Single Radial Immunodiffusion Assay Research Articles

Polyclonal increase in certain IgG subclasses in mice persistently infected with the 87V strain of scrapie

Eight different combinations of seven strains of scrapie agent and the three known Sinc genotypes of mice were screened for changes in the concentration of IgG in serum. A single radial immunodiffusion assay was used to measure IgG throughout the incubation period which in different models ranged from an average of 125 days to longer than the maximum observation period of about 600 days. The only major changes occurred with the 87V strain of scrapie injected intracerebrally (i.c.) or intraperitoneally (i.p.) into mouse strains of the Sinc genotype p7p7. IgG concentration reached 1·5 to 2·0 times the control values in i.c. infected mice, which developed clinical disease after 270 to 320 days and also in i.p. infected mice, which did not develop the disease within the 600-day observation period. At very high IgG concentration, the increase was polyclonal; it involved the IgG1 subclass more than the others and was accompanied by an increased rate of IgG clearance from serum. It is suggested that some scrapie infections of mice (and sheep) may upset the control of IgG production. The underlying mechanism may involve cell-pathogen interactions which are common to all scrapie infections, but only lead to gross changes in IgG in some combinations of agent strain and host genotype.

Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7α-hydroxylase in rat liver microsomes and its regulation by thyroid hormones

In the preceding paper, evidence was presented that rat liver microsomes contain two structurally related isozymes of cytochrome P450, namely cytochromes P450a and P450m, that can both catalyze the 7α-hydroxylation of testosterone. The aim of the present study was to determine the extent to which these two P450 isozymes are responsible for the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes. Four monoclonal antibodies against cytochrome P450a, designated A2, A4, A5, and A7, were prepared in BALB/c mice. Monoclonal antibodies A2 (an IgM), A4 (an IgG 2b), and A5 (an IgG 1) were determined to be distinct immunoglobulins, whereas A7 could not be distinguished from A5. All of the antibodies were highly specific for cytochrome P450a; none cross-reacted with cytochrome P450m or with 10 other P450 isozymes purified from rat liver microsomes. Competition experiments between unlabeled and horseradish peroxidase-conjugated antibodies revealed that each of the monoclonal antibodies recognized the same epitope on cytochrome P450a. None of the monoclonal antibodies bound to denatured cytochrome P450a, suggesting that they each bound to a spatial epitope. A monospecific, polyclonal antibody against cytochrome P450a was also prepared, as described in the preceding paper. The levels of cytochrome P450a in liver microsomes were determined by single radial immunodiffusion, Western immunoblot (with polyclonal antibody), and enzyme-linked immunosorbent assay with monoclonal antibody. The levels of cytochrome P450a declined with age in male but not female rats, and were inducible up to 10-fold by treatment of rats with various xenobiotics. The levels of cytochrome P450a (but not cytochrome P450m) were also elevated (~3-fold) by thyroidectomy of mature male rats. Near normal levels of cytochrome P450a were restored by treatment of athyroid rats with triiodothyronine, whereas treatment with thyroxine was less effective in this regard. These changes in the levels of cytochrome P450a were highly correlated ( r = 0.995) with changes in testosterone 7α-hydroxylase activity. None of the monoclonal antibodies inhibited the catalytic activity of cytochrome P450a when reconstituted with NADPH-cytochrome P450 reductase and lipid. In contrast, the polyclonal antibody not only inhibited the catalytic activity of purified cytochrome P450a, but also completely inhibited (>96%) the 7α-hydroxylation of testosterone catalyzed by liver microsomes from immature and mature rats of both sexes and by liver microsomes from male rats treated with a variety of cytochrome P450 inducers. Cytochrome P450m did not appear to contribute to the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes. Although cytochrome P450m catalyzed the 15α-hydroxylation of testosterone in a reconstituted system, this reaction was a minor pathway of testosterone oxidation catalyzed by liver microsomes from mature male rats, which contain the highest levels of cytochrome P450m. In contrast to 7α-hydroxylation, the 6α-hydroxylation of testosterone was inhibited less than 40% by antibody against cytochrome P450a, except in liver microsomes from mature female rats (where the reaction was inhibited >95%). These results indicate that cytochrome P450a is solely responsible (>96%) for the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes and suggest that enzymes other than cytochrome P450a can contribute significantly to the 6α-hydroxylation of testosterone.

Quantification of Apolipoprotein A-1 in Cow Serum by Single Radial Immunodiffusion

A single radial immunodiffusion assay was developed to determine the concentration of the major apolipoprotein A-I in bovine serum. Assay was performed using agarose gel plates with monospecific rabbit antiserum. Standards made with bovine high density lipoproteins or serum 10 times diluted were applied in 3-μl amounts to each well in the gel. The assay working range was 2 to 22mg of apolipoprotein A-I. The intraassay and interassay CV were 3.7 and 4.9%, respectively. Using protein A-I. The intraassay and interassay CY were 3.7 and 4.9%, respectively. Using radial immunodiffusion assay, 23 serum samples from cows at various stages of lactation were analyzed. Apolipoprotein A-I was significantly increased in mid lactation compared with concentrations in early lactation or dry period.

Daytime foaling management of the mare 2: Induction of parturition

Summary Fifty-six mares of lighthorse breeding were utilized in a controlled management scheme for induced daytime foaling. All mares received pre-foaling mammary secretions sampling for evaluation of total hardness (ppm) or calcium carbonate content (ppm). Sampling began 10 days prior to expected foaling date for each mare and was performed once daily for 3 days followed by twice daily until foaling occurred. Mares were then either induced (i) to foal (No.= 33) according to readiness for parturition as indicated by the mammary secretion total hardness (≥250 ppm by Sofchek™ test strips a ), or allowed to foal spontaneously (s) (No.=23). Inductions were performed by one of two synthetic prostaglandin-analogues : prostalene b , given as one 4 mg dose subcutaneously (No.= 16), or fenprostalene c , given as two 0.5 mg doses at 2 hour intervals subcutaneously (No. = 17). All mares receiving fenprostalene for induction (No. = 17/17) responded and delivered viable foals a mean of 3.9h from the initial injection; 75% of the mares receiving prostalene for induction (No.=12/16) responded and delivered viable foals a mean of 3.7h from the injection time (mares failing to respond produced viable foals spontaneously 30–56h later). No differences (p>0.05) were noted in the mean response intervals to foaling between the prostalene and fenprostalene treated groups. Two foaling complications (6%) were experienced in the induced group (1 mare with premature separation of the placenta, 1 with dystocia due to retained fetal head). Three foaling complications (13%) were encountered in the spontaneously foaling group (2 stillbirths with placentae intact surrounding the dead foals, 1 with premature placental separation). All foals delivered from mares induced to foal were judged healthy, full-term and viable; none experienced failure of passive transfer as determined by serum samples taken at 18–24h and analysed by either CITE Foal IgG test d or single radial immunodiffusion assay for IgG and IgM.

N-acetylcysteine added to saliva does not affect IgA concentration or the agglutination of Streptococcus mutans in vitro.

N-acetylcysteine (NAC) or placebo were mixed with parotid or whole saliva to a final concentration of 0.004-10 mg/ml saliva. Placebo and NAC-containing parotid saliva had the same bacterial agglutinating capacity for 4 strains of Streptococcus mutans. Immunoglobulin A (IgA) concentration in whole saliva, using ELISA and single radial immunodiffusion assays, did not reveal any differences between NAC and placebo-treated samples. NAC did not affect the immunoelectrophoretic pattern of IgA.

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman—Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6·3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman—Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.

The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.

IgG Subclass Responses and Immediate Skin Sensitivity in Guinea-Pigs with Chronic Relapsing Experimental Allergic Encephalomyelitis

In strain-13 guinea-pigs inoculated for chronic relapsing experimental allergic encephalomyelitis (CR-EAE), IgG1 and IgG2 subclass antibody responses were investigated using single radial immunodiffusion and enzyme-linked immunosorbent assays (ELISA) for IgG1 and IgG2 specific for whole cord, myelin, myelin basic protein and Mycobacterium tuberculosis. The early acute stage revealed no increase in IgG1 but was associated with increased levels of IgG2 specific for neural and adjuvant components. Throughout the chronic phase of the disease, there were increased levels of IgG of both subclasses specific for the antigens tested but a preferential synthesis of IgG1. Levels of both IgG1 and IgG2 specific for neuroantigens were lowest in those guinea-pigs which did not develop signs of chronic disease. Immediate skin sensitivity against a wide range of neural antigens was not demonstrated though positive results may have been masked by the ability of myelin basic protein to induce non-specific mast cell degranulation and by altered histamine responsiveness in disease. Guinea-pigs with chronic paralysis had a lower skin sensitivity to histamine, compound 48/80 and M. tuberculosis than those in remission.

Plasma fibronectin and microvascular damage in essential mixed cryoglobulinaemia.

Plasma fibronectin (FN) was measured in 17 patients with essential mixed cryoglobulinaemia (EMC) and in 17 normal subjects by single radial immunodiffusion (RID) and enzyme-linked immunosorbent assay (ELISA). In 9 patients the presence of FN in the cryoprecipitates was also assessed by immunoblotting. In the EMC group, plasma FN levels were significantly lower than in control subjects, using both methods, and FN was constantly demonstrated in EMC cryoprecipitates. Capillaroscopic observation of the capillary bed in skin and bulbar conjunctiva, performed in all cases, showed severe alterations of microcirculation in EMC patients. A negative correlation between plasma FN and capillaroscopic abnormalities of skin capillaries was observed. These data support the hypothesis that plasma fibronectin plays a role in the pathogenesis of systemic vasculitis in EMC.

A double antibody radioimmunoassay for human α1-microglobulin

Using purified α 1-microglobulin from urine and specific rabbit antiserum, a double antibody radioimmunoassay has been developed. The assay is sensitive to 3 ng/ml and covers a working range from 25–800 ng/ml. The recovery rates on the average were 91.8% in serum and 97.7% in urine. The coefficients of intra-assay variation ranged from 3.6–4.5% and those of inter-assay variation from 4.0–8.8%. Correlations in serum and urine between the present assay and single radial immunodiffusion ( r = 0.972; r = 0.978) and enzyme-linked immunosorbent assay ( r = 0.935; r = 0.971) were satisfactory.

Susceptibility of Salmonids to Furunculosis: Differences between Serum and Mucus Responses against Aeromonas salmonicida

Abstract At the Reeds Creek (West Virginia) state hatchery, mortality from enzootic furunculosis is most severe among brown trout Salmo trutta, intermediate in brook trout Salvelinus fontinalis, and virtually nonexistent in rainbow trout Salmo gairdneri. In each species, survivors of epizootics had developed similar levels of protective antibodies in their sera as demonstrated by microtiter agglutination and passive immunization experiments. We concluded that serum antibodies were not related to graded differences in resistance to furunculosis. These same fishes, however, produced a mucus precipitin that reacted with cell-free bacterial extracts in single-radial immunodiffusion assays. The mucus precipitin activity correlated with the resistance of the three species to furunculosis. Breeding studies with brown trout also showed that fish selected for a high level of mucus precipitin activity against Aeromonas salmonicida, the cause of fish furunculosis, produced progeny that were more resistant to furuncu...

Immunochemical properties and intracellular localization of two molecular forms of arginine aminopeptidase in Streptococcus mitis ATCC 9811

Streptococcus mitis contains two multiple forms of arginine aminopeptidase (I and II) which differ from each other with respect to their content, immunochemical properties and cellular localization. Immunological analyses by Ouchterlony double immunodiffusion and immunoprecipitation showed an antigenic difference between each form by the use of antisera specific for each enzyme. The amounts of enzymes I and II within the cell were estimated to be 230 +/- 4.3 and 646 +/- 20 ng/mg protein (+/- S.D.), respectively, using a standard curve of purified enzyme in a single radial immunodiffusion assay. When intact cells were treated with the cell wall lytic enzyme, N-acetylmuramidase, though both enzymes were solubilized, a time lag was observed for the solubilization of enzyme II. Enzyme I was detected only in the cell wall fraction and showed no detectable associated with the membrane. Although most of the enzyme II activity was recovered in the cell wall fraction, a slight amount (7.5%) of the total activity was also found in the membrane fraction.

Quantitation of apolipoprotein A-I in pooled human serum by single radial immunodiffusion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Apolipoprotein A-I was released from human HDL particles by treatment with 8 m urea, and the free apolipoprotein exhibited identical antigenicity and the same low mobility as purified apolipoprotein A-I in electrophoresis. Treatment of serum with 8 m urea enabled quantitation of apolipoprotein A-I by single radial immunodiffusion assay, as judged by comparison with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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.

Simple method for analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography.

A simple and rapid method has been developed for the separation of apolipoproteins in high density lipoprotein (HDL) fractions by high performance liquid chromatography (HPLC) with gel permeation columns (G3000SW TSK GEL). The HPLC pattern monitored by A280 for a mixed solution of the HDL fraction (10 microliter) and an eluent buffer (200 microliter, 0.1 M sodium phosphate buffer containing 0.1% SDS) incubated at 60 degrees C for 5 min showed two completely separated peaks which corresponded to the major components of human HDL, apolipoprotein A-I and A-II. Moreover, quantitation of apolipoprotein A-I by our method was found to correlate well with that by a single radial immunodiffusion (SRID) assay.

The single radial immunodiffusion assay highlights small antigenic differences among influenza virus hemagglutinins.

The results of single radial immunodiffusion assays of influenza virus hemagglutinin were found to be greatly altered by small antigenic differences between test and reference strains. When such differences were present, the precise specificity of the antiserum used had a critical effect on the measured hemagglutinin antigen content obtained.

Immunoassay of poliovirus antigens by single-radial-diffusion: development and characteristics of a sensitive autoradiographic zone size enhancement (ZE) technique.

The reactions of polioviruses in single-radial-immunodiffusion (SRD) tests were investigated with a view to developing accurate and sensitive antigen assay systems. In direct SRD tests, employing high concentrations of immune poliovirus serum in agarose gels, poliovirus D-antigens produced clear reaction zones demonstrated by protein staining. The reactions were type-specific for polioviruses of types 1, 2 and 3 but the tests were of low sensitivity, being applicable only to the assay of virus concentrates. A novel autoradiographic zone size enhancement (ZE) test was developed which increased the sensitivity of the SRD assay 40- to 100-fold. The ZE test was dependent upon the ability of unlabelled poliovirus to co-migrate with the radioactive marker virus and so enhance the zone size detected autoradiographically. The areas of the autoradiographic zones were directly proportional to the concentration of unlabelled antigen. The ZE test was capable of detecting poliovirus D antigens in diluted cell culture fluid harvests in amounts corresponding to 10(3.3) to 10(4.3) TCID50 of infectious virus. Studies with poliovirus type 3 strains indicated that the ZE test was narrowly strain-specific for the D-antigen of poliovirus type 3 strains when homologous type 3 D-antigen was used as radioactive marker, but broadly cross-reactive for the D-antigen of type 3 viruses when heterologous poliovirus type 3 D-antigen was used as marker.

Isolation and partial characterization of two immunologically similar vitamin D-binding proteins in rat serum.

Two immunologically similar, probably identical, binding proteins for vitamin D and its metabolites (DBP1 and DBP2) were isolated separately from rat serum after approximately 180-fold purification by novel procedures using Blue Sepharose CL-6B chromatography. The freshly purified DBP1 and DBP2 each showed a single band of protein on polyacrylamide gel electrophoresis, and had alpha-mobility, although DBP1 moved slightly faster than DBP2. DBP1 and DBP2 had the same molecular weight, which was estimated as approximately 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric points of DBP1 and DBP2 were estimated as 4.9 and 5.0, respectively, from the results of isoelectric focusing experiments. DBP1 and DBP2 both appeared to have one binding site for 25-hydroxyvitamin D3 per molecule of protein, with apparently similar association constants at 4 degrees C of 5--7 x 10(9) M-1. The amino acid compositions of DBP1 and DBP2 were also determined and compared. A monospecific antiserum against rat DBP2 was prepared in a rabbit and was used for immunological studies of rat DBP. On double immunodiffusion, anti-DBP2 antiserum produced precipitin lines of complete reaction-of-identity against the purified DBP1, the purified DBP2, and rat whole serum. There was no immunological cross-reactivity between rat DBP and sera from man, dog, and rabbit, but mouse serum showed a pattern of partial identity with rat DBP. When rat serum samples were analyzed by immunoelectrophoresis using anti-DBP antiserum, three patterns of precipitin line were observed: a pattern showing the existence of only DBP1, designated as DBP 1-1; a pattern showing the existence of only DBP2, designated as DBP 2-2; and a pattern showing the existence of both DBP1 and DBP2, designated as DBP 2-1. Using single radial immunodiffusion assay for rat serum DBP, the mean (+/- S.D.) serum DBP concentrations were found to be 461 +/- 59 microgram/ml in adult male rats and 328 +/- 16 microgram/ml in adult female rats, and the difference was significant (p < 0.001). In molar terms, DBPs are present in normal rat serum in large excess relative to vitamin D and its metabolites, and most of the serum DBP, therefore, circulates as apo-DBP, not containing a bound molecule of vitamin D or of its metabolites. The immunoprecipitation studies of DBP in rat serum showed that DBPs were common main transport proteins for naturally occurring vitamin D and its metabolites, and that DBP played some, but not a principal, role in the transport of synthetic 1 alpha-hydroxyvitamin D3.

Net biosynthesis of antithrombin III by the isolated rat liver perfused for 12–24 hours compared with rat fibrinogen and α-2 (acute-phase) globulin, antithrombin III is not an acute phase protein

Antithrombin III-heparin cofactor has been isolated from normal rat plasma, purified to homogeneity on acrylamide gel electrophoresis and used to prepare a monospecific antiserum in rabbits. Measurements of rat antithrombin III were made by a single radial immunodiffusion assay. Net synthesis of antithrombin III was investigated during 12- or 24-h perfusions of the isolated rat liver. In perfusions performed under basal conditions cumulative synthesis of antithrombin-III was observed to occur at a rate sufficient to replace the total circulating plasma antithrombin III in about 6 h. In perfusions performed under full supplementation conditions which greatly enhanced synthesis of fibrinogen and α-2 (acute-phase) globulin (known acute-phase reactant proteins) net synthesis of antithrombin III was not significantly greater than that observed in control perfusions. Although these prolonged perfusion studies conclusively demonstrate net synthesis of antithrombin III by the isolated rat liver, they afford no evidence that this protein is an acute-phase reactant.

Characteristics of hiproly barley II. Quantification of two proteins contributing to its high lysine content

Quantitative extraction of salt and water soluble proteins from Hiproly, CI 4362 and Bomi barley was performed and their content of the lysine rich SP II albumin determined by a single radial immunodiffusion assay. The elevated content of SP II albumin in Hiproly accounted for 37% of the difference in crude protein lysine between Hiproly and Bomi and for 19% between Hiproly and its low lysine sisterline CI 4362. Apart from the lysine rich SP II albumin, other proteins contribute to the high lysine content of Hiproly.