Chicken antibodies: taking advantage of evolution--a review.

Laying hens are highly cost-effective as producers of antibodies compared with the mammals traditionally used for such production. Also, chicken antibodies have biochemical advantages over mammalian antibodies due to the phylogenetical differences between avian and mammalian species, resulting in increased sensitivity as well as decreased background in immunological assays. In contrast to mammalian antibodies, chicken antibodies do not activate the human complement system nor will they react with rheumatoid factors, human anti-mouse IgG antibodies, or bacterial and human Fc (fragment crystallizable)-receptors. Thus, chicken antibodies offer many advantages over mammalian antibodies and may replace such antibodies in the future.

Increased phosphate content of fibrinogen in vivo correlates with alteration in fibrinogen behaviour.

Fibrinogen was purified from five patients admitted for hip-replacement surgery the day before (day 0), the day after (day 2) and and one week after the operation (day 8). The behaviour of each patient's three fibrinogens was compared in thrombin gelation assays and plasmin degradation experiments to investigate whether the reported increase in protein-bound phosphate at day 2 and day 8 had any effect on the functional behaviour of fibrinogen as has been demonstrated in vitro. It was found that the thickness of the fibrin fibres produced by thrombin increased markedly at day 2 and declined thereafter. Susceptibility to plasmin appeared to decrease post-operatively by 50% and remained at that level on day 8 despite the phosphate content returning to normal. This has also been shown for fibrinogen phosphorylated in vitro. We conclude, after testing the fibrinogens with and without alkaline phosphatase pretreatment, that our data most resemble the published findings for in vitro phosphorylation of fibrinogen by casein kinase II.

Chicken antibodies: a tool to avoid interference by complement activation in ELISA.

MicroELISA plates coated with mammalian IgG will activate the human complement system. It has been shown that this activation of the complement system may interfere in solid-phase immunometric assays, and that there is a difference between IgG from different species and between different IgG subclasses in their ability to activate the human complement system. We have studied the ability of mammalian IgG and avian IgG to activate the human complement system. We show that chicken IgG do not activate the human complement system, and chicken IgG can thus be used in solid-phase immunometric assays to reduce interference by complement activation.

The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen.

The alpha-chain of human fibrinogen was found to be phosphorylated in EDTA-anticoagulated whole blood when trace amounts of (gamma-32P)ATP and 7.5 mM Mg2+ ions were added. Fibrinogen was not phosphorylated if only the ATP was added. The thrombin-induced gelation of fibrinogen phosphorylated by protein kinase A, casein kinase I or II was studied spectrophotomerically. It was found that phosphorylation by protein kinase A caused the formation of thinner fibrin fibres, whereas phosphorylation by casein kinase II resulted in fibres slightly thicker than those of the control fibrinogen (equivalent to a 20% increase in the control fibrinogen concentration). Phosphorylation with casein kinase I did not significantly affect the fibrin fibre thickness. Dephosphorylation by alkaline phosphatase removed 50% of the 32P-labelled phosphate from protein kinase A-phosphorylated fibrinogen and over 90% from the casein kinase I or II-phosphorylated fibrinogens. This dephosphorylation resulted in a general increase in fibre thickness in the gelation assay in all samples, although the fibres of the phosphorylated fibrinogens remained substantially thinner than the dephosphorylated control fibrinogen. Plasmin digestion of the phosphorylated fibrinogens showed that they were more resistant to cleavage, being cleaved at only 30% to 70% of the rate of control fibrinogen and that this resistance was unaltered by dephosphorylation, in contrast to the thrombin gelation experiments.

Plasmin digestion of human fibrinogen previously phosphorylated by protein kinase C or dephosphorylated by alkaline phosphatase in vitro.

Human fibrinogen, either untreated or previously phosphorylated by protein kinase C, was incubated with plasmin generated by streptokinase, urokinase or tissue plasminogen activator and the resulting fragments were separated by gel electrophoresis. Plasmin degradation resulted in the expected X, Y and D fragments, but the degradation rates differed. In vitro phosphorylation of fibrinogen was seen to inhibit the plasmin digestion. Treatment with alkaline phosphatase did not reverse the inhibition.

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.

Dephosphorylation of human fibrinogen, previously phosphorylated in vitro by protein kinase C, by whole blood or intestinal alkaline phosphatase. Effects on thrombin-induced gelation of in vitro dephosphorylated human fibrinogen.

Human fibrinogen, a phosphoprotein, was either left untreated or phosphorylated by protein kinase C. Then both were dephosphorylated by calf intestinal alkaline phosphatase. The dephosphorylated fibrinogen gave an increased fibre thickness during thrombin-induced gelation. Whole blood anticoagulated by heparin, EDTA or sodium citrate, contained dephosphorylating activity against 32P-labeled fibrinogen, although there were significant differences in activity among the three anticoagulants.

Aspects on the phosphorylation of muscle phosphofructokinase by protein kinase C--inhibition by phosphofructokinase stabilisers.

Our report presents data on the phosphorylation of muscle phosphofructokinase by Ca2+-activated, phospholipid-dependent protein kinase. We have found a stoichiometrical phosphorylation (about 1.5 mol per mol subunit), and a low apparent Km (about 0.7 microM). These data speak in favor of a physiological role for the reaction, as does the fact that phosphofructokinase from a new species (rat) was successfully phosphorylated. On the other hand we present the hitherto unpublished circumstance that the phosphorylation is inhibited by conditions that stabilise the activity of phosphofructokinase. This fact makes us question the true significance of this reaction.

Incidence of arthritis and autoreactivity of anti-collagen antibodies after immunization of DBA/1 mice with heterologous and autologous collagen II.

Native type II collagens of rat, chick, bovine, human and murine origin have been used for immunization of male and female DBA/1 mice of different ages. The four heterologous collagens were able to induce arthritis in both male and female mice although the incidence of arthritis was higher in males. The onset of arthritis was sudden with severe lesions starting to appear 5- to 6-weeks after immunization. Mouse collagen II, on the other hand, caused arthritis exclusively in males. The onset of arthritis was in the latter case less dramatic and usually delayed until 12- to 14-weeks post immunization. Antibody production against both the immunogen and other type II collagens, including mouse collagen was seen in both arthritic and non-arthritic animals, and antibody titres against both the collagen used for immunization and mouse collagen were higher in the sera from arthritic than in identically immunized non-arthritic animals. Total amounts of auto-anticollagen II antibodies were, however, more dependent on which collagen preparation was used for immunization than on whether the immunized mice developed arthritis or not. These results indicate that arthritis induction in mice is not dependent solely on the levels of autoreactive anti-collagen II antibodies.

Phosphorylation in vitro of fibrinogen from three mammalian species with four different protein kinases.

Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated.

Phosphorylation of human fibrinogen in vitro by calcium-activated phospholipid-dependent protein kinase from pig spleen.

Human plasma fibrinogen rapidly incorporated stoichiometric amounts of [32P]-phosphate when incubated with [32P]ATP and calcium-activated, phospholipid-dependent protein kinase purified from pig spleen. Half-maximal fibrinogen kinase activity was attained at less than 0.1 mM calcium acetate. The optimum concentration of phosphatidylserine was about 50 micrograms per ml. Diolein slightly potentiated the stimulatory effect of phosphatidylserine. The alpha-chain of fibrinogen, which is reported to contain endogenous phosphate (Blombäck, B., Blombäck, M., Edman, P., & Hessel, B. (1962) Nature 193, 883-884 and Doolittle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D., & Riley, M. (1979) Nature 280, 464-468) was phosphorylated by the protein kinase. The apparent Km value for the phosphorylation reaction (0.3-0.6 microM fibrinogen) was comparable with the Km values reported for the hitherto most effective substrate proteins for protein kinase C. Up to 5 mol phosphate per mol fibrinogen could be incorporated, indicating at least three phosphorylatable sites per half molecule.