Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 5 Proteins, Proteomics, and the Dysproteinemias
or luminescent labels ( Parra et al. , 2005a, 2005b ). These
labels extend the sensitivity and reproducibility of assays.
Most immunoassays for specific serum proteins are based
on ELISA formats and include assays for canine CRP, porcine
and bovine Hp and a cross-species SAA immunoassay
that can be used in most species as the antibody shows
cross-species specificity for SAA ( Eckersall et al. , 1989 ;
Eckersall et al. , 2001 ; Hiss et al., 2003 ; Sheffield et al. ,
1993 ; Yule et al. , 1997 ). Immunoassays based on ELISA
or related formats have been developed for low-abundance
proteins found in serum that are used as biomarkers for
disease in particular tissues. Thus, assays have been developed
for biomarkers such as troponin I ( Spratt et al. , 2005 )
as a cardiac biomarker and trypsin-like immunoreactivity
as a biomarker for pancreatic disease and intestinal malabsorption
( Fetz et al. , 2004 ; Steiner et al. , 2000 ; Williams
and Batt, 1988 ).
d . Immunochromatography
Attempts have been made to produce immunoassays in
formats that can be used in practice or on a farm as pointof-care
assays. Latex agglutination has been used in tests
in which visible agglutination can be observed and has
been used in assays for IgG for confirmation of transfer
of antibody from colostrum. A more recent innovation has
been the development of immunochromatography in which
application of a sample to a test slide leads to diffusion of
a sample and reagent along a membrane and appearance of
a colored line for a positive result. A test method based on
this principle for canine CRP has been produced and was
assessed for identification of animals with an acute phase
response ( McGrotty et al. , 2004 ). This technology should
be able to produce rapid, in practice, testing for proteins,
particularly when there is a large difference in concentration
between health and disease states.
2 . Biochemical Assays
Apart from albumin and fibrinogen (see Sections V.A and
V.B), assays for a number of other serum proteins have
been developed based on their chemical, physical, or biological
activity. These generally have the advantage that
they can be performed on automated biochemical analyzers,
do not need the instrumentation required for the more
sensitive immunoassays, and are usually applicable in all
species.
Haptoglobin, an acute phase protein, can be measured
by making use of its high affinity for hemoglobin
and subsequent preservation of the peroxidase activity
of this protein at low pH. Interference by albumin in this
assay was eliminated by use of a novel reagent, which
also incorporated the chromogen ( Eckersall et al. , 1999 ).
Ceruloplasmin, a copper-containing acute phase protein,
can be estimated by measuring its endogenous oxidase
activity ( Ceron and Martinez-Subiela, 2004 ). Methods
have been described for measuring the protease inhibitors,
α 1 -antitrypsin and α 1 -antichymotrypsin (Conner et al. ,
1988a, 1988b, 1989 ), based on the specificity of their
action, though these assays have not been automated.
3 . Quality Assurance and Quality Control
An impediment to the greater use of specific protein assays
is the lack of primary reference standards for calibration,
quality control material, and the availability of quality
assurance schemes. None of these is presently available
from commercial sources. However, the European Union
has funded a project to establish reference material for
bovine and porcine serum proteins and to establish a quality
assurance scheme, but at the time of the project too few
laboratories were running specific protein assays to enable
a scheme to be viable ( Skinner, 2001 ). As more interest
in the use of specific assays expands, this could be reactivated.
Laboratories running the tests routinely should prepare
their own material for internal quality control.
VI . NORMAL PLASMA AND SERUM
PROTEINS
Although 289 proteins have been reported in human
plasma ( Anderson and Anderson, 2002 ), only 70 assays
have been validated to the extent of reporting reference
intervals. Of these, only about 10 are currently employed
for diagnostic testing in domestic animals. Table 5-3 gives
an overview of serum proteins, but as proteins are under
genetic control, variations occur between individuals and
especially between species. Biochemical and pathophysiological
features of albumin and several globulins that
are being used for diagnosis of disease are described next,
grouped by their function(s).
A . Albumin
1 . Biochemistry
Albumin is the major single protein found in serum and
constitutes 35% to 50% of the total serum protein. Bovine
serum albumin, when synthesized and secreted by the
hepatocytes, is a nonglycosylated protein of 583 amino
acids with a molecular weight of 66.4 kDa and a pI of
pH 5.6 (accession number P02769, UniProt database at
www.ebi.uniprot.org ). Based on X-ray crystallographic
studies on human serum albumin, the structure is a heartshaped
protein with three homologous domains ( Fig. 5-5 )
( Nakajou et al., 2003 ) containing 67% of the protein as
α -helix with no β -sheet ( Curry et al. , 1998 ). A notable feature
of the primary structure of albumin is that there is an
odd number of cysteine residues (35 in total) so that, after
the formation of 14 cysteine-cysteine internal disulfide