. The final porcine somatotropin concentration was 
determined by absorbance at 278 nm. Biotinylated porcine somatotropin was 
diluted 1:100 and stored frozen at -70.degree. C. 
EXAMPLE 3 
Production of Hybridomas: Mice--Six to eight week old female Balb/c mice 
were obtained from Charles River Laboratories. For the duration of the 
studies, they were allowed food and water ad libitum. 
Immunizations--For the primary immunizations, N27 was dissolved in 
carbonate buffer (pH 9.6) and then emulsified in Freund's complete 
adjuvant (Gibco). Mice were inoculated subcutaneously with 100 .mu.g of 
N27 in 0.2 ml. Two to three weeks later, mice were boosted by 
intrapertioneal injection of 10 to 50 .mu.g N27 in PBS. Subsequent boosts 
were given in a similar manner with a minimum interval of three weeks 
between boosts. 
EXAMPLE 4 
Preparation of SP2/0 cells for Fusion: SP2/0 cells (obtained from the 
American Type Culture Collection) were grown in Dulbecco's modified Eagle 
media (DMEM) containing 1500 mg glucose/liter (Gibco) supplemented with 
10% fetal calf serum (Hyclone), 1 mM sodium pyruvate, 2 mM glutamine and 
1% penicillin-streptomycin (all obtained from Gibco). Cells were 
subcultured at a ratio of 1:4 to 1:10 when confluent. 
Three days preceding the cell fusion, cells were seeded at a concentration 
of 5.times.10.sup.4 cells/ml which allowed the cells to enter log phase 
growth before the fusion. 
EXAMPLE 5 
Preparation of Spleen Cells for Fusion: Three days following a boost, the 
immunized mouse was sacrificed and the spleen was aseptically removed. The 
spleen was placed in a petri dish containing DMEM and the lymphocytes were 
gently teased out of the spleen with sterile forceps. Cells obtained in 
this manner were centrifuged at 160.times.g for 10 minutes. The spleen 
cell pellet was resuspended in DMEM and pelleted at 160.times.g for 5 
minutes. This was repeated two times. An aliquot of spleen cells was 
removed and counted using a Coulter counter. Zapoglobin (Coulter 
Diagnostics) was used to lyse the red blood cells. Routinely, 
approximately 1.times.10.sup.8 spleen node cells were obtained. 
EXAMPLE 6 
Fusion Protocol: Spleen cells were mixed with SP2/0 cells at a 4:1 ratio. 
The cell mixture was pelleted at 200.times.g for 5 minutes and the 
supernatant was carefully removed. One ml of PEG-1500 (American Type 
Culture Collection) and various volumes of growth media were added 
dropwise in the following sequence: 
______________________________________ 
Addition Time 
______________________________________ 
1 ml PEG 1 minute 
Gently stir 1 minute 
1 ml media 1 minute 
1 ml media 1 minute 
8 ml media, stirring 2 minutes 
______________________________________ 
Touching the sides and bottom of the centrifuge tube with the pipette was 
avoided when stirring. 
The cells were centrifuged at 200.times.g for 5 minutes and resuspended in 
approximately 53 ml of growth media. Cells were dispensed into 5 96-well 
plates (Costar) at 0.1 ml/well and incubated at 37.degree. C. (5% carbon 
dioxide) overnight. 0.1 ml HAT (Sigma) medium (hypoxanthine, aminopterin, 
thymidine) was added to each well the next day. Cells were refed with HAT 
media twice a week for three weeks and then fed with HT (Sigma) medium 
(hypoxanthine, thymidine) for an additional two weeks. Afterwards, cells 
were maintained on standard growth media. 
Ascites Fluids--Balb/c mice were injected intrapertioneally with 0.5 ml 
pristane (2, 6, 10, 14-tetramethyl pentadecane, Sigma). Ten days later 
mice were given 5.times.10.sup.5 hybridoma cells i.p. using a 21 gauge 
needle. Swelling of the abdomen of the inoculated mouse was observed in 
1-3 weeks. The ascites fluid was harvested by holding the mouse with the 
abdomen side up and inserting an 18 gauge needle with no syringe attached 
into the abdomen. The mouse was tilted to allow fluid to collect into a 15 
ml centrifuge tube. Ascites fluid was centrifuged at 200.times.g for 15 
minutes and the clarified fluid was removed and frozen for later use. 
Fluid was collected from a mouse on alternate days till the mouse 
succumbed to the tumor. 
EXAMPLE 7 
Evaluation of Hybridomas by ELISA: Miscellaneous Solutions: 10xPBS: 12.36 g 
Na.sub.2 HPO.sub.4, 1.8 g NaH.sub.2 PO.sub.4, 85 g NaCl. Add distilled 
water to make 1 liter. OPD substrate: 30 ml 0.2M Na.sub.2 HPO.sub.4 +25 ml 
0.1M citric acid, correct to pH 5.0. Add 25 .mu.g o-phenylenediamine 
(Sigma) and stir until dissolved. Add 40 .mu.l 30% H.sub.2 O.sub.2 (Sigma) 
just prior to use. 
Colonies were assayed as early as two weeks after the fusion. 
Ninety-six-well ELISA plates (Nunc) were coated with 600 ng/well native 
porcine somatotropin overnight at 4.degree. C. or for 4 hours at 
37.degree. C. Plates were washed three times using PBS+0.1% Tween-20 and 
blocked with 2% BSA in PBS for 1-2 hours at 37.degree. C. or overnight at 
4.degree. C. Plates were then washed as before. One hundred (100) .mu.l of 
fusion supernatants were added to the wells and allowed to incubate 3-4 
hours at 37.degree. C. Plates were washed and 100 .mu.l of an appropriate 
dilution of horseradish peroxidase conjugated goat anti-mouse antibody 
(Cappel, Cat. #3211-0081) was added to each well. Plates were incubated 
for 2-4 hours at 37.degree. C. and then washed. One hundred (100) .mu.l 
well OPD substrate was added. A color change was observed within 20 
minutes and the development stopped by addition of 50 .mu.l well 12% 
sulfuric acid. Plates were read on an ELISA reader (Titertek Multiskan) 
using a filter with a wavelength of 492 nm. Wells positive to native 
porcine somatotropin were reassayed by ELISA on native and recombinant 
porcine somatotropin. 
EXAMPLE 8 
Capture Assays: These assays were used with either biotinylated porcine 
somatotropin or radiolabeled porcine somatotropin. Ninety-six-well plates 
made by Nunc were used in the biotin-pST assays. Flexible assay plates 
(Falcon 3911 Micro Test III) were used when using radiolabeled materials. 
Ninety-six-well ELISA plates were coated with 2 .mu.g/ml affinity purified 
goat anti-mouse immunoglobulin overnight at about 4.degree. C. Plates were 
washed three times using PBS+ 0.1% Tween-20 and blocked with 2% BSA in PBS 
for 1-2 hours at 37.degree. C. or overnight at 4.degree. C. Plates were 
then washed as before. One hundred (100) .mu.l/well of antibodies 
(supernatants or ascites fluids in appropriate dilutions) were added and 
incubated for 3-4 hours at 37.degree. C. Plates were washed. One hundred 
(100) .mu.l/well biotinylated porcine somatotropin or radiolabeled porcine 
somatotropin were added and allowed to bind for 2.5-3 hours at 37.degree. 
C. Plates were washed. 
When radiolabeled porcine somatotropin was used, wells from flexible plates 
were cut and individual wells placed into test tubes for counting on a 
gamma counter. 
When biotinylated porcine somatotropin was used, one hundred (100) 
.mu.l/well of an appropriate dilution of streptavidin-horseradish 
peroxidase conjugate (Bethesda Research Laboratories) was added and 
incubated at 37.degree. C. for 30 minutes. Plates were washed thoroughly. 
One hundred (100) .mu.l/well OPD substrate was added. Upon completion of 
color change, 50 .mu.l/well of 12% sulfuric acid was added and the 
absorbance was quantified using a Titertek MultiSkan. Several wells were 
positive for pST and were chosen for cloning. 
EXAMPLE 9 
Cloning of Hybridomas: Cells in a desired well were carefully suspended. A 
small aliquot was removed and cells were counted using a hemocytometer. 
Cells were diluted to a concentration of 5 cells/ml in growth media 
containing 10% conditioned media. Cells were dispensed into 96 well trays 
at 0.2 ml/well. Plates were left undisturbed to allow growth of distinct 
colonies. Antibody production was evaluated by ELISA. Positive wells 
containing a single colony were picked and cells were further subcloned to 
determine their clonality. Desired cells which proved to be monoclonal 
were frozen for future use. 
EXAMPLE 10 
Monoclonal antibodies produced from N27-immunized mice are capable of 
differentiating between native and recombinant porcine somatotropin. In 
solid phase assays, supernatants bound specifically to native porcine 
somatotropin at dilutions below 1:4000 (Table 1), while ascites bound at 
dilutions less than 1:10.sup.6 (Table 2). None of the monoclonal 
antibodies had significant binding towards recombinant porcine 
somatotropin in these assays. 
EXAMPLE 11 
Capture assays were performed to assess relative affinity of the monoclonal 
antibodies toward labeled porcine somatotropin in a liquid phase. In these 
assays, 9A5 ascites had a high affinity for native porcine somatotropin 
and no significant affinity for recombinant porcine somatotropin. An 
anti-pST positive control bound identically to both somatotropins (FIG. 
2). 
EXAMPLE 12 
The ability to bind to labeled porcine somatotropin in porcine serum was 
assessed in capture assays where serum was "spiked" with porcine 
somatotropin conjugated to biotin. Significant binding was observed (FIG. 
3). 9A5 Ascites bound to porcine somatotropin at concentrations as low as 
6 ng/ml (0.6 ng/well). Detectability is similar to that obtained with 
radioimmunoassays. 
Monoclonal antibodies specific to native but not recombinant porcine 
somatotropin were shown to bind in solid phase assays and liquid phase 
capture assays. The antibodies were able to bind to labeled porcine 
somatotropin in spiked serum at low concentrations. No binding was 
detected in unspiked samples. 
The hybridoma producing the antibodies 9A5 was deposited with The American 
Type Culture Collection (ATCC), Rockville, Md. and assigned Accession No. 
HB10308. 
TABLE 1 
______________________________________ 
Nat pST Rec pST Nat pST Rec pST 
1/dil Mean SD Mean SD Mean SD Mean SD 
______________________________________ 
Positive Control 9A5 
4 2.25 .30 2.18 .33 1.81 .03 0 .06 
16 2.73 .23 2.71 .26 2.77 .01 .18 0 
64 2.46 .36 1.27 .14 3.10 .46 .20 .04 
256 1.49 .25 .56 .04 2.45 .08 .20 .04 
1024 0.47 .11 .15 0 .95 .08 .08 .07 
4096 0.18 .01 .06 .15 .40 .03 .25 .04 
16384 0.05 0 .08 .14 .14 .01 .18 .06 
5F1 9C7 
4 1.80 .05 0 .03 0.28 .29 0 .03 
16 2.73 .05 .16 .10 2.14 .03 .13 .06 
64 2.83 .10 .18 .08 1.34 .14 .23 .11 
256 1.96 .08 .22 .12 .53 .02 .23 .11 
1024 .56 .05 .07 .09 .13 .02 .13 .12 
4096 .28 .03 .23 .11 .13 .01 .19 .05 
16384 .12 .01 .14 .10 .07 .03 .17 .08 
Negative Control 
4 0 .01 0 .02 
16 .02 0 .13 .02 
64 .07 0 .17 .01 
256 .07 .01 .18 .08 
1024 0 .01 .09 .06 
4096 .04 .01 .18 0 
16384 .07 .05 .15 .02 
______________________________________ 
SD = standard deviation 
TABLE 2 
______________________________________ 
Nat pST Rec pST Nat pST Rec pST 
1/dil 
Mean SD Mean SD Mean SD Mean SD 
______________________________________ 
Positive Control 9A5 
10.sup.3 
1.78 .06 1.48 .32 1.85 .03 0 .1 
10.sup.4 
2.63 .15 2.39 0.07 2.76 .04 .29 .1 
10.sup.5 
1.60 .62 1.05 .12 2.41 .04 .15 .03 
10.sup.6 
0.27 .1 0.46 .08 0.64 .05 .13 .02 
10.sup.7 
0 .02 0.10 .07 0.09 .02 .17 .1 
10.sup.8 
0.08 .01 0.27 .06 0.09 .02 .24 .01 
10.sup.9 
0 .02 0.16 .03 0 .01 .11 .07 
5F1 Negative Control 
10.sup.3 
1.94 .05 .14 .03 .76 .04 0 .05 
10.sup.4 
2.70 .04 .13 .03 .06 .04 .24 .04 
10.sup.5 
1.73 .16 .26 .05 .08 .05 .11 0 
10.sup.6 
0.45 .06 .26 .03 .08 .02 .10 .01 
10.sup.7 
0.08 .07 .15 .10 0 .02 .12 .14 
10.sup.8 
0.10 .03 .31 .05 .12 .12 .31 0 
10.sup.9 
0.01 .01 .19 .11 0 0 .19 .16 
______________________________________ 
SD = standard deviation