Determination of Haptoglobin in Bovine Serum using Polyclonal and Monoclonal Anti-human Haptoglobin Antibodies

Two ELISA procedures to determine haptoglobin (Hp) in bovine serum were developed. Equine haemoglobin was used as the solid phase. Self-developed goat polyclonal antibody (variant I) and monoclonal antibody (variant II) raised against human Hp were used. The results were compared with the guaiacol method. High correlation was found (r = 0.96 and r = 0.90, respectively) based on the results of 548 bovine serum samples, of which 357 were from clinically healthy cows and 191 from cows and calves monitored during treatment for the most common diseases. The Hp detection limit of ELISA using polyclonal Ab was 0.1 mg/l and using MoAb 0.21 mg/l. The addition of 2% PEG 6000 at the antibody-binding steps enabled major shortening of the incubation periods. The relatively short time, low cost of reagents, and high correlation with the reference method support the use of these ELISA variants in bovine diagnostics. Cattle, acute-phase protein, enzyme immunoassay, haptoglobin antibody The acute-phase response (APR) is a local and systemic nonspecific response to inflammatory agents. During APR, acute-phase proteins (APPs) are produced mainly in the liver and released to the blood plasma. The plasma concentrations of the majority of APPs are known to increase during an acute-phase response, but some, including albumin, decrease (Eckersall and Conner 1988; Heinrich et al. 1990; Stefaniak et al. 1995). During the acute-phase response in cattle, the concentrations of many major APPs, such as haptoglobin (Hp), serum amyloid A, and fibrinogen, and minor APPs, such as ceruloplasmin, α1-antitrypsin, and α1-acid glycoprotein, show dynamic alterations (Conner et al. 1986; Eckersall and Conner 1988; Horadagoda et al. 1993; Dowling et al. 2002). In the serum of the Bovidae and Cerviadae families, Hp is composed of 2 to 20 polymerized forms of α2β2 tetramer and its structure is similar to human Hp type 2-2 (Travis and Sanders 1972). The molecular weight of bovine Hp ranges from 200 to 2000 kDa. The N-terminal amino-acid sequence of the α chain is 56% and of the β chain 90% identical to human Hp (Morimatsu et al. 1991). In contrast to monogastric mammals, about 50% of healthy cattle show an undetectable Hp level in the blood plasma, and in the remaining healthy animals the concentration is below 0.1 g/l. Moreover, the Hp concentration in cattle is not affected by age, sex, pregnancy, or lactation (Richter 1974). A rise in concentration follows injury, inflammation, and infection and is related to the intensity of inflammation (Conner et al. 1988; Deignan et al. 2000; Dowling et al. 2002; Godson et al. 1996; Gruys et al. 1993). About 10 to 24 h after induction of the inflammatory response, the Hp concentration rises rapidly, a high concentration being achieved between days 2-3 with the maximum usually on the 4th day, and decreases to normal levels after about 11 days (Conner et al. 1988). The determination of Hp concentration in blood serum may be a valuable diagnostic tool for assessing problems occurring in cows during puerperium (Chan et al. 2004) as well ACTA VET. BRNO 2010, 79:105–112; doi:10.2754/avb201079010105 Address for correspondence: Paulina Jawor Department of Immunology, Pathophysiology and Veterinary Prevention Wroclaw University of Environmental and Life Sciences, Wrocław, Poland Phone: +48 71 3205 240 Fax: +48 71 3205 246 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm as for determining fatty liver (Yoshino et al. 1992). Hp is commonly used in monitoring the treatment of limb diseases (Jawor et al. 2008) and the health of the udder (Kováč et al. 2007), detecting diseases of clinical or subclinical course in calves, and in monitoring herd health (Ganheim et al. 2003). The broad availability of Hp in cattle diagnostics has resulted in this APP becoming the most commonly determined in this species. It is especially important in cattle because classical inflammatory indicators (sedimentation rate, leukocytosis) show poor usefulness. Many methods of Hp determination were designed for cattle. Connell and Smithies (1959) described a method based on the peroxidase activity of the Hp-MetHb complex and Spooner and Miller (1971) developed a method of detecting the Hp-Hb complex in gel. Other methods utilize the differences in electric field migration between free Hb and the Hp-Hb complex using agar gel electrophoresis and zone capillary electrophoresis (Mazur 1980; Pirlot et al. 1999). Methods of immunological determination of Hp using radial immunodiffusion were described by Morimatsu et al. (1992). In that study, a large molecule of bovine Hp was reduced by cysteine to diminish differences in polymerization degree among individuals. The competitive (McNair et al. 1995) and noncompetitive (Sheffiled et al. 1994; Godson et al. 1996) variants of ELISA to determine Hp were described, both based on anti-Hp monoclonal antibodies (MoAbs). Kątnik et al. (1989) produced and characterized the unique monoclonal antibody clone no. 2.36.71.41 which was able to recognize not only human Hp type 2-2, but also goat, equine, and cow Hp with high affinity as well as the Hp of many wild ruminants of the Bovidae and Cervidae families (Kątnik et al. 1991; Gospodarek et al. 1997; Gospodarek 1998). The 2.36.71.41 MoAb clone was shown to recognize the epitope in haptoglobin located around the disulphide bond linking subunits of Hp. Monoclonal antibody 2.36.71.41 was used in ELISA for the quantitative determination of Hp in goats (Kątnik et al. 1991). The aim of the study was to design, standardize, and compare two ELISA variants for the quantitative determination of Hp in cattle serum. The ELISAs are based on the mouse monoclonal antibody (clone 2.36.71.41) described earlier (Kątnik et al. 1989; Kątnik et al. 1991) and a goat polyclonal antibody against human Hp produced by us and showing cross-reactivity with bovine Hp. Materials and Methods The study design was approved by the Second Local Ethics Commission for Experiments on Animals at the Agricultural University of Wrocław (no. 91/04 II). Sampling There were 548 bovine serum samples examined, of which 357 were from two dairy herds during one year of herd health monitoring and 191 from cattle monitored during the treatment of different diseases (ketosis, mastitis, displacement of the abomasum, limb diseases, and bronchopneumonia and diarrhoea in calves). No haemolysis was observed in the serum samples. Antibodies Human serum Hp was isolated according to Kątnik and Jadach (1993) using an affinity column filled with Sepharose 4B coupled with two MoAbs against Hp (clones 2.36.71.41 and 7.60.66.55). The hyperimmune antiserum was produced by immunizing a goat with purified human Hp. Five injections of 100 μg of Hp diluted in 0.5 ml of PBS and emulgated with 0.5 ml of Freund’s incomplete adjuvant (Sigma) were administered subcutaneously in 14-day intervals. Blood was taken 12 days after the last injection and the serum was prepared and stored at -20 °C until use. Anti-Hp polyclonal antibody was isolated from the goat antiserum on a column filled with Sepharose 4B coupled with human Hp 2-1. The anti-Hp antibody was conjugated with horseradish peroxidase according to Farr and Nakane (1981) (modified by Stefaniak 1993). Mouse ascites containing MoAb produced by hybridoma clone 2.36.71.41 was obtained from Middle-European Diagnostics, Wroclaw. Hp concentration measurements Guaiacol method Hp concentration was determined using the guaiacol method (Jones and Mould 1984). ELISA for human 106