Single Radial Immunodiffusion Assay Research Articles

Bovine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period.

Colostrum and milk samples from 60 Holstein-Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 59.8 +/- 28.5, beta-lg 14.3 +/- 4.6, alpha-la 2.04 +/- 0.6, BSA 1.21 +/- 0.44. Large variations were recorded for IgG concentrations (15.3-176.2 mg/ml) and yields (0.2-925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-gamma-globulinaemia was found in 18.3% of the cows. The concentrations of IgG, beta-lg and BSA dropped abruptly in subsequent milkings and alpha-la concentration decreased slowly. The mean IgG concentration was < 2 mg/ml after eight milkings and < 1 mg/ml after fifteen milkings. However, IgG concentration did not differ significantly, at the 1% level, during milkings 11-15. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to the milk supply.

Quantification of C3 and C4 in infertile men with antisperm antibody in their seminal plasma.

Previous studies showed that some infertilities are caused by antisperm antibodies (ASAs). It was shown that some major complement (C) components are present in seminal fluid. Due to the role of C in the pathogenesis of ASAs, the existence and amount of two key C components (C3 and C4) were investigated in seminal plasma (SP). Single radial immunodiffusion assay and a sandwich-type enzyme-linked immunosorbent assay (ELISA) were used for C3 and/or C4 quantification, respectively, in serum and SP, and the tray agglutination test was used for ASA detection in 12 fertile and 53 infertile men (18 ASA-positive [ASA+] and 35 ASA-negative [ASA-] men). Of the 18 ASA + infertile men, 61.11% had positive C3, whereas 27.77% showed positive C4 levels. ASA + infertile men showed significant differences in seminal plasma C3 mean values compared with ASA- infertile (P < 0.01) and fertile (P < 0.05) men, but the seminal plasma C4 values only showed differences compared with ASA- infertile men (P < 0.05). No significant differences were observed in serum C3 and/or C4 levels of ASA+ infertile men compared with other groups. No significant correlation was found between ASA titer and C3 and C4 levels in SP. A significant correlation existed between SP and serum C3 levels of ASA+ (r = 0.522, P < 0.01) and ASA- (r = 0.451, P < 0.01) infertile men, but no correlation was observed between C4 levels. In the presence of ASAs, the C system has no definitive activity in blood serum or outside the male genital tract. In SP, and in association with ASAs, C has no lytic activity by the classical pathway. The excess of C3 in SP of ASA+ infertile men may participate in other C-mediated activities in the male reproductive tract.

Characterization of plasma immunoglobulin G concentrations of llamas

Abstract Objective To develop appropriate reference ranges for plasma IgG concentrations of llamas. Animals 643 llamas on 5 farms. Procedure Plasma IgG concentration was measured by using a single radial immunodiffusion assay kit. Farm of origin, age, body condition score, and sex were recorded for each llama. The effect of each factor on plasma IgG concentration was evaluated separately, using ANOVA; the association between age and IgG concentration was evaluated, using linear regression. Multivariable regression models were developed to examine concurrent effects of age, sex, body condition score, farm, and various interactions on IgG concentration. Results The IgG concentrations were between 127 and 3,969 mg/dl. In llamas &lt; 12 months old, farm of origin accounted for 29% of variability for IgG concentration. Reference range for plasma IgG concentrations in llamas &lt; 12 months old was 391 to 2,357 mg/dl; for llamas &gt; 12 but &lt; 28 months old was 771 to 2,796 mg/dl; and for llamas &gt; 28 months old was 570 to 3,264 mg/dl. These ranges were applicable only for the kit used in this study. Conclusions Healthy llamas have a wide range of IgG concentrations. Determinants of IgG concentration are multifactorial, and their importance varies with age of the llamas. Clinical Implications The wide range of IgG concentrations observed in healthy llamas and the influence that age and farm may have on IgG concentrations indicate that a result for one specific llama should be interpreted in relation to those of its herdmates. (Am J Vet Res 1998;59:406–409)

Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand.

To characterize Fas antigen expression on the cell surface, and to determine the effect of this expression in rheumatic diseases using a newly established gld-congenic MRL strain of mice (MRL/gld), which is defective in its functional Fas ligand (Fas-L). Flow cytometric analyses of lymphoid cells and macrophages were performed using anti-Fas and other cell surface markers. Histopathologic manifestations were examined using immunochemistry and light and electron microscopy. Serum levels of IgG and anti-DNA antibodies were measured by single radial immunodiffusion and enzyme-linked immunosorbent assay, respectively. MRL/gld mice developed systemic lymphadenopathy with an accumulation of Thy1.2+, B220+ and CD4-, CD8- T cells, which both express the Fas antigen. Splenic B cells positive for surface IgM and/or surface IgD, and resident peritoneal macrophages exhibited up-regulated expression of the Fas antigen, at much higher levels than those observed in MRL/MpJ-+/+ (MRL/+) mice. Forms of rheumatic disease were observed in these mice, although not in C3H/HeJ-gld/gld mice. These forms included diffuse glomerulonephritis, granulomatous arteritis, and arthritis, and were associated with the infiltration of mononuclear cells expressing the Fas antigen. Serum levels of IgG and anti-DNA antibodies were significantly increased in MRL/gld mice compared with MRL/+ mice. Rheumatic disease was generated by the gld gene in mice with an MRL background, as it is by the lpr gene, which is a Fas deletion mutant, associated with autoimmune traits. Rheumatic disease in this MRL strain was initiated by an incapacity for Fas/Fas-L-induced apoptosis, resulting in the development of autoimmunity and allowing for a persistent immune response in the affected lesions.

Immunochemical quantification of myoglobin heat denaturation: Comparative studies with monoclonal and polyclonal antibodies

Solutions of bovine myoglobin in 0.1 M‐phosphate buffer, pH 6.0, were heated over the temperature range 55–85°C for 60 min. Their immunological reactivity was tested after 0.2 μm filtration using polyclonal antibodies in the single radial immunodiffusion assay (SRID) and six monoclonal antibodies (MAbs) in a competitive and a sandwich ELISA. Reproducible results were obtained in the SRID assay (coefficient of variation (CV) = 4.0%), with a mid‐point denaturation of 79.0 ± 0.4°C. Similar results were obtained with two MAbs (mid‐point denaturation at 79.4 ± 0.5°C and 78.3 ± 0.8°C for competitive and sandwich ELISAs respectively), but the CV% values were higher (8 and 13% respectively). With the remaining four MAbs, quantification was unreliable as immunoreactivity increased during heating with large inter‐assay variations. A careful control of MAb immunoreactivity in situations covering all possible denaturation states of the test protein is an absolute prerequisite to their use in ELISAs for the immunoquan...

Biotin-avidin amplified enzyme-linked immunosorbent assay (ELISA) for the measurement of canine serum IgA, IgG and IgM

An amplified capture enzyme-linked immunosorbent assay (ELISA) has been developed by the use of the biotin-avidin detection system, for the measurement of canine plasma immunoglobulins (Ig) A, G and M. Test responses of dilutions of both the Ig standards and test plasma samples were consistently linear (r >0·987) for the three Ig classes. The within-assay variation was 3·53 per cent IgG, 5·84 per cent for IgM and 6·34 per cent for IgA. The analytical recoveries were 95 per cent for IgA, 97 per cent for IgG and 98 per cent for IgM. The lower detection limits of the assay were 38·4 ng ml −1 for IgG, 20·3 ng ml −1 for IgM and 41·2 ng ml −1 for IgA. The results indicate that this ELISA has a much higher sensitivity than the single radial immunodiffusion assay or the non-amplified ELISA for measurements of canine Igs, but has a comparable specificity and precision.

Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog

A capillary reversed passive latex agglutination test (capillary RPLA) was developed which allows quantification of serum C-reactive protein (CRP) within approximately 15 min. The logarithmic regression line (calibration curve) obtained after measuring each CRP concentration three times in twofold dilutions of a standard canine serum containing 222 micrograms/ml of CRP was y = 6.394 + 0.030x (r = 0.995). Capillary RPLA permitted quantification of CRP in the range 6.9-222 micrograms/ml. The coefficients of variation ranged from 10.28% to 12.40%. The recovery rates (percentage recovery) of CRP by capillary RPLA were within the range 87% to 106%. On measuring the CRP concentrations in sera from 78 dogs by capillary RPLA, single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), close correlations were demonstrated between SRID and capillary RPLA (y = 7.250 + 1.109x, r = 0.978), between SRID and ELISA (y = 3.042 + 1.059x, r = 0.967), and between capillary RPLA and ELISA (y = 1.778 + 0.929x, r = 0.962). Capillary RPLA may be considered useful as a routine biochemical technique for measurement of serum CRP concentration in the dog.

Comparison of methods for measuring serum immunoglobulin concentrations in neonatal llamas

Summary Blood samples were collected from 25 neonatal llamas before suckling and at 24, 48, 72, 96, and 120 hours of age. Nine tests for determining serum immunoglobulin concentrations were performed on each sample, to compare within-test variation and correlations among tests. The single radial immunodiffusion assay was the only quantitative test and therefore, was judged the most accurate test for determining the status of passive transfer of immunoglobulins in neonatal llamas. Measurement of globulin concentration and total serum protein, and the sodium sulfite precipitation test were accurate when compared with radial immunodiffusion assay results. Measurements of total serum solids by use of a hand-held refractometer and of albumin concentration, and the zinc sulfate turbidity and 10% glutaraldehyde coagulation tests were not reliable for detecting failure of passive transfer in neonatal llamas. Ten of 25 neonatal llamas were suspected of having partial to complete failure of passive transfer on the basis of results of the single radial immunodiffusion assay. The other tests used in this study identified between 4 and 7 of these 10 llamas at 24 hours of age. On follow-up communication 4 to 6 months after the last blood sample collection, none of the 25 llamas were identified as having been clinically ill or had received any treatments for illness associated with low immunoglobulin concentrations.

Influence of calf genotype on colostral immunoglobulins in Bos taurus and Bos indicus cows and serum immunoglobulins in their calves.

Purebred Bos indicus calves are documented to have lower survival rates than Bos taurus calves. Thus, this study was designed to investigate the possibility that this decreased survival rate may be attributed to dam colostral immunoglobulin (Ig) concentrations and subsequent calf serum Ig concentrations. The specific objective was to determine the effect of breed type of calf on colostrum production, immunoglobulin concentrations in colostrum and calf serum, and availability and absorption efficiency of Ig. Brahman (B) and Angus (A) cattle were reciprocally mated to produce calves of the following types: A x A (n = 8), A x B (n = 9), B x B (n = 11), and B x A (n = 11). At birth, calves were separated from their dams and a blood sample was collected before feeding pooled colostrum (30 mL/kg birth weight) at 1 and 6 h of age. From 6 to 12 h of age, each calf was placed in a box that allowed interaction with the dam but prevented suckling. At 12 h of age, each calf was fed its dam's colostrum and placed with the dam. Additional blood samples were collected at 12, 24, and 48 h after birth. Serum and colostrum samples were analyzed for IgG, IgG1, IgG2, IgM, and IgA using single radial immunodiffusion (RID) assay techniques. The cows were hand-milked after induction of milk letdown with oxytocin at 1 and 12 h after calving. Colostrum volume was recorded, and samples were collected. Brahman cows produced more (P < .001) colostrum at 1 and 12 h than A cows. Total Ig concentrations were obtained by summing IgG, IgG1, IgG2, IgM, and IgA concentrations. Total Ig (P < .02), IgG (P < .005), and IgA (P < .01) concentrations in colostrum were greater in cows producing crossbred calves. Total Ig (P < .006), IgG (P < .02), IgG1 (P < .004), and IgG2 (P < .02) available in colostrum were affected by B x B and A x B breed types of calf. Brahman cows had more Ig available at 1 and 12 h than A cows due to increased production of colostrum. Breed type influenced colostral Ig in cattle. Serum concentrations of total Ig, IgG, IgG1, IgG2, IgM, and IgA in the calf and efficiency of absorption at 6 and 12 h were not affected by breed type, sex of calf, or any interaction.

Fluorescein isothiocyanate-labeled protein G as an affinity ligand in affinity/immunocapillary electrophoresis with fluorescence detection.

Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10(-6) mol/L was used, h-IgG concentrations between 10(-6) and 10(-9) mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r2, of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h-IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r2 = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG1 antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG1, in human sera.

Single-radial-immunodiffusion as an in vitro potency assay for human inactivated viral vaccines

Single-radial-immunodiffusion (SRID) assays have been used to determine the potency of all human inactivated influenza virus vaccines licensed by the Food and Drug Administration for use in the United States since 1978. SRID replaced less reliable tests which were based on the aggregation of erythrocytes by the hemagglutinins of influenza viruses. Similar SRID assays have been used experimentally to determine the potency of inactivated polio and rabies vaccines. In each case, the assays are based on the diffusion of viral antigen into an agarose gel containing specific antibodies to the antigen being measured. For influenza and rabies, disruption of the virions with a detergent is necessary to permit the diffusion of the appropriate antigens, where as with polio, intact virions are allowed to diffuse. The interaction between antigen and antibody produces a zone of precipitation whose size is directly proportional to the amount of antigen applied. A potency value for unknowns is obtained by comparing the sizes of zones produced by unknown preparations to the sizes of zones obtained with a calibrated reference of known antigen content. Once the specific reference antigens and antibodies are prepared and the test standardized, it is a remarkably simple technique which unlike agglutination assays is very reproducible, relatively unaffected by minor variations in test conditions and is far less time consuming and cumbersome than in vivo assays for potency such as those done by inoculating mice or monkeys. More importantly, clinical studies demonstrate that standardization of influenza vaccines by SRID provides a better correlate of human immunogenicity than previous methods.

Correction factors for single radial immunodiffusion and electroimmunodiffusion assays of IgA in secretory fluids

While the assay of immunogiobulin A (igA) in serum is well standardized, the determination of secretory IgA (slgA) remains a problem [1]. The presence of free secretory component (SC) complicates direct assay when anti-SC sera are used [2]. In addition, slgA polymorphism interferes with methods based on anti-or chain sera, and a correction factor must be used to calculate the concentrations of total IgA obtained with a monomeric IgA (mlgA) standard [3]. The value of this correction factor differs for single radial immunodiffusion (SRID), electroimmunodiffusion (EID), radioimmunologic assay and immunonephelometry [4-7]. We propose correction factors for use in determining IgA in colostrum and feces. They are given for both commercial IgA standards and for those prepared in our laboratory.

Serum immunoglobulin (G, A and M) classes and IgG subclasses in sickle cell anaemia.

Serum concentrations of immunoglobulins G, A, M and IgG subclasses were determined by single radial immunodiffusion assay in a population of sickle cell anaemia patients resident in the tropics. Fifty apparently healthy subjects of haemoglobin genotype AA, of comparable age, sex and socioeconomic status (SES), and in the same environment as the patients, were included as controls. Three indices of morbidity in SCA, namely frequency of crisis, degree of anaemia and the number of organ complications, were used to derive a severity score for each patient; and thus categorize the subjects into severity groups. Immunoglobulin levels were then correlated with the indices of morbidity as well as the derived severity score. IgG, IgA, IgM, IgG1 and IgG3 levels were significantly raised in the SCA subjects when they were compared as a group with the controls. When separated into disease severity groups, the mildly affected patients were found to have virtually normal levels of immunoglobulins. Total IgG concentration and level of the IgG3 subclass showed significant positive correlation with frequency of crisis and derived severity score. Markedly raised levels of IgG and IgG3 may be predictive of severity in sickle cell anaemia.

Bovine haptoglobin: Single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera

Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.

Impaired blood superoxide dismutase in the traumatic paraplegic patients.

Blood superoxide dismutase (SOD) activity and blood copper, zinc-superoxide dismutase (Cu.Zn-SOD) content were measured by luminol chemiluminescence assay and by single radial immunodiffusion assay, respectively, in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976, compared with 20 age-matched healthy subjects. We found that blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than those in healthy subjects (p < 0.01). In healthy subjects, blood Cu.Zn-SOD fully expressed the enzymatic activity, whereas only 77% of blood Cu.Zn-SOD in the paraplegic patients expressed the enzymatic activity, indicating that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. Also the serum lipid peroxide level in the paraplegic patients was higher than that in healthy subjects (p < 0.05). These findings suggest decreased endogenous blood protection against oxygen derived free radicals in these paraplegic patients.

Immunoquantitation of rat erythrocyte superoxide dismutase: Its use in copper deficiency

Two immunoassays have been developed for the determination of rat erythrocyte dismutase (Cu,Zn-SOD). An enzyme-linked immunosorbent assay (ELISA) was very sensitive down to 4 ng/ml with a coefficient of variation (CV) of 18% while the single radial immunodiffusion assay (SRID) permitted an adequate detection level (5 μg/ml) with far better accuracy (CV = 4.2%). The latter was thus selected for the determination of Cu,Zn-SOD in the red blood cells of normal and copper-depleted rats. The average value of Cu,Zn-SOD in normal adult rat erythrocytes was 1142 ± 120 ng/mg hemoglobin. When compared to activity measurements, good correlation was obtained between enzyme content and enzyme activity ( r = 0.803, P < .001). In an experimental copper deficiency followed by supplementation, good correlation was observed in the course of depletion ( r = 0.848, P < .001) and repletion ( r = 0.896, P < .001). During depletion, the loss of enzyme activity was mainly related to a loss of enzyme. However, enzymatically inactive protein was formed which would be activated when copper was added. These results indicate the importance of a combined use of Cu,Zn-SOD immunoquantitation and activity measurements to enable a better understanding of changes occuring with respect to enzyme activity.

20] Design, construction, and characterization of poliovirus antigen chimeras

Publisher Summary This chapter presents the techniques involved in the construction and use of poliovirus complementary DNA (cDNA) cassette vectors and in the recovery and exploitation of poliovirus antigen chimeras. It presents the detailed knowledge of the structure of the poliovirus in experiments aimed at redesigning the antigenic characteristics of the virus particle. This work started as part of a research into the designing of new improved polio vaccines but has also opened up the possibility of constructing vaccines against other infectious diseases, based on the poliovirus particle. The studies have resulted in the development of poliovirus as an epitope expression vector into which short, well-defined antigenic peptides can be inserted. In practice, the antigenic characterization involves two well documented approaches: (1) single radial immunodiffusion or antigen-blocking assays and (2) the neutralization of virus infectivity. Both assays can be used to demonstrate an interaction between the chimeric virus and antibodies raised against the foreign protein from which the expressed epitope is derived.

Serum Immunoglobulin Concentrations in Cattle Naturally Infected with Bovine Leukemia Virus

In order to elucidate whether natural infection of BLV in cattle might induce humoral immunological responses, changes in IgG1, IgG2, and IgM concentrations in the sera of infected cattle were determined. Twelve BLV-infected cattle were used. Cattle of different breeds were classified serologically and haematologically into BLV + PL+, BLV + PL- and BLV-free groups. Ig concentrations in the sera of the three groups were quantitated using a commercial single radial immunodiffusion assay. The findings were compared to those of BLV-free cattle. The serum IgM concentrations were significantly lower in cattle with PL (P less than 0.001) than in BVL + PL- and BLV-free cattle. The IgM concentrations tended to be lower in BLV+ PL- than those of BVL-free cattle. There were no significant differences in IgG1 and IgG2 serum concentrations between the three experimental cattle groups. IgG1 was the predominant subtype in the sera of all cattle groups.

A modified zinc sulfate turbidity test for the detection of immune status in newlyborn foals

The accuracy of a modified zinc sulfate turbidity test, in estimating a serum IgG concentration of 400 mg/dl, was evaluated. Sera from 161 foals <18 hours of age were incubated for 15 and for 60 seconds with zinc sulfate. Turbidity was assessed both visually, in a veterinary practice and a laboratory, and with a spectrophotometer. Serum IgG concentration was determined by single radial immunodiffusion assay. Twenty five of the 161 foals (15.5%) had <400 mg of IgG/dl of serum. Failure of passive transfer (serum IgG concentration <400mg/dl) was correctly identified visually in 21 of the sera (84%) in the laboratory, and in 20 of the sera (80%) in the veterinary practice. Spectrophotometric values (light transmittance 550 nm) from zinc sulfate assays were inversely proportional to the serum IgG content (r= −0.88; p<0.0001). Serum containing <400 mg/dl transmitted >80% light. Evidence of adequate passive transfer was detected visually in 100% of the samples (136 serum samples) and 96% of these samples transmitted <80% light (131 of 136). Seventy four percent of those foals sampled by 10 hours after foaling had >400 mg of IgG/dl. The modified zinc sulfate turbidity test detected accurately all foals with failure of passive transfer when sera was assessed visually and by spectrophotometer. Serum containing <400 mg of IgG/dl had no precipitate or slight precipitate and transmitted >80% light.

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.