Effect of storage on the measurement of apolipoproteins A-I and A-II by radial immunodiffusion

We studied the effect of storage time and conditions on the measurement of apolipoprotein A-I and A-II by radial immunodiffusion. Purified A-I and A-II standards were stable for at least 6 months before any change in immunoreactivity was detected if stored at 4 degrees C at concentrations of 0.06-0.24 mg/ml for A-I and 0.016-0.064 mg/dl for A-II in 0.84 M tetramethylurea, 6.4 M urea, and 8 mM Tris-hydrocholoride, pH 8.0. Purified A-I (0.8-1.6 mg/ml) and A-II (0.5-1.0 mg/ml) were stable for 1 year if stored at -60 degrees C in 5 mM NH4HCO3 with or without 4.2 M tetramethylurea. Serum or plasma could be stored at 4 degrees C (under conditions where evaporation and bacterial growth were minimized) for at least 46 days or at -20 degrees C for up to 3 years without any change in A-I or A-II levels. For four serum samples stored at -20 degrees C for 2 to 3 years, the coefficient of variation of measurement ranged from 6.3 to 9.8% for A-I and from 6.7 to 10.6% for A-II. Samples stored at 4 degrees C had comparable apolipoprotein levels to those stored at -20 degrees C. However, apolipoprotein levels in serum samples were 3-5% higher than those obtained on plasma samples. We conclude that purified A-I or A-II and serum and plasma can be stored for long periods without any change in the measurement of the A-I or A-II by radial immunodiffusion.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

Quantitative Measurement of Precipitating Antibodies in Streptococcal Grouping Antisera by the Single Radial Immunodiffusion Technique

A comparative study was made of the single radial immunodiffusion test and the classical quantitative precipitin test for determining the amount of precipitable antibodies present in streptococcal groups A and C antisera. The potency of 21 group A and 54 group C antisera was determined by both methods; purified group-specific carbohydrates were used as antigens. The coefficient of correlation between the results from the two methods was 0.976 for group A antisera and 0.946 for group C antisera. When the concentration of antigen, the volume of antiserum used, and the depth of the antigen-agar mixture are kept constant, the diameter of the precipitin disc is directly related to the concentration of precipitable antibodies present in the antiserum. The use of the radial immunodiffusion test for evaluating and standardizing streptococcal grouping antisera is discussed as well as the advantages and disadvantages of using a concentrated vaccine for producing these antisera.     

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcgamma receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels > 150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.     

Detection and quantitation in plasma and synovial fluid of a fragment of human C4 with alpha mobility generated during the activation of the complement system.

A newly identified fragment of human C4 was detected, using a particular antiserum, in human serum after activation by heat-aggregated immunoglobulins, but not after activation of the complement alternative pathway. This fragment was shown to have a sedimentation velocity of approximately 2.5S, to be heat stable, and to exhibit alpha mobility in immunoelectrophoresis. This C4alpha mobility fragment was not generated in human C4 deficient serum but was generated in human C2 deficient serum after incubation with heat-aggregated immunoglobulin. After precipitation of native C4 and its higher molecular weight fragments from serum by polyethylene glycol, it was possible to quantitate the lower molecular weight C4 alpha mobility fragment by radial immunodiffusion. In kinetic experiments, it was shown that the C4alpha mobility fragment was generated after some delay when compared to the disappearance of C4 hemolytic activity. Quantitation of the C4alpha mobility fragment may be of further use in human diseases for the evaluation of the catabolism of C4: joint fluids of patients suffering from rheumatoid arthritis contained high levels of the C4alpha mobility fragment, and low concentrations were found in patients with degenerative joint disease.     

The composition of immune sera against Neisseria meningitidis studied by high-performance liquid chromatography

The comparative study of the composition of immune rabbit sera to N. meningitidis, as well as nonimmune sera, has been made by the methods of HPLC and radial immunodiffusion. The quantitative evaluation of the main serum proteins by the two methods has shown the coincidence of the results yielded by these methods. To study the total level of IgM and IgG in the sera under study, a simple and rapid HPLC technique is proposed. The study of the stability of sera during storage (at 4-6 degrees C) has revealed that immune sera show greater stability during storage under such conditions in comparison with sera obtained from nonimmune animals.     

Preliminary evaluation of four immunological tests for the early diagnosis of Hypoderma tarandi causing hypodermosis in reindeer

294 serum samples from five Norwegian reindeer herds were examined for antibodies against Hypoderma tarandi L. The first and second larval instars of H. tarandi were tested as antigens in immunodiffusion tests, passive haemagglutination and an Enzyme-Linked Immunosorbent Assay (ELISA). The latter technique, using first instar antigens, produced the best results. A significant difference (P less than 0.1%) was observed between the antibody value of naive reindeer bred in France and those from infected Norwegian herds. No correlation was observed between the antibody titre and the number of warbles recovered at necropsy from the 294 Norwegian reindeer.     

Effect of the nude mutation on alpha-fetoprotein synthesis and hemopoiesis in the mouse liver in the postnatal period of development]

The dynamics of population of alpha-fetoprotein (AFP)-containing cells in the liver and the level of AFP in the blood of C3H/HeJ+/+ and thymus-less mutant C3H/HeJnu/nu mice during postnatal development was studied by means of indirect immunofluorescence and radial immunodiffusion. The content of AFP-positive hepatocytes and AFP concentration in the blood serum of C3H/HeJnu/nu mice were shown to exceed markedly those in C3H/HeJ+/+ mice beginning from the age of 2 weeks. The histological analyses has revealed the foci of hemopoiesis in the liver of adult C3H/HeJnu/nu mice, unlike in the liver of normal mice. The neonatal thymectomy of C3H/HeJ+/+ mice did not influence the parameters under study. A possible relationship between the increased AFP level and the preservation of hemopoiesis in the liver of the mice homozygous by the mutation nude is discussed.     

Isolation of Corynebacterium diphtheriae from sperm fluid

The isolation of toxigenic Corynebacterium diphtheriae from sperm is reported. The organism was identified through the investigation of fluorescence under the UV light, the presence of pirazinecarboxilamidase enzyme (Pyz), in vitro and in vivo and virulence methods (single radial immunodiffusion, cell culture, guinea pig intradermic test). The strain was initially considered nontoxinogenic by single radial immunodiffusion, but its virulence was observed afterwards, when we applied the tests already mentioned. The strain could be considered a "Diphtheroid" without adequate specification.     

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja)

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek''s Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek's disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek's disease.     

Rapid and simple method for the determination of α1-acid glycoprotein in serum by column liquid chromatography

A rapid and simple method for the determination of α₁-acid glycoprotein (AAG) in serum was developed by using an anion-exchange column for clean-up of serum and a hydroxyapatite column for high-performance liquid chromatography (HPLC). A good correlation was observed between this HPLC method and the conventional radial immunodiffusion method. The method may also be used to determine the AAG concentration in the serum of experimental animals.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek's Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek''s disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek''s disease.     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Quantitative determination of human apolipoprotein D by electroimmunoassay and radial immunodiffusion.

1. An electroimmunoassay and a radial immunodiffusion procedure are described for the quantitative determination of human serum apolipoprotein D. Purified apolipoprotein D and antisera to both lipoprotein D and apolipoprotein D were used to standardize the assays. The assays are applicable to measurement of apolipoprotein D in serum and density classes. The electroimmunoassay is more sensitive (50 ng apolipoprotein D quantitatively detectable), rapid (time required for completion of assay is 5 h) and precise (the within- and between-assay coefficients of variation are 4 and 7%, respectively) than radial immunodiffusion. However, comparable results were obtained by both methods (r = 0.85). 2. Serum apolipoprotein D levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb, III, IV and V were in the same range (10 to 12 mg/dl). In contrast, patients with hyperchylomicronemia (type I) had decreased apolipoprotein D levels (5 mg/dl; P less than 0.001). The apolipoprotein D in serum of normolipidemic subjects was detectable in all density classes but measurable only in HDL2 (21%), HDL3 (43%) and VHDL (36%). 3. Rocket electrophoresis is also a valuable tool for assessing the structural relationships among apolipoproteins or their constituent polypeptides. Interaction between serum and a mixture of antibodies to A-I, A-II and apolipoprotein D resulted in the formation of separate lipoprotein A and lipoprotein D rockets indicating that apolipoprotein D is not a constituent polypeptide of apolipoprotein A. This observation confirms the existence of lipoproteins A and D as separate lipoprotein families.     

Immunological studies of the anticryptococcal factor of normal human serum

Preliminary studies have shown a very high inhibitory activity in the alpha₂ and gamma zone of human serum towards the growth of Cryptococcus neoformans. These findings are now corroborated by single radial immunodiffusion tests, which showed the some loss of IgA and IgM globulins and of the other three globulin fractions (ceruloplasmin, alpha₂ macroglobulin and alpha₂ HS glycoprotein) which migrate in the alpha₂ zone. The data was obtained by single radial immunodiffusion tests. The losses were not statistically significant however. No change in the immunoglobulin content of the sera kept for 6 days in contact with a heat-killed suspension of C. neoformans was noted. These findings suggest, that the inhibitory activity of the normal human serum on the in-vitro growth of C. neoformans is due to the above mentioned globulin fractions and not to a single specific factor.     

Weed competition in drilled summer cabbage

A single immunodiffusion drop test has been developed for the serological detection of lily symptomless virus in lilies. The presence of virus is demonstrated by intra-gel precipitation in drops of an antiserum-agar mixture which are contiguous with droplets of pyrrolidine-treated leaf extracts covered with paraffin oil. Single immunodiffusion drop tests require c. twelve times less antiserum than radial diffusion plate tests. The results of virus detection by this technique agree closely with those obtained by electron-microscopical screening of negatively stained leaf extracts.     

The identification of diphtheria, tetanus and pertussis vaccines by single radial and double immunodiffusion techniques

The identification tests for adsorbed diphtheria, tetanus and pertussis vaccines, which are required by the European Pharmacopoeia to be undertaken in animals, may be replaced by precipitation tests, for instance in agaros gels. Such in vitro tests eliminate the use of animals and are less expensive and time-consuming. The single radial immunodiffusion technique is a suitable semiquantitative test, while the double diffusion test is necessary for the investigation of complete or partial identity. The precipitates obtained in the single radial diffusion tests and in double diffusion tests with diphtheria toxoid were visible without staining; those obtained in the double diffusion tests with tetanus toxoid were weaker and staining was sometimes needed.     

Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidyl-AH-sepharose.

L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique.