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Althouse GC. 1999;2:65-68 Use of commercially available ELISAs

   

Original research

March and April, 1999
Use of commercially available ELISAs to help determine estrous status in female swine

Gary C. Althouse, DVM, PhD, Dipl. ACT; James E. Hixon, MS, PhD

GCA: College of Veterinary Medicine, Department of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois 61802; JEH: College of Veterinary Medicine, Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802

Summary

Objective: To determine the accuracy of two commercially available ELISAs in discriminating between high and low serum progesterone (P4) concentrations in female swine.

Methods: Serum was randomly harvested from 65 crossbred females of various ages and parities. Progesterone concentrations were categorized as "high" (> 2.5 ng per mL) or "low" (<= 2.5 ng per mL) using the Progestassay(TM) (Synbiotics Corp., San Diego, California) ELISA assay. Similarly, P4 concentrations were categorized as "high" (> 5.0 ng per mL) or "low" (<= 5 ng per mL) using the Target (BioMetallics, Princeton, New Jersey) ELISA assay. The specificity and sensitivity for each ELISA were determined by comparing the results of both test kits to radioimmunoassay (RIA) methods.

Results: Serum P4 concentrations using RIA were either <= 0.42 ng per mL, or ranged from 3.35-17.87 ng per mL. Both ELISA test kits produce a blue color that is inversely related to serum P4 concentration. Both test kits allowed a clear distinction between high and low concentrations of P4 in swine serum. The Progestassay was 100% specific and 95.1% sensitive for differentiating low versus high P4 concentrations. The Target test kit was 100% specific and 86.9% sensitive. For the serum samples included in the present study, the lowest concentration, as measured by RIA, that tested as "high" by the Progestassay was 3.35 ng per mL, and the lowest RIA concentration that tested as "high" by the Target assay was 5.57 ng per mL. Statistically, no differences were found between the RIA and Progestassay (P=.25) but differences between the RIA and Target were significant (P=.007). Differences between the Progestassay and Target approached significance (P=.06).

Implications: Both the Progestassay and Target ELISA test kits work well at semi-quantitative measurement of serum P4 concentrations in swine. Both kits are simple to run and take only 15-25 minutes to perform on the farm. The ability to run these tests sow-side should make them a practical aid when investigating herd reproductive problems.

 

Keywords: serum, progesterone, ELISA, swine, reproduction

Received: August 11, 1998
Accepted: December 22, 1998

Progesterone (P4)-based enzyme-linked immunosorbant assays (ELISAs) have been used to assess various aspects of reproduction in cattle,1 horses,2 dogs,3 and swine.4-8 The ELISA is a semi-quantitative test which is simple to run in the field. It discriminates between high and low P4 concentrations through color development. In swine, ELISAs have been investigated for their usefulness in determining pregnancy via P4 concentrations in serum, plasma, blood, saliva, and feces.4-8

Serum P4 concentrations measure luteal function and, thus, the pregnancy and estrous status of the sow. During the estrous cycle, luteal tissue forms rapidly after ovulation, causing a dramatic increase in serum P4 concentration (Figure 1).9 By the second day post-ovulation, high serum P4 concentrations induce behavioral changes in the female,9 including nonreceptivity to the boar. P4 secretion by the corpora lutea (CL) continues to increase, reaching maximum serum concentrations as high as 33 ng per mL by day 11-12.9 High P4 secertions are maintained for an additional 2-3 days. At day 14-15 of the estrous cycle, if the uterus is nongravid, the CL rapidly regress within a 48-hour period, causing a drop in serum P4 concentrations to baseline levels (< 1 ng per mL).9 If, however, the animal is pregnant, the CLs and P4 secretion are maintained and the serum P4 concentration is expected to be>5.0.

Functional luteal tissue accompanied with high P4 production is therefore present throughout gestation. It can also accompany certain pathologic conditions (e.g., ovarian cysts) in swine. Partial or total luteinization of the cyst wall is not uncommon with multiple, large ovarian cysts (2-6 cm diameter).10-13 Plasma P4 concentrations of 5-15 ng per mL can be observed in animals with multiple, large ovarian cysts.11,13

Progesterone ELISAs were devised to assess pregnancy status indirectly by determining whether high P4 concentrations are present in serum at days 17-25 after service.4-8 However, nonreturn to estrus and ultrasound diagnosis can successfully determine pregnancy, thus diminishing the diagnostic usefulness of the P4-based ELISA for pregnancy checking. Progesterone ELISAs can still be of service to a clinician diagnosing management-related reproductive problems when there is a primary complaint of anestrus in the gilt pool or postweaned sows. In these situations, it is helpful to know the status of the ovaries.

The objective of this study was to evaluate two commercially available ELISAs for their accuracy in detecting high P4 concentrations in swine serum.

Materials and methods

Animals and blood sample collection

A total of 65 randomly selected crossbred females of various ages and parities from two commercial herds were used in this study. Blood was collected in the morning from all animals via jugular venipuncture directly into silicon-coated clot tubes. Samples were allowed to clot at room temperature over a 20-minute period, and the serum decanted from the clot tubes into two separate sterile tubes. One serum tube was immediately used for P4 testing using the commercial ELISA kits, and the second serum tube frozen at -70 degrees C for later analysis with radioimmunoassay (RIA).

Evaluation of the assays

The two commercially available P4 ELISA kits tested in this study were:

Assay of P4 concentrations in serum was performed with both ELISA test kits at room temperature per manufacturers’ instructions. Both ELISA test kits produce a blue color, which is inversely related to P4 concentrations. For the Progestassay kit, P4 concentration was interpreted as "high" or "low" by comparing test well color development to color development with the P4 cutoff standard (2.5 ng per mL) provided with the kit.

The Target test kit distinguishes between a "low" (< 1.0 ng per mL), "intermediate" (1.0-5.0 ng per mL), or "high" (> 5.0 ng per mL) P4 concentration. To facilitate comparability between assays and with the RIA, we interpreted Target ELISA results by comparing the test well color development to a printed color guide card provided by the manufacturer, except that we combined the "intermediate" and "low" categories so that results were regarded either as "low" (blue, <= 5.0 ng per mL) or "high" (clear, >5.0 ng per mL) (Figure 2).

Conventional RIA procedures were used to quantitate serum P4 concentrations in all samples.10,11 The interassay coefficient of variation (CV) for the measurement of P4 was 11.6%, with an intraassay CV of 7.8%. The lowest concentration of serum P4 the RIA can detect is 0.25 ng per mL. For this study, those serum samples with RIA values of < 1.0 ng per mL were categorized as "low" P4 concentrations, while P4 concentrations > 1.0 ng per mL were categorized as "high."5,9,14,15

Data analysis

The specificity of each ELISA kit was calculated as the percentage of samples identified as "low" (negative) by the ELISA test that were <1.0 ng per mL by the RIA. Sensitivity of each ELISA kit was calculated as the percentage of samples identified as "high" (positive) by the ELISA test that were >=1.0 ng per mL by the RIA. A McNemar’s test was used to compare the RIA to the Progestassay and the Target, and between the Progestassay and Target.

Results

Of the 65 blood samples collected, eight serum samples showed various degrees of hemolysis. When comparing the ELISA test kit P4 concentrations to their respective RIA values, no false readings were found, indicating that hemolysed serum does not appear to effect either test kit.

Manufacturer’s directions on both ELISA kits were easy to follow. Color development could more easily be assessed for the Progestassay kit against a nonreflective white background (e.g., notebook paper) for the test wells. The Target test provided its own white background.

Serum P4 concentrations measured using RIA ranged from the lowest sensitivity limit of the assay (0.25 ng per mL) to 17.87 ng per mL. Four samples (6.2%) fell into the "low" P4 concentration category by RIA (<= 0.42 ng per mL)(Figure 2). The remaining 61 serum samples (93.8%) ranged from 3.35-17.87 ng per mL (c +/- sd = 10.56 +/- 3.19 ng per mL), and therefore fell into the "high" category by RIA. None of the P4 concentrations found in this study group fell between 0.42-3.35 ng per mL.

When compared to the RIA, the lowest P4 concentration identified by Progestassay as "high" was 3.35 per mL, and the lowest P4 concentration identified by Target as "high" was 5.57 ng per mL. Both kits, however, falsely identified as "low" serum samples (three for Progestassay, seven for Target) that RIA identified as high.

The Progestassay had a 100% specificity and 95.1% sensitivity for differentiating low versus high serum P4 concentrations compared to the RIA standard (Figure 2). The Target test kit was also 100% specific, but sensitivity was slightly lower at 86.9% in comparison to the RIA standard. Statistically, no differences were found between the RIA and Progestassay (P=.25) but were significant (P=.007) between the RIA and Target. Differences between the Progestassay and Target approached significance (P=.06).

Discussion

Both ELISA test kits examined in this study clearly distinguished between high and low concentrations of serum P4 in swine. Both the Progestassay and Target kits are acceptably sensitive and specific, as assessed by their comparability to the RIA "gold standard."

Our decision to combine the three results categories for which the Target assay was designed into two ("high" and "low") categories enlarged the "gray zone" between high for the Target ELISA (> 5.0 ng per mL) and high for the RIA (> 1.0 ng per mL, Figure 2). This decision caused the 3.35 ng per mL sample to be called "low" by the Target test but "high" by RIA, thereby affecting the test’s overall sensitivity score.

Because none of the P4 concentrations in the serum samples from this study (as measured by the RIA) fell between 0.42-3.35 ng per mL, it is possible that the sensitivity of these ELISA kits may be greater or less than what we have reported. The chance for false positives with either kit, however, seems unlikely, because the ELISAs set their cutoffs higher than the standard RIA cutoff.

Because the ELISA assay cutoffs were between 2.5-5.0 ng per mL higher than the RIA cutoff, there is a 9.5%-15% chance that, if choosing normally cycling sows randomly, one would take a serum sample from a sow whose P4 concentration would fall within the "gray zone" between high and low, as defined by both ELISAs. For this reason, the results of these ELISAs, when used to assess P4 concentration in an individual animal, should be interpreted with caution. Either of these assays, however, will provide reliable evidence to the clinician in diagnosing management-related problems when used on a group of anestrus females (10 animals or 10% of the females, whichever is greater). Alternatively, sequential samples taken from the same female at a 3-day interval can provide a reliable evaluation of her serum P4 concentrations.

Although statistically the Target test was less accurate than the Progestassay, the sensivity and specificity of both ELISAs were still adequate to justify their use in swine.

Application in practice

Veterinarians are frequently confronted with complaints about noncycling females. Knowing P4 concentrations can help when addressing reproductive management problems related to anestrus in the gilt pool or postweaned sows. With the ELISA P4 assays, serum can be taken from a representative portion of the gilt pool and tested on the farm.

High serum P4 concentrations strongly indicate that sows are cycling. If the majority of animals show high P4 concentrations, changes in the heat detection program are warranted because these animals probably cycled but were missed by the heat detection regime.

Low serum P4 concentrations can indicate either gilts in estrus or can help confirm the producer’s observations of noncycling gilts. Estrus gilts can be easily identified by performing heat detection with a good teaser boar on these animals at the time of the visit. Once estrus is ruled out as a cause of low serum P4 concentrations, suggested changes in gilt pool management can be made. In this situation, it would first be prudent to determine the onset of puberty in these gilts. Running ELISA P4 assays on an adequate number of gilts across age defines an age window for onset of puberty for the herd. Once age of puberty is identified for a given producer’s gilts, strategies to control puberty–such as controlled stressors (e.g., movement, re-grouping, change in environment, etc.), boar exposure, and heat detection–can be strategically implemented.

Suspicion of postweaning anestrus in sows can also be confirmed or refuted using an ELISA P4 assay. If P4 concentrations are low, then she has problably not cycled, and therefore management suggestions can be made to enhance the probability of normal wean-to-estrus intervals (e.g., different weaning times, postweaning handling, changes in lactation or gestation management, nutrition, etc.). If P4 concentrations are high, it suggests the sows are cycling, and therefore management attention should first be directed at honing the heat detection program.

Progesterone ELISAs can contribute to the diagnosis of cystic ovaries in the herd. In swine, the prevalence of multiple ovarian cysts has been reported at <10%.10-12 In a subpopulation of animals culled specifically for reproductive disturbances, there was a 14% prevalence of multiple, large ovarian cysts.16 Thus, females with cystic ovaries make up a small percentage of the breeding population, including those culled specifically for reproductive problems. In the past, the ability to diagnose this pathologic condition antemortem was limited, allowing these sterile animals to persist in the breeding herd.

Determining estrous cycle activity is very important when trying to differentiate a cystic sow from one in diestrous or one that is pregnant. While the ELISA P4 assay alone cannot definitively diagnose animals with cystic ovaries, it can be used in conjunction with realtime ultrasound (RTU) to make a definitive diagnosis. The large, cystic structures are easily visualized transcutaneously or transrectally when viewing the ovaries.17 Multiple, large cysts are usually found present on both ovaries. Partial luteinization of the cyst walls can frequently be seen on the monitor.17 With anamnesis followed by RTU, these sterile animals can be culled earlier from the breeding herd, reducing nonproductive sow days.

Reproduction is the foundation of production. As such, clinicians are frequently confronted with the task of identifying the cause(s) of poor reproductive performance in a swine herd. A typical approach to this problem includes analyzing the herd’s reproductive records, an on-site examination of the herd and its management practices, and selected diagnostic tests that provide objective data on the breeding herd. Through this approach, a clinician can usually distinguish between an infectious, chemical, or noninfectious cause for the poor reproductive performance.

When the cause is infectious or chemical, therapeutic and/or preventive herd management strategies are usually implemented and producer compliance is usually high. If, however, the problem is believed to be noninfectious (e.g., the result of poor heat detection, poor breeding technique, inappropriate timing of breeding, etc.), and changes in management are recommended to aid in diagnosing as well as resolving the problem(s), producer compliance often dwindles, decreasing the clinician’s chances of diagnosing and, more importantly, resolving the problem. The P4 ELISA can provide the simple on-farm evidence that will optimize producer compliance with the recommended change in management.

Implications

Acknowledgements

Thanks to Dr. David Schaeffer for statistical analysis.

References

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