Development and analytical validation of a radioimmunoassay for the measurement of alpha₁-proteinase inhibitor concentrations in feces from healthy puppies and adult dogs

Abstract

-PI for 3 sampling days of > 13.9 µg/g or >21.0 µg/g, respectively, should be considered abnormal in dogs >1 year of age. Fecal cα 1 -PI concentrations in dogs <1 year of age were significantly higher and should be carefully interpreted in this age group.Key words: Alpha 1 -proteinase inhibitor; canine; fecal; immunoassay; puppies.Radioimmunoassay for fecal canine α 1 -proteinase inhibitor 477 methods usually are of higher analytical sensitivity, RIAs are usually easier to control and standardize than ELISAs. Although RIAs require special equipment and the use of a radioactive tracer, they have the advantage of not requiring purification and labeling of large amounts of antibodies from ideally 2 different individuals of a heterologous species, but usually only require a small volume of a heterologous antiserum. An ELISA for the measurement of cα 1 -PI concentration in fecal samples from dogs has previously been developed and analytically validated, 10 but due to the large amount of anti-cα 1 -PI antibodies needed, this smallscale manufacturing methodology was difficult to maintain long-term and has been discontinued, as the original source material was no longer available. Therefore, the current study was aimed at the development and analytical validation of a new RIA suitable for quantifying Fcα 1 -PI (as a clinical marker for GI protein loss in dogs) in a large number of samples, the de novo determination of a reference interval from a large number of healthy dogs, and to compare Fcα 1 -PI concentrations measured in healthy dogs above 1 year of age to that in healthy puppies, as it had been previously noticed that Fcα 1 -PI concentrations are occasionally increased in puppies, yet Fcα 1 -PI has never been studied in healthy puppies. Materials and methods Collection of fecal specimens from dogsSamples of naturally passed feces were collected from 73 healthy adult pet dogs of various breeds (median age: 3.8 years, range: 1.0-17.3 years), and from 28 healthy pet dog puppies of various breeds (mean age: 6.1 months; range: 1.8-10.7 months). All dogs had been vaccinated and dewormed regularly prior to fecal sample collection, did not show clinical signs of GI disease, and did not receive any medications or had any medical conditions known to affect the GI tract. As a high intra-individual variability of cα 1 -PI in fecal samples has been reported (Steiner JM, Ruaux CG, Miller MD, et al.: 2003, Intra-individual variability of fecal α 1 -proteinase inhibitor concentration in clinically healthy dogs. J Vet Intern Med 17:445. Abstract), samples were collected from 3 bowel movements from 3 consecutive days for each dog, placed into pre-weighed polypropylene tubes, a and immediately frozen until extraction. For extraction, feces were then thawed and extracted as previously described. 10 Briefly, aliquots of approximately 1.0 g wet weight were diluted 1:5 in phosphate buffered saline b (PBS) supplemented with 5% (v/v) newborn calf serum, c 1% (v/v) Triton X-100, d and 0.25 mM thimerosal. After homogenization by vortexing...

Serum concentrations of canine alpha1-proteinase inhibitor in cobalamin-deficient Yorkshire Terrier dogs

“…Hypocobalaminemia and hypoalbuminemia can occur concurrently, and both have been shown to be associated with chronic GI diseases (e.g., PLE and IBD) in dogs. 1 However, to the authors' knowledge, serum albumin and fecal and/or serum canine alpha 1 -proteinase inhibitor (cα 1 -PI) 9,10 have not yet been reported in COB-deficient dogs of breeds frequently diagnosed with PLE (e.g., Yorkshire Terriers).…”

Purification and partial characterization of α1-proteinase inhibitor in the common marmoset (Callithrix jacchus)

“…Fecal α 1 -PI may be increased even before hypoalbuminemia is observed , making it an early marker for intestinal protein loss (SL Vaden, 2002). Validated assays for measurement of fecal alpha 1 -proteinase inhibitor for detection of intestinal protein loss are available for humans (Karbach et al, 1983), dogs (Heilmann et al, 2011; Melgarejo et al, 1998), and cats (Burke et al, 2012). …”

Development and analytical validation of a radioimmunoassay for the quantification of alpha1‐proteinase inhibitor in serum and feces from the common marmoset (Callithrix jacchus)

“…The reason for this is unknown, however a similarly high CV has also been reported for dogs (median: 29.2%, range 0.0% to 102.0%). 15 One possible explanation for this could be the effect of water content, as the fecal samples were normalized to volume. This perhaps could have potentially been reduced by normalization to an independent variable like dry weight and this was not further explored in this study.…”

“…Sample collection and storage conditions used in dogs 15,16 and cats 7 were presumed to be appropriate. As the samples were collected, stored, extracted, and assayed around the same time from the two marmoset colonies, we do not expect this to affect our reference interval, however, changes in serum concentrations have been reported in dogs where samples were stored over a period of one year.…”