Haptoglobin in Pigs: Development and Validation of an Enzyme Immunoassay for Various Body Fluids and Establishment of Physiological Reference Levels

Haptoglobin (Hp) is an acute phase protein. Quantifying its concentrations is presently discussed as being useful to monitor animal health and welfare. To obtain a simple, rapid and robust assay system we developed a competitive enzyme-immuno assay which is species specific and also sensitive enough to be applied in matrices in which low concentrations are to be expected, e.g. in saliva.

Hp was purified from porcine serum after sodium sulphate precipitation by affinity chromatography on hemoglobin crosslinked to sepharose followed by gel filtration. Purity and identity were confirmed by SDS-PAGE. The purified protein was used to immunise rabbits. In all antisera obtained, the titres were determined and the specificity was evaluated by Western blotting. In the assay biotinylated porcine (p) Hp was used as tracer and was incubated together with either Hp standard or sample in microtiter plates coated with anti rabbit sheep IgG Fc fragments. After adding the specific rabbit antiserum, plates were incubated for 1 hour, washed and evaluated via a streptavidin peroxidase system with TMB as substrate. The limit of detection was 0.02 mg/L, parallelism of serum dilutions was proven and recovery of pHp added to serum samples was 99.6%. The coefficients of intra and inter assay variation were 3.3 (n = 5) and 10.2 (n = 16), respectively. Comparing the new assay with an established nephelometric assay system using antibodies against human Hp [1], the concentrations measured in 86 serum samples were identical in both assays (r = 0,96, p < 0.01). In contrast to biochemical or nephelometric systems, there is no interference in haemolytic or turbid samples in our assay and significantly lower amounts of antiserum are needed. In addition, it can validly be applied in blood serum, plasma, whole blood, saliva and in meat juice, the latter being possibly of particular relevance for slaughter line controls.

Hp was measured in blood sera from male and female pigs at various physiological stages. Pigs from private and from experimental farms were clinically inspected and diagnosed as healthy. The following ages were monitored: suckling piglets (2 weeks old, n = 10), weaned piglets (4 weeks, n = 10), fattening pigs (6 months, n = 10), gilts (n = 10) and sows (n = 14), gilts (n = 6) and sows (n = 19) within 14 days after conception, sows being pregnant for 2 months (n = 30), sows being pregnant more than 100 days (n = 6) and lactating sows (n = 24).

Hp concentrations were lower in suckling piglets than in weaned or fattening pigs. In sows from different reproductive stages, highest concentrations were observed in lactating and non-pregnant sows. The assay presented herein provides a powerful tool to monitor Hp concentrations in various body fluids irrespective of sample condition. Based on the reference values for blood provided herein, it can be effectively used to control health status at the different stages of pig production.