Spurious capillary zone electrophoresis pattern in hypercholesterolemic dogs

Abstract

Capillary zone electrophoresis (CZE) is a relatively new serum protein electrophoresis method with higher resolution than other electrophoretic techniques. Hypercholesterolemic dogs exhibit a peculiar CZE pattern. Specifically, they have a shoulder or peak immediately next to the albumin peak. We investigated the prevalence of this spurious peak in hypercholesterolemic dogs and its correlation with the serum cholesterol concentration. Moreover, possible discrepancies between the CZE and spectrophotometric (bromocresol green [BCG] method) albumin concentrations in those animals were evaluated, as well as the accuracy in measuring albumin by a different CZE fractionation system. We retrospectively enrolled 500 hypercholesterolemic and normotriglyceridemic dogs. Each electrophoretic curve was inspected visually to identify a spurious peak (prevalence of 68.8%). We chose 120 dogs to further investigate the albumin concentration; CZE albumin was significantly higher than measured using the BCG method. A weak but significant correlation (r = 0.412; p <0.0001) was observed between the magnitude of the spurious peak and the serum cholesterol concentration. Finally, the significant difference between CZE and BCG albumin measurement disappeared (p = 0.92) when the spurious peak was considered as α1-globulins instead of albumin.

Serum protein electrophoresis (SPE) is considered a reference method for the evaluation of serum protein classes (namely albumin, and α1-, α2-, β-, and γ-globulins). ¹⁶ SPE is used widely in veterinary medicine for research and diagnostic purposes, as well as to monitor treatment. Indeed, variations in protein fraction concentrations, together with an abnormal electrophoretic pattern, can help to identify various pathologic conditions, such as acute and chronic inflammation, infectious diseases,^20,44 and neoplasia.^18,30 Moreover, it can help to identify immunodeficiencies that are characterized by low levels of immunoglobulins, which usually migrate in β- and γ-globulin regions. ⁸ Furthermore, in canine species, electrophoretic flattening is considered a positive prognostic factor in evaluation of the treatment response in chronic infectious diseases, such as leishmaniasis and ehrlichiosis. ⁸

Three electrophoretic methods can be used to evaluate serum proteins: cellulose acetate, agarose gel (AGE), and capillary zone (CZE). The CZE system is a relatively new technique that has been validated for dogs and cats. ²⁰ Even though CZE is more expensive than the other methods, it offers some advantages, such as higher resolution, ¹⁸ high degree of automation, and similar sensitivity to AGE in detecting monoclonal peaks. ²²

In SPE, albumin is the most prominent peak on the anodal side of the electrophoretogram, ⁴⁰ and its concentration can be quantified by converting its electrophoretic percentage from the total protein concentration measured by the biuret method. ³² However, albumin concentration can also be measured using spectrophotometric methods. Indeed, the bromocresol green (BCG) method is a dye-binding method that is used routinely to measure the albumin concentration in both human ²¹ and veterinary medicine. ¹⁷ Nevertheless, the BCG dye is not an albumin-specific reagent; thus, it can also bind other serum proteins, such as the acute-phase proteins, ²¹ leading to false overestimation of the albumin concentration, which seems more severe in hypoalbuminemic dogs and horses. ³⁵ Overestimation of the albumin concentration was reported as well using the BCG method in heparinized canine plasma samples because of fibrinogen binding. ⁴³ In human medicine, overestimation has been reported for the CZE albumin concentration compared to bromocresol purple and immunoturbidimetric methods.^14,32 However, there are no similar reports in veterinary medicine, probably because of the difficulties to run nephelometry and immunoturbidimetry, which are considered the gold standard methods for serum albumin measurement.^14,32 Hypoalbuminemia is a common type of dysproteinemia, ¹⁶ and the accuracy of serum albumin concentration measurement is pivotal, especially to allow recognition and severity assessment of hypoalbuminemia, as well as for monitoring purposes.

Cholesterol, the main sterol in animals, can be obtained from a diet based on animal products or it can be synthesized, mostly by the liver, endocrine glands, and other tissues. Pure cholesterol and cholesterol esters are hydrophobic and insoluble in plasma, and they must be transported as lipoproteins. Plasma lipoproteins are very large molecules composed of lipids, phospholipids, and proteins, ⁷ and they can be classified according to their electrophoretic mobility (α–β lipoprotein regions) or, more frequently, according to their chemical and physical aspects (density, size, composition) determined by ultracentrifugation, in which case, they are classified as chylomicrons, very low–density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). ¹⁶ Chylomicrons are the main transporters of dietary lipids and serum triglycerides; VLDL, LDL, and HDL mainly contain cholesterol and are involved in the metabolism of endogenously produced lipids.^4,46,47

Some diseases (e.g., nephrotic syndrome) or comorbidities (e.g., cholestasis associated with inflammation) can lead to concurrent hypoalbuminemia and hypercholesterolemia. Indeed, it is pivotal to recognize any possible interference of hypercholesterolemia with the serum albumin concentration measurement to avoid clinical misclassification. ¹¹ Hypercholesterolemia, as well as the presence of different lipoprotein profiles, have been investigated in dogs.^46,47,48 Primary hyperlipidemia is reported in various breeds, such as Briards, ⁴⁵ Rough Collies, ²³ Shetland Sheepdogs, ³⁹ and Miniature Schnauzers.^38,49 Endocrine disorders, such as hypothyroidism, ³⁷ diabetes mellitus, and hyperadrenocorticism, ¹⁹ as well as other diseases, such as protein-losing nephropathy, ²⁵ cholestasis, ¹² and obesity,^10,31 are among the most frequent conditions associated with hypercholesterolemia.

During routine analyses in our diagnostic laboratory (BiEsseA Laboratorio Analisi Veterinarie, Milano, Italy), we observed a shoulder on the right side of the albumin peak (cathodal side) of serum CZE in dogs with hypercholesterolemia. To our knowledge, this electrophoretic pattern has not been reported previously in veterinary medicine. In human medicine, a cathodic shoulder on the albumin peak using CZE is reported with severe hyperlipidemia, especially with hypertriglyceridemia.^6,36 In veterinary medicine, interference as a result of hypertriglyceridemia has been reported as a cathodic peak in the α2-globulin region. ²⁸ The inclusion of this spurious peak within the albumin fraction could cause albumin overestimation and consequent underestimation of the globulin fractions, with increased risk of clinical misclassification.

Our aims were 1) to assess the prevalence of this spurious electrophoretic pattern in hypercholesterolemic dogs, 2) to compare CZE and BCG albumin measurement in hypercholesterolemic dogs, 3) to investigate correlation between the observed spurious CZE peak and serum cholesterol concentration, and 4) to propose an electrophoretic fractionation system to improve the accuracy of CZE albumin measurement in hypercholesterolemic dogs.

Materials and methods

Sample selection

We retrospectively searched the database of the commercial veterinary laboratory BiEsseA Laboratorio Analisi Veterinarie, from November 2019 to March 2021, for 500 canine serum samples with the following inclusion criteria:

• Availability of serum biochemistry, performed using an automated spectrophotometer (AU 480; Beckman Coulter) and including the following parameters: urea, creatinine, calcium, potassium, sodium, chloride, glucose, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyl transferase, amylase, lipase, total bilirubin, total protein (TP), albumin, globulin, albumin:globulin ratio, cholesterol, triglycerides, C-reactive protein.

• Serum triglyceride concentration (measured using the glycerol phosphate oxidase method) within-the-laboratory RIs (WRIs; 0.34–1.24 mmol/L).

• Serum cholesterol concentration (measured using the cholesterol esterase method) higher than the laboratory upper RI limit (URL; >7.51 mmol/L).

• SPE evaluated on fresh serum samples and performed by a CZE automated analyzer (Minicap; Sebia) using reagents provided by the manufacturer (Protein(E) 6 kit) with standard setting.

As part of our QA procedure, we monitored the reproducibility of results from 2 reference canine serum samples obtained from routine submissions and stored properly (with normal and pathologic electrophoretic patterns, respectively) run together with routine samples. ¹ Specifically, instrument analytical performance was judged as acceptable if CVs were <10% for each electrophoretic fraction. The CZE curves were inspected visually by 2 qualified operators (S. Rossi, ECVCP Diplomate; G. Mangiagalli, ECVCP resident) for a spurious peak on the cathodic side of the albumin peak. Both operators were blinded to the sample cholesterol concentration. The spurious peak might appear as an inflection point of variable amplitude and shape (Fig. 1).

Figure 1.

Examples of capillary zone electrophoresis (CZE) patterns in normocholesterolemic and hypercholesterolemic dogs, with different spurious peak shapes. A. CZE of a normal canine serum sample without a spurious peak (cholesterol concentration: 4.39 mmol/L). B. Right shoulder within the albumin peak (cholesterol concentration: 9.82 mmol/L). C. Isolated peak on the right side of the albumin peak (cholesterol concentration: 9.26 mmol/L). D. Distinct isolated peak on the right side of the albumin peak (cholesterol concentration: 18.67 mmol/L).

From the 500 samples selected, we selected 120 more-recent cases with the spurious peak (group A) and used them to assess the correlation between the magnitude of the spurious albumin peak and the concentration of cholesterol, as well as for comparison with BCG-measured albumin. As well, we selected 50 serum samples from a control population (group B), which included normocholesterolemic and normotriglyceridemic dogs selected in the same retrospective period as group A, according to the following inclusion criteria:

• Availability of the same serum biochemical parameters reported for group A.

• Serum triglycerides concentration WRI 0.34–1.24 mmol/L.

• Serum cholesterol concentration WRI 3.62–7.51 mmol/L.

• SPE performed using the same CZE automated analyzer as for group A. CZE curves were visually evaluated for the absence of the spurious peak.

For each serum sample, TP values (measured using the biuret method) and BCG albumin, globulins (calculated by subtraction of albumin from TP), cholesterol, and triglycerides were retrieved from the biochemistry panel. The concentrations (g/L) of the spurious electrophoretic peak (when present) and of the other protein fractions were calculated using electrophoresis software (Phoresis; Sebia) based on the TP concentration and the percentage of each fraction, calculated as the area under the curve (AUC) of each electrophoretic peak. Then, each electrophoretic curve from group A was modified manually to separate the spurious peak from the major albumin peak at the inflection point. The spurious peak was then included in the α1-globulin fraction, and the AUC of the modified albumin and the combined spurious peak plus α1-globulin fractions were automatically recalculated by the CZE instrument software.

Statistical analysis

Statistical analyses were performed (Analyse-it v.4.97 software) in Excel (v.2209; Microsoft). The distribution of data (cholesterol and albumin measured with both BCG and CZE) was assessed through the Shapiro–Wilk test. Because the distribution of data was not normal for group A, subsequent analyses were performed using nonparametric statistical tests. The correlation between BCG and CZE albumin in both groups, and the correlation between serum cholesterol and the spurious peak AUC in group A, were performed using the Spearman test. The comparison between the Δalbumin (i.e., the difference between albumin measured using CZE and BCG) in the 2 groups was performed using the Wilcoxon–Mann–Whitney U test. The same test was used to compare the results of the α1-globulin concentration before and after modification of the electrophoretogram in group A. The agreement between methods (BCG vs. CZE) for the evaluation of albumin concentration was performed using Passing–Bablok regression analysis and Bland–Altman difference plot testing in both groups. Statistical significance was set at p ≤ 0.05.

Results

Among the canine samples with hypercholesterolemia enrolled retrospectively, 344 of 500 (68.8%) had a spurious CZE peak; the remaining 156 dogs had hypercholesterolemia without a spurious electrophoretic peak. Moreover, dogs with a spurious peak had a higher median cholesterol concentration (median: 9.32 mmol/L; min–max: 7.50–17.92 mmol/L) compared to those without a spurious CZE peak (median: 8.39 mmol/L, min–max: 7.50–11.74 mmol/L; p <0.0001).

Based on the inclusion criteria, we further evaluated the most recent 120 of the 344 hypercholesterolemic dogs with the spurious peak (group A). Purebred dogs (n = 85; 70.8%) were mainly Golden Retrievers (n = 16; 13.3%) and German Shepherds (n = 6; 5%); the remaining dogs were crossbred dogs (n = 35; 29.2%). Of these 120 dogs, 52 (43.3%) were females and 65 (54%) were males (sex was not reported for 3 dogs); neutered or intact status was not reported. The median age was 8.8 y (range: 0.4–18 y). The control group (group B, n = 50), had a breed composition similar to that of group A (34 purebred, 68%; 16 crossbred, 32%). Females were 30 (60%); males were 20 (40%; sex was not reported for 4 dogs); neutered or intact status was not reported. The median age was 6.8 y (range: 0.5–14 y). None of the dogs belonging to group B had a spurious electrophoretic peak.

There was no statistical difference in albumin concentration between groups A and B measured with either BCG (p = 0.68) or CZE (p = 0.052; Table 1). Both groups had a strong significant positive correlation between CZE albumin and BCG albumin concentrations, which was higher in group B (r = 0.933; p <0.0001) than in group A (r = 0.881; p <0.0001).

Table 1.

Median and minimum–maximum values for albumin (g/L) measured both with bromocresol green (BCG) and capillary zone electrophoresis (CZE) in hypercholesterolemic dogs with the spurious electrophoretic peak (group A) and in normocholesterolemic dogs without the spurious electrophoretic peak (group B). No significant differences were observed in albumin concentration between groups A and B measured with either BCG or CZE.

Method	Group	Minimum	Median	Maximum
BCG	A	16.0	29.8	36.7
	B	18.0	29.6	41.3
CZE	A	18.2	36.6	48.2
	B	21.3	34.8	45.0

Passing–Bablok regression analysis of the albumin methods calculated for group A revealed a proportional bias when including the spurious peak within the albumin fraction (intercept: −0.12, 95% CI: −3.58 to 2.87; slope: 1.23, 95% CI: 1.12–1.34; Fig. 2). The Bland–Altman test highlighted significantly higher values of CZE albumin when including the spurious peak within the albumin fraction (bias 6.5, 95% CI: 6.08–6.91; p <0.0001; Fig. 2). For group B, Passing–Bablok regression analysis of the albumin methods revealed proportional bias (intercept: −0.47, 95% CI: −4.49 to 3.98; slope: 1.17, 95% CI: 1.04–1.29; Suppl. Fig. 1). The Bland–Altman test highlighted significantly higher values of CZE albumin compared to the BCG method (bias 4.65, 95% CI: 4.22–5.07; p <0.0001; Suppl. Fig. 1). However, the Δalbumin was significantly higher in group A than in group B (group A median: 6.4 g/L, min–max: −1.6 to 15.4 g/L; group B median: 4.9 g/L, min–max: 1–7.2 g/L; p <0.0001).

Figure 2.

Passing–Bablok regression analyses (left) and Bland–Altman difference plots (right) of albumin measurement with capillary zone electrophoresis (CZE) compared to the bromocresol green (BCG) method in hypercholesterolemic dogs with a spurious CZE peak A. before and B. after the removal of the spurious albumin peak.

A weak but significant correlation was observed between the spurious peak (expressed in g/L) and the cholesterol concentration in group A (r = 0.412; p <0.0001). In contrast, no significant correlation was observed between the spurious peak and BCG albumin (r = 0.094; p = 0.30) or TP (r = 0.108; p = 0.24) concentrations.

Removing the spurious peak from the CZE albumin fraction resulted in a slightly increased correlation between BCG albumin and corrected CZE albumin concentration in group A (r = 0.894; p <0.0001). Passing–Bablok regression analysis of the albumin methods revealed both a constant and a proportional bias after the removal of the spurious peak from the CZE albumin fraction in group A (intercept: −6.31, 95% CI: −10.24 to 3.66; slope: 1.21, 95% CI: 1.12–1.34; Fig. 2). Conversely, the Bland–Altman test highlighted the absence of significant differences between CZE and BCG albumin measurements when removing the spurious peak from the albumin fraction (bias −0.02, 95% CI: −0.4 to 0.36; p = 0.92; Fig. 2).

The spurious peak was then included in the α1-globulin fraction, resulting in a significant increase of the latter fraction (original α1 median: 3.3 g/L, min–max: 1.7–6.8 g/L; α1 + spurious peak median: 9.7 g/L, min–max: 7.1–14.5 g/L; p <0.0001). Specifically, observing the original data, only 6 of 120 (5%) dogs had an α1-globulin concentration that exceeded the URL for this specific fraction, whereas the inclusion of the spurious peak into the α1-globulin peak resulted in a concentration of this fraction above the URL in all dogs enrolled in group A. In group A, 35 of 120 (42%) dogs were hypoalbuminemic according to BCG results. However, considering the original data using CZE, only 9 of 120 (10.8%) dogs were classified as hypoalbuminemic. After removal of the spurious peak, 31 of 120 (37.2%) dogs had an albumin value that fell below the lower reference limit (LRL), meaning that inclusion of the spurious peak in the albumin fraction would result in misclassification of 22 (26.4%) dogs. Some discordant results were observed between CZE and BCG. Specifically, 4 dogs were classified as hypoalbuminemic with CZE but normoalbuminemic with BCG (however, all of the BCG results were close to the LRL). Eight dogs were classified as hypoalbuminemic with BCG but as normoalbuminemic using CZE even after removal of the spurious peak.

Discussion

We observed a peculiar electrophoretic pattern with a spurious peak located in the cathodic side of the albumin fraction in 344 of 500 (68.8%) hypercholesterolemic dogs, but never in normocholesterolemic dogs. The observed correlation between the spurious peak concentration and serum cholesterol concentration may suggest that cholesterol (most likely HDL cholesterol) is the main component of this peak, even though other possible components of the spurious peak could not be excluded completely. It is worth noting that, in our prevalence study, some hypercholesterolemic dogs did not have the spurious peak, thus suggesting that a different lipoprotein-bound cholesterol (namely HDL and LDL) could possibly affect the presence and the shape of the peak. Indeed, the spurious peak could appear in different shapes, even in samples with similar serum cholesterol concentrations. In some cases, it appeared as a cathodic shoulder on the albumin peak, whereas in others it was a more defined and isolated peak. Further studies, investigating the lipoprotein composition in hypercholesterolemic dogs with and without the spurious CZE albumin peak, may help in elucidating these aspects. These differences may rely on different lipoprotein migration properties based on their classes (e.g., VLDL, LDL, HDL), or on the classes involved in a specific pathologic process. Indeed, it is well known that lipoprotein classes and their concentrations can vary depending on the underlying disease. ⁴⁸

In our caseload, CZE albumin of dogs with the spurious peak was significantly higher than BCG albumin. Given that BCG may have low specificity in measuring serum albumin in dogs, ⁴³ the observation of higher CZE albumin values compared to BCG increased the suspicion of a possible overestimation of CZE albumin, most likely the result of increased cholesterol concentrations, leading to an inaccurate CZE albumin measurement. Thus, a correction of this electrophoretic alteration might be needed to obtain a more reliable albumin concentration in these patients. To reduce the interference of cholesterol with CZE albumin measurement, we considered different fractionation systems. Complete removal of the spurious peak was considered inappropriate given that it would lead to rearrangement of the electrophoretic fraction concentrations with subsequent overestimation of the globulin fractions (Suppl. Table 1). For this reason, we preserved the spurious peak and included it in the α1-globulin fraction; regardless of the electrophoretic method used, this region is not considered important diagnostically. ²⁴ In healthy dogs, α1-globulins are represented by a weak band or a flat region between the albumin and the α2-globulin fraction, and it is assumed that α1-lipoprotein (also called HDL), α1-antitrypsin, and α1-antichymotrypsin migrate in this region. ⁴²

Note that most of the information about the contribution of specific proteins to the different electrophoretic regions is based on human medicine. Indeed, studies about CZE α1 fraction protein migration are lacking in veterinary medicine, and it has been simply presumed that the protein migration may be similar to that reported in people. In human medicine, α1-lipoprotein may overlap the albumin fraction (Sebia Capillarys) or may appear as a diffuse increase between albumin and α1-acid glycoprotein (orosomucoid) band (Paragon CZE 2000; Beckman Coulter), depending on the analyzer.^24,26 We used a Minicap analyzer (Sebia) in our study, and, according to the manufacturer, α1-lipoprotein, which is the most prominent lipoprotein fraction reported in dogs,^9,27 could migrate as part of the albumin band as it does in human samples. The use of specific methods, such as immunoelectrophoresis^15,41 or lipoprotein electrophoresis,^5,29 would allow identification of the proteins that migrate in the spurious peak. However, given the retrospective nature of our study, it was not possible to perform specific additional evaluations. Nevertheless, based on reports in people, a major component of this peak could likely be represented by α1-lipoproteins in hypercholesterolemic dogs. Another hypothesis that would explain the presence of the spurious peak in hypercholesterolemic dogs could be that the binding between serum cholesterol and albumin may give a different electrophoretic mobility to the albumin itself. In human medicine ³⁴ and in rats, ¹³ it has been hypothesized that serum cholesterol (especially the non-esterified form) could be a serum albumin ligand, acting as a transport protein. Thus, the spurious peak could be considered a portion of the albumin fraction with different migration properties; similarly, it has been reported that drugs and hormones bound to albumin can lead to a different migration pattern of the albumin itself in SPE.^2,3 However, if the spurious peak was composed of molecule-bound albumin, the removal of this peak from the albumin fraction would possibly have resulted in a negative bias in CZE albumin compared to BGE albumin. On the contrary, the removal of the spurious peak from the albumin fraction resulted in better agreement, supported by the Bland–Altman test, with no significant differences between CZE and BCG albumin measurement, making the hypothesis of albumin-bound cholesterol less likely.

A positive bias has been reported in the measurement of human serum albumin concentration with CZE compared to bromocresol purple, turbidimetry, and nephelometry methods.^32,33 In the control group of our study (group B), the comparison of the 2 methods revealed a significant difference between CZE and BCG albumin concentrations, with higher CZE albumin concentrations. In dogs, it was suggested that the serum albumin concentration measured with the BCG method tended to be higher compared to SPE. ³⁵ However, in our study, the serum albumin concentration was lower when measured with the BCG method compared to CZE in control dogs. Further studies should be performed to better investigate this result. However, given our results, it is advisable to use the same method when comparing serial albumin measurements (e.g., for treatment monitoring).

One of our aims was to evaluate the agreement between CZE and BCG methods for the measurement of the albumin concentration. The inclusion of the spurious peak in the α1 fraction instead of the albumin fraction increased the correlation between CZE and BCG albumin concentration, and the Bland–Altman test revealed the absence of significant differences between these methods. Thus, the proposed electrophoretogram modifications would decrease the risk of clinical misclassification of the albumin concentration using CZE in hypercholesterolemic dogs, improving the agreement and correlation between CZE and BCG albumin concentrations. The risk of misclassification was further supported observing the noteworthy number of group A dogs (15 of 120) that would have been erroneously considered as normoalbuminemic rather than hypoalbuminemic if the spurious peak had been included in the albumin fraction. Moreover, the inclusion of the spurious peak among α1-globulins avoids the rearrangement of the globulin fractions, given that the α1 region was the only fraction affected. Nevertheless, the clinical relevance of an increase of the α1 fraction has not been reported in veterinary medicine.

Our study has some limitations. Lipoprotein electrophoresis and immunoelectrophoresis would be suggested to confirm our hypothesis about the presence of lipoproteins and to evaluate which classes of lipoproteins migrate in the albumin region in CZE in dogs. Moreover, we performed only the CZE method in our study. The use of different electrophoretic methods or instruments could increase the robustness of the results obtained. Even though no reports are present in the literature about spurious peaks in hypercholesterolemic dogs using AGE, investigations are warranted to verify if the higher albumin concentrations in hypercholesterolemic dogs found in our study can be detected also using other SPE techniques. Another limitation is that a standardized cutoff allowing isolation of the spurious peak was not feasible, and removal of the spurious peak was performed only on visual inspection of the curves. This is because of both the different shapes of the CZE albumin peak in normal samples ²⁰ and also the different appearances of the spurious peak in hypercholesterolemic dogs. Our approach, especially in samples with only an albumin right-shoulder spurious peak, may lead to false, slightly lower, CZE albumin concentrations, given that the manual cutoff could include a very small portion of the albumin peak. Nevertheless, our results highlighted better correlation between the 2 albumin measurements after removal of the spurious peak, thus supporting the methodology employed. Discordant results should be interpreted cautiously, especially in those cases in which only CZE albumin was lower than the RIs (considering that, in healthy animals, CZE albumin concentrations were higher than BCG albumin). Laboratory results should always be interpreted considering the history and other clinical findings. Finally, although we investigated the agreement between 2 methods that are used routinely to measure the albumin concentration, neither of these is the gold standard for albumin measurement. Nevertheless, the use of immunoturbidimetry or nephelometry is not feasible in routine testing, given the need for species-specific antibodies.