An immunoturbidimetric assay for canine C-reactive protein.

Antiserum was raised in sheep against canine C-reactive protein (CRP) and antibody, which was not specific for CRP, was removed by absorption with normal canine serum protein linked to agarose beads. The antiserum was used to develop an immunoturbidimetric assay for canine CRP on a MIRA (Roche Diagnostics) automated clinical biochemical analyser and assessed for routine analysis of CRP in canine serum samples. The assay gave standard curves with each standard having a coefficient of variance (CV) between 4.8 and 11%, interassay CVs below 11% and intra-assay CVs of less than 5%. Parallel dilution curves were obtained with purified CRP diluted in buffer and with endogenous CRP in serum diluted with buffer or with a serum with a negligible CRP content. The immunoturbidimetric assay results correlated with the results obtained using an ELISA method, r = 0.88. The immunoturbidimetric assay of canine CRP proved to be suitable for the routine analysis of canine CRP.

Plasma haptoglobin in cattle (Bos taurus) exists as polymers in association with albumin.

1. Plasma haptoglobin (Hp) in cattle (Bos taurus) has a molecular mass so large that it is virtually unable to penetrate 4% polyacrylamide gels and is excluded from gel filtration media with an upper exclusion limit of greater than 1000 kDa. 2. In most species, apart from ruminants, Hp has a molecular mass of 100 kDa, consisting of two subunits of 40 kDa and two subunits of 9 kDa, although in a few species, such as man, a genetic variant of Hp forms polymers of higher mass. 3. Bovine Hp was purified by (i) salt precipitation, gel filtration and ion exchange chromatography and (ii) by affinity chromatography and gel filtration, with final purification by (iii) preparative electrophoresis. 4. Bovine Hp could only penetrate sodium dodecyl sulphate polyacrylamide gels following denaturation with mercaptoethanol, when it was shown to be composed of subunits of 40 and 16 kDa, but a protein of 67 kDa was consistently present. 5. Western blotting identified the 67 kDa protein as bovine serum albumin. 6. The polymeric form of Hp found in bovine plasma is formed by association of 40 and 16 kDa subunits with albumin.

Acute phase response in calves following infection with Pasteurella haemolytica, Ostertagia ostertagi and endotoxin administration.

The concentrations of bovine acute phase proteins were monitored in plasma following experimental infection with Pasteurella haemolytica and Ostertagia ostertagi and after endotoxin administration. Raised levels of haptoglobin, alpha 1 proteinase inhibitor and seromucoid were detected after pasteurella infection and endotoxin administration. Ceruloplasmin levels increased after endotoxin administration but not during pasteurella infection. Raised levels of the four acute phase proteins were found in eight of 19 calves infected with ostertagia but showed a variable pattern and did not correlate with plasma pepsinogen increases. Bovine alpha 1 antichymotrypsin and alpha 2 macroglobulin were identified as acute phase reactants.

Acute phase response in the dog following surgical trauma.

The levels of five acute phase reactants and serum glycoproteins were measured in dogs following surgical trauma. Canine C-reactive protein peaked 24 hours after surgery and peak levels of ceruloplasmin, haptoglobin and seromucoid were detected on the fourth to sixth days. Levels of serum glycoproteins varied in a similar manner to ceruloplasmin, haptoglobin and seromucoid. There was very little variation in the levels of alpha 1 antitrypsin.

Inhibition of elastase by canine serum: demonstration of an acute phase response.

Canine serum alpha 1-proteinase inhibitor is not a major acute phase reactant in the dog, unlike the equivalent protein in humans. The possibility that an alternative protease inhibitor system is stimulated during the acute phase response in the dog was investigated. alpha 2-macroglobulin was not an acute phase reactant, but an inhibitor of elastase was identified in canine serum which could be separated from proteinase inhibitor by gel filtration and which was shown to be an acute phase reactant. This protein has been named canine elastase inhibitor.

Bovine acute phase response following turpentine injection.

The serum levels of five proteins, alpha 1 antitrypsin, ceruloplasmin, fibrinogen, haptoglobin and seromucoid, were measured daily in calves after the subcutaneous injection of oil of turpentine. Raised concentrations were detected on the second and third days after injection with peak levels occurring on the fourth to seventh days and returning to normal by the 17th day. Levels of four of these proteins, alpha 1 antitrypsin, ceruloplasmin, haptoglobin and seromucoid were compared in the same calves following three different doses of turpentine. Levels of haptoglobin and seromucoid varied with the dose whereas ceruloplasmin and alpha 1 antitrypsin levels did not.

Bovine and canine acute phase proteins.

Acute phase proteins are serum proteins which increase in concentration during the acute phase response to inflammation or infection. The response occurs in all animals, but in different species the response of individual proteins can be significantly different. Of the numerous acute phase proteins which have been identified in humans, a number have been examined in cattle and dogs but usually on an individual basis with little reference to their part in the acute phase response. Biochemical, physiological and clinical investigations into haptoglobin, fibrinogen, alpha 1-proteinase inhibitor, ceruloplasmin, seromucoid and C-reactive protein of cattle and dogs have therefore been reviewed with the emphasis on their role in this response to tissue damage.

Acute phase response and mastitis in the cow.

The levels of three plasma proteins, which are classed as acute phase reactants, were compared in a group of cows which suffered from mastitis with those in a group of cows which were clinically normal. The plasma levels of haptoglobin, ceruloplasmin and alpha 1 antitrypsin were higher in the cows with mastitis than in normal cows.

The prevention of distortion in ultrathin-layer polyacrylamide gel isoelectric focusing.

Although isoelectric focusing patterns in ultrathin layers of polyacrylamide gel are distorted by the presence of salts, such as ammonium persulfate, this reagent is commonly used to promote polymerization of the gel. The amount of ammonium persulfate can be reduced but this causes the formation of sloppy gels or incomplete polymerization. A method is described here in which an ultrathin polyacrylamide gel is formed on a polyester sheet using ammonium persulfate in the absence of ampholyte. After complete polymerization, the ammonium persulfate is washed out and ampholyte is allowed to diffuse into the gel. Subsequent isoelectric focusing is then free from distortion caused by the presence of ammonium persulfate.