Common marmosets (Callithrix jacchus) are New World primates that are used in biomedical research.1 Marmosets are an attractive species for use in biomedical research because of their small size, low cost of maintenance, multiparous reproduction, lack of susceptibility to certain zoonotic diseases, and relatively short life span (compared with that of other primates).2 However, gastrointestinal tract inflammation is commonly seen in common marmoset colonies, with a prevalence as high as 60% in laboratory settings.3 The cause of this disease remains unknown. Clinical signs include diarrhea, chronic progressive weight loss in adults despite a good appetite, failure of juveniles to thrive, anemia, and hypoproteinemia. The disease currently is believed to be a variant of inflammatory bowel disease, particularly when there is lymphocytic inflammation. The disease currently is known as CLE; however, it was previously referred to as marmoset wasting syndrome3 and bone and gastrointestinal disease of marmosets.4 The disease leads to death, and diagnosis is made during necropsy.
Hypoproteinemia associated with this condition is presumably attributable to intestinal loss of protein. Thus, detection of enteric protein loss could facilitate screening and removal of affected marmosets, thereby reducing the negative impact of this disease on studies involving the use of marmosets. Excretion of 51Cr-albumin in feces is the test of choice for detecting enteric protein loss, but this modality is only available at a few institutions because it is associated with radiation hazards.5 α1-Proteinase inhibitor is a serum protein that is lost into the intestinal tract at a rate similar to that of albumin because it has a molecular weight similar to that of albumin. It is resistant to degradation by digestive and bacterial enzymes within the intestinal lumen; therefore, α1-PI in the feces can be detected with immunoassays, and this method has been used to detect enteric protein loss in cats,6,7 and dogs.8–10
In another study11 conducted by our research group, the purification and partial characterization of α1-PI in marmoset serum were described. The aims of the study reported here were to develop and analytically validate an ELISA for the measurement of α1-PI concentrations in serum and fecal samples from marmosets and to calculate reference intervals for α1-PI concentrations in serum and feces from healthy marmosets.
Materials and Methods
Sample
Leftover serum (n = 42) and fecal (23) samples of common marmosets that had been submitted to the Gastrointestinal Laboratory at Texas A&M University for diagnostic testing as part of routine management procedures at 2 research coloniesa,b were used for assay development and validation. The marmosets had been euthanized. Serum samples from the euthanized marmosets were stored at −80°C until shipped on dry ice to the gastrointestinal laboratory. Paired serum samples and naturally passed fecal samples were obtained from 30 marmosets at the 2 research colonies and used to calculate reference intervals for concentrations of serum and fecal α1-PI concentrations in healthy marmosets. Collection of samples was approved by each institution's animal use protocols (protocols 1259CJ and 06120X, respectively). Paired fecal and serum samples were obtained to ensure that the individual common marmosets did not have α1-PI deficiency, as has been described for humans.12 Fecal samples were obtained for 3 consecutive days because there can be considerable day-to-day variation in fecal α1-PI concentrations in other species.c Fecal samples were collected into preweighed polypropylene tubesd as soon as possible after voiding and were stored at −80°C until shipped on dry ice to the gastrointestinal laboratory.
α1-PI in marmoset serum
For the present study, α1-PI was purified from pooled marmoset serum by use of immunoaffinity chromatography and ceramic hydroxyapatite chromatography, as described elsewhere.11 Polyclonal antibodies were raised in a New Zealand White rabbit by inoculation of purified marmoset α1-PI emulsified in Freund complete and Freund incomplete adjuvants by a commercial antibody production service,e and specificity of antibodies was evaluated with western blot analysis, as described elsewhere.11 Rabbit anti-marmoset α1-PI antibodies were purified by use of affinity chromatography with a chromatography columnf as per the manufacturer's instructions. Lipoprotein precipitation was performed, and antiserum then was applied to the column. Buffer exchange (75mM Tris-HCl and 150mM NaCl; pH, 8.0) was performed by use of a disposable gel filtration column.g Buffer exchange of purified antibodies was performed with PBS solution (pH, 7.2); purified antibodies were concentrated to approximately 1 mg/mL and stored at −80°C.
One aliquot of the purified antibodies was dialyzed with 200mM carbonate-bicarbonate (pH, 9.4) and coupled with horseradish peroxidase by use of a commercially available kith as per the manufacturer's instructions. The conjugate was dialyzed against 25mM Tris-HCl and 150mM NaCl (pH, 8.0) and purified by use of a commercially available purification kit with nickel-chelated agarose.i Purification was followed by another buffer exchange with 100mM sodium phosphate and 150mM NaCl (pH, 7.2). The resultant antibody solution was mixed with 2 parts of a commercial conjugate stabilizer solutionj as per the manufacturer's recommendations and stored at −20°C.
Extraction of fecal samples
Fecal samples were thawed to room temperature (approx 22°C) before extraction. Fecal samples (approx 1.0 g of feces [wet weight]) were extracted in PBS solution supplemented with 5% newborn calf serum, 1% Triton X-100, and 0.25mM thimerosal (dilution, 1 part feces:5 parts extraction solution). Samples were mixed in a vortex device for 20 minutes and then centrifuged at 2,100 × g and 5°C for 20 minutes. Supernatants (ie, fecal extracts) were harvested with serum filtersk and stored frozen at −80°C until further analysis.
Preparation of serum samples, fecal extracts, and standards
Serum samples were diluted in 0.05M sodium phosphate, 0.02% sodium azide, and 0.5% bovine serum albumin (pH, 7.5; final dilution, 1:64,000). Fecal extracts were thawed and diluted in 0.05M sodium phosphate, 0.02% sodium azide, and 0.5% bovine serum albumin (pH, 7.5; final dilution, 1:200). A 1-mg/mL solution of purified marmoset α1-PI was diluted with 0.05M sodium phosphate, 0.02% sodium azide, and 0.5% bovine serum albumin (pH, 7.5) to achieve concentrations of 100, 50, 20, 10, 5, 2, and 1 μg/L.
ELISA
We added 100 ng of affinity-purified anti-marmoset α1-PI antibodies diluted in 100 μL of 200mM carbonate-bicarbonate (pH, 9.4) to each well of a 96-well enhanced-binding ELISA platel; plates were incubated at 37°C for 1 hour. Each well was washed 3 times with wash buffer (200 μL of 25mM Tris-HCl, 150mM NaCl, and 0.05% polyethylene glycol sorbitan monolauratem; pH, 8.0). Then, blocking buffer (200 μL of 25mM Tris-HCl, 150mM NaCl, 0.05% polyethylene glycol sorbitan monolaurate, and 10% bovine serum albumin; pH, 8.0) was added to block nonspecific binding sites; plates were incubated again at 37°C for 1 hour, which was followed by 3 washes with wash buffer. Coated plates were stored at 4°C for up to 2 weeks.
An aliquot (100 μL) for each of 7 standard solutions, blanks, and diluted test samples of serum or fecal extracts was applied to wells of each plate (in duplicate). Blanks consisted of assay buffer (25mM Tris-HCl, 150mM NaCl, 0.05% polyethylene glycol sorbitan monolaurate, and 0.5% bovine serum albumin; pH, 8.0). Plates were incubated at 37°C for 1 hour, which was followed by 3 washes with wash buffer. Then, horseradish peroxidase-conjugated antibody diluted in assay buffer (50 ng/well) was added, and plates were incubated at 37°C for another hour. Plates were again washed 3 times with wash buffer.
Plates were developed by the addition of a 3,3′,5,5′-tetramethyl benzidinen substrate. Plates were incubated for 5 minutes, and the reaction then was stopped by the addition of a stopping buffer (4M acetic acid and 0.5M sulfuric acid). Measurements for plates were obtained at 450 nm by use of an automated plate reader.o A commercial software packagep was used to calculate a 5-variable logistic curve fit. The calculated curve was used to determine α1-PI concentrations in the test samples. During the initial developmental phase, several variables of the assay, including titration of the primary and secondary antibody concentrations, were optimized to yield the best performance.
Assay validation
The ELISA was validated by determining the lower limit of detection, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. Validation procedures were performed separately for serum samples and fecal extracts. The lower limit of detection of the assay was determined by loading 10 sets of blank control samples as unknowns and calculating the mean + 3 SD for 20 replicates. Four serum samples and 4 fecal extracts were used to determine dilutional parallelism. Two-fold dilutions of serum samples (1:64,000 to 1:512,000) and fecal extracts (1:1,000 to 1:8,000) were used, and the percentage of standard antigen recovery (ie, O:E; calculated as [observed concentration/expected concentration] × 100) was determined for each dilution. Spiking recovery was determined by adding pure marmoset α1-PI (10, 20, 50, and 100 μg/L) to 4 serum samples and 4 fecal extracts. The percentage of standard antigen recovery was calculated for each sample. Intra-assay variability was determined by evaluating 4 serum samples and 4 fecal extracts multiple times within the same assay. The intra-assay CV (CV = [SD/mean] × 100) was calculated for each sample. Interassay variability was determined by evaluating 4 serum samples and 4 fecal extracts within multiple consecutive assays; the interassay CV was then calculated for each sample.
Position effect
Four serum samples and 4 fecal extracts were randomly assigned (in duplicate) by use of online softwareq to 32 positions on an ELISA plate. The CV was calculated and used to compare concentrations of the samples.
Statistical analysis
Commercially available softwarer was used for the statistical analysis. Significance was set at values of P < 0.05. Concentrations for the 32 positions of each of the 4 serum samples and 4 fecal extracts were compared with a Kruskal-Wallis test by ranks.
A reference interval for α1-PI concentrations in serum samples and for 3-day mean and 3-day maximum concentrations in fecal extracts from healthy marmosets with no signs of CLE was calculated by use of the central 95th percentile. Additionally, the mean CV for the 3 days of fecal sample collection.
Results
Standard curves
Standard curves for the marmoset α1-PI ELISA were reproducible (Figure 1). Lower limit of detection of the assay was 0.006 μg/L.
Representative standard curve of absorbance measured at 450 nm for the α1-PI ELISA used to measure concentrations in serum and fecal samples obtained from common marmosets (Callithrix jacchus). The curve was based on a 5-variable logistic fit. Concentrations of the 7 standards were 100, 50, 20, 10, 5, 2, and 1 μg/L.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.259
Dilutional parallelism
The O:E values of serial dilutions for dilutional parallelism were determined. Mean ± SD value for the 4 serum samples was 117.1 ± 5.6% (range, 112.2% to 123.0%; Table 1). Mean value for the 4 fecal extracts was 106.1 ± 19.7% (range, 82.6% to 130.2%; Table 2).
Results of dilutional parallelism for an α1-PI ELISA for 4 serum samples obtained from common marmosets (Callithrix jacchus).
| Sample | Dilution | Observed (μg/mL) | Expected (μg/mL) | O:E (%) | Mean (%) |
|---|---|---|---|---|---|
| 1 | 1:64,000 | 18.3 | NA | NA | — |
| 1:128,000 | 10.0 | 9.2 | 109.5 | — | |
| 1:256,000 | 5.2 | 4.6 | 114.4 | — | |
| 1:512,000 | 2.6 | 2.3 | 113.6 | 112.5 | |
| 2 | 1:64,000 | 18.7 | NA | NA | — |
| 1:128,000 | 10.5 | 9.4 | 112.5 | — | |
| 1:256,000 | 5.4 | 4.7 | 115.5 | — | |
| 1:512,000 | 2.5 | 2.3 | 108.5 | 112.2 | |
| 3 | 1:64,000 | 19.5 | NA | NA | — |
| 1:128,000 | 11.0 | 9.7 | 112.6 | — | |
| 1:256,000 | 6.2 | 4.9 | 126.7 | — | |
| 1:512,000 | 3.2 | 2.4 | 129.8 | 123.0 | |
| 4 | 1:64,000 | 17.4 | NA | NA | — |
| 1:128,000 | 10.0 | 8.7 | 115.3 | — | |
| 1:256,000 | 5.3 | 4.4 | 121.7 | — | |
| 1:512,000 | 2.7 | 2.2 | 124.7 | 120.6 |
NA = Not applicable.
Results of dilutional parallelism for an α1-PI ELISA for 4 fecal extracts obtained from common marmosets.
| Sample | Dilution | Observed (μg/mL) | Expected (μg/mL) | O:E (%) | Mean (%) |
|---|---|---|---|---|---|
| 1 | 1:1,000 | 7.8 | NA | NA | — |
| 1:2,000 | 3.9 | 3.9 | 100.1 | — | |
| 1:4,000 | 1.8 | 1.9 | 93.4 | — | |
| 1:8,000 | 0.5 | 1.0 | 54.2 | 82.6 | |
| 2 | 1:1,000 | 24.9 | NA | NA | — |
| 1:2,000 | 15.4 | 12.5 | 123.6 | — | |
| 1:4,000 | 8.4 | 6.2 | 134.5 | — | |
| 1:8,000 | 4.1 | 3.1 | 132.6 | 130.2 | |
| 3 | 1:1,000 | 17.4 | NA | NA | — |
| 1:2,000 | 9.9 | 8.7 | 114.5 | — | |
| 1:4,000 | 5.0 | 4.3 | 114.6 | — | |
| 1:8,000 | 2.2 | 2.2 | 100.8 | 110.0 | |
| 4 | 1:1,000 | 12.2 | NA | NA | — |
| 1:2,000 | 6.9 | 6.1 | 112.9 | — | |
| 1:4,000 | 3.4 | 3.0 | 112.6 | — | |
| 1:8,000 | 1.2 | 1.5 | 79.0 | 101.5 |
See Table 1 for key.
Spiking recovery
The O:E values of spiking recovery were determined. Mean ± SD value for the 4 serum samples was 102.9 ± 12.1% (range, 86.8% to 115.8%; Table 3). Mean value for the 4 fecal extracts was 97.9 ± 19.0% (range, 83.0% to 125.1%; Table 4).
Results of spiking recovery for an α1-PI ELISA for 4 serum samples obtained from common marmosets and spiked with 3 concentrations of α1-PI.
| Sample | Amount added (μg) | Observed (μg/mL) | Expected (μg/mL) | O:E (%) | Mean (%) |
|---|---|---|---|---|---|
| 1 | 0 | 17.6 | NA | NA | — |
| 10 | 29.1 | 27.6 | 105.4 | — | |
| 25 | 48.4 | 42.6 | 113.7 | — | |
| 50 | 86.8 | 67.6 | 128.4 | 115.8 | |
| 2 | 0 | 21.2 | NA | NA | — |
| 10 | 32.4 | 31.2 | 104.2 | — | |
| 25 | 49.2 | 46.2 | 106.7 | — | |
| 50 | 76.6 | 71.2 | 107.7 | 106.2 | |
| 3 | 0 | 34.0 | NA | NA | — |
| 10 | 45.0 | 44.0 | 102.3 | — | |
| 25 | 60.0 | 59.0 | 101.8 | — | |
| 50 | 87.2 | 84.0 | 103.8 | 102.6 | |
| 4 | 0 | 32.7 | NA | NA | — |
| 10 | 38.5 | 42.7 | 90.2 | — | |
| 25 | 50.5 | 57.7 | 87.4 | — | |
| 50 | 68.5 | 82.7 | 82.8 | 86.8 |
See Table 1 for key.
Results of spiking recovery for an α1-PI ELISA for 4 fecal extracts obtained from common marmosets and spiked with 3 concentrations of α1-PI.
| Sample | Amount added (μg) | Observed (μg/mL) | Expected (μg/mL) | O:E (%) | Mean (%) |
|---|---|---|---|---|---|
| 1 | 0 | 10.0 | NA | NA | — |
| 10 | 23.4 | 20.0 | 117.0 | — | |
| 25 | 43.2 | 35.0 | 123.4 | — | |
| 50 | 81.0 | 60.0 | 135.0 | 125.1 | |
| 2 | 0 | 60.2 | NA | NA | — |
| 10 | 73.1 | 70.2 | 104.2 | — | |
| 25 | 82.9 | 85.2 | 97.4 | — | |
| 50 | 96.9 | 110.2 | 88.0 | 96.5 | |
| 3 | 0 | 10.9 | NA | NA | — |
| 10 | 18.0 | 20.9 | 85.8 | — | |
| 25 | 28.7 | 35.9 | 80.0 | — | |
| 50 | 50.7 | 60.9 | 83.2 | 83.0 | |
| 4 | 0 | 43.1 | NA | NA | — |
| 10 | 52.4 | 53.1 | 98.8 | — | |
| 25 | 59.7 | 68.1 | 87.7 | — | |
| 50 | 69.7 | 93.1 | 74.9 | 87.1 |
See Table 1 for key.
Intra-assay and interassay variability
Intra-assay variability was determined for 4 serum samples and 4 fecal extracts. The CV for the serum samples was 2.4%, 2.4%, 2.6%, and 4.1%, respectively, and the CV for the fecal extracts was 2.6%, 5.5%, 6.6%, and 6.8%, respectively. Interassay variability was determined for 4 serum samples and 4 fecal extracts. The CV for the serum samples was 1.7%, 3.1%, 3.1%, and 6.3%, respectively, and the CV for the fecal extracts was 5.1%, 8.1%, 10.4%, and 13.8%, respectively.
Position effect
No significant (P = 0.964) position effect was evident for the ELISA plate. Median CV among the positions was 3.5% (range, 2.3% to 6.8%).
Reference interval
Reference intervals for α1-PI concentrations in serum and 3-day mean and 3-day maximum concentrations in fecal extracts were calculated. The reference interval for serum concentrations was 1,254 to 1,813 μg/mL. The reference interval was 11.5 to 42.2 μg/g of feces for the 3-day mean fecal concentration and 13.2 to 51.2 μg/g of feces for the 3-day maximum fecal concentration.
Median CV of α1-PI concentrations in fecal extracts among the 3 days of fecal sample collection was 23.0% (range, 3.3% to 77.0%).
Discussion
An ELISA for the quantification of α1-PI in serum samples and fecal extracts from healthy marmosets was successfully developed. The assay was precise and reproducible for both types of biological samples, as determined by use of currently accepted criteria.13 However, the assay had limitations with respect to linearity and accuracy. Mild deviations (O:E > 120% but < 130%) from the generally acceptable O:E range of 80% to 120% were detected for dilutional parallelism and spiking recovery of serum samples. However, more pronounced deviations were detected for both dilutional parallelism and spiking recovery of fecal extracts. This discrepancy in performance between serum samples and fecal extracts may have been attributable to a possible matrix effect when fecal extracts were used. The greatest underestimation of α1-PI concentrations occurred when the expected concentration was < 2 ng/g of feces during dilutional parallelism (O:E, 54.2% and 79.0% for fecal samples 1 and 4, respectively). However, given that the assay is intended to be used to detect increases in fecal concentrations of marmoset α1-PI as evidence of gastrointestinal protein loss, issues with linearity in samples with a concentration < 2 ng/g of feces should not detract from the clinical usefulness of the assay. The specific reason for overrecovery by the assay is unknown, but this problem is generally attributed to the presence of high-affinity antibodies.14
The reference interval for concentrations of α1-PI in serum of marmosets was 1,254 to 1,813 μg/mL. This is comparable to serum α1-PI concentrations reported for dogs15 (732 to 1,802 μg/mL), cats16 (250 to 600 μg/mL), and humanss (900 to 1,200 μg/mL).
Fecal α1-PI concentrations of healthy cats range from 0.04 (or undetectable) to 1.72 μg/g of feces (median, 0.51 μg/g of feces), and fecal concentrations of healthy adult dogs range from < 2.2 to 26.8 μg/g of feces (median, 4.7 μg/g of feces). Fecal α1-PI concentrations were much higher for the 30 healthy marmosets of the study reported here; they ranged from 11.4 to 48 μg/g of feces (median, 27.1 μg/g of feces) for samples collected on 1 day. A reason for this higher concentration in marmosets is unknown.
Similar to results reported for dogs9 and cats,7 a high CV was calculated for common marmosets for the 3-days of fecal sample collection (median, 23.0%; range, 3.3% to 77.0%). Thus, there was considerable day-to-day variation of fecal α1-PI concentrations. This would appear to justify the need to obtain 3 consecutive fecal samples from common marmosets.
An important limitation of the present study was the low number of marmosets in the population used to determine the reference interval for both fecal extracts and serum samples. Ideally, reference intervals are calculated on the basis of results from approximately 120 healthy subjects. However, samples from only 30 marmosets were available for our use. Because the number of samples was limited, we did not conduct further investigations into the effects of storage or repeated freeze-thaw cycles on the recovery of marmoset α1-PI concentrations in serum samples or fecal extracts. Because there were only a limited number of samples available for validation purposes, a relatively narrow range of concentrations was represented that did not span the entire working range of the assay. Validation with samples that have a wider range of concentrations would have been preferable, but this was not feasible.
For the study reported here, the α1-PI ELISA described here was sensitive, precise, and reproducible, although there was suboptimal linearity for fecal samples that contained < 2 ng/g of feces and suboptimal accuracy (as assessed with spiking recovery) at both ends of the assay working range. Additional studies are necessary to determine the clinical use of this assay for the detection of protein-losing enteropathy in marmosets.
Acknowledgments
Supported by a grant from the National Institutes of Health (NIH R24 No. 1R24RR023344-01A2).
The authors thank Drs. Steven N. Austad and Corrina Ross for technical support and for providing marmoset serum and fecal samples.
ABBREVIATIONS
| α1-PI | α1-Proteinase inhibitor |
| CLE | Chronic lymphocytic enteritis |
| CV | Coefficient of variation |
| O:E | Observed-to-expected ratio |
Footnotes
Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, Tex.
Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Tex.
Steiner JM, Ruaux CG, Miller MD, et al. Intra-individual variability of fecal α1-proteinase inhibitor concentration in clinically healthy dogs (abstr). J Vet Intern Med 2003;17:445.
Fecal collection tube (101 × 16.5 mm; including spatula), Sarstedt AG & Co, Nümbrecht, Germany.
Lampire Biological Laboratories, Pipersville, Pa.
HiTrap NHS-activated HP chromatography column, GE Healthcare, Uppsala, Sweden.
Disposable PD-10 desalting column, GE Healthcare, Uppsala, Sweden.
EZ-Link plus activated peroxidase, Thermo Fisher Scientific Inc, Rockford, Ill.
Pierce conjugate purification kit, Thermo Fisher Scientific Inc, Rockford, Ill.
SuperFreeze conjugate stabilizer, Thermo Fisher Scientific Inc, Rockford, Ill.
Fisherbrand serum filter sytem (IB model), Fisher Scientific Inc, Pittsburgh, Pa.
MaxiSorp Nunc-Immuno plates, Thermo Fisher Scientific Inc, Rockford, Ill.
TWEEN-20, Sigma-Aldrich Corp, St Louis, Mo.
1-Step Ultra TMB-ELISA, Thermo Fisher Scientific Inc, Rockford, Ill.
Synergy 2 alpha microplate reader, BioTek, Winooski, Vt.
Gen5 data analysis software, version 1.05, BioTek Instruments Inc, Winooski, Vt.
Random integer generator, Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.org. Accessed Sep 24, 2013.
GraphPad Prism, version 5.0, GraphPad Software, San Diego, Calif.
Mela M, Smeeton W, Alexander G. The limitations f serum alpha-1 antitrypsin levels in patients with chronic liver disease and heterozygous alpha-1 antitrypsin deficiency (abstr). Gut 2011;60:A57–A58.
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