Single Radial Immunodiffusion Technique Research Articles

Gut-associated IgA deficiency in lepromatous leprosy.

Sera, intestinal secretions and intestinal biopsies were collected from twelve biopsy-proved lepromatous leprosy patients with the help of a capsule invented by Roy Choudhury. Sera from another twenty-five proved lepromatous cases were also included. Sera and intestinal aspirates from twenty-five normal subjects and twenty patients with intestinal tuberculosis were also taken as controls. Acid-fast organisms, morphologically resembling Mycobacterium leprae, were detected in the intestinal aspirates of only two leprosy patients. Immunoglobulin levels in their sera and elevation secretions were estimated by the single radial immunodiffusion technique. Significant elevation of serum IgG, IgA and IgM, selective IgA deficiency in intestinal aspirates and flattening of intestinal villi along with mononuclear cell infiltration were conspicuous observations in the leprosy group. On the other hand, the patients with intestinal tuberculosis showed elevation of the IgG level in serum as well as in the intestinal secretions. It was postulated that persistent challenge by M. leprae or its antigens to the IgA immunocytes of the intestinal epithelium might have induced tolerance leading to IgA deficiency and subsequent subtotal atrophy of the intestinal villi in the patients with lepromatous leprosy.

Antithrombin III deficiency in diabetes mellitus: influence on vascular degenerative complications.

Plasma Antithrombin III (At III), a natural inhibitor of coagulation, was determined using a single radial immunodiffusion technique. In 116 diabetics, plasma At III levels were significantly decreased (26.6 +/- 0.4 mg/100 ml) compared with those in 64 controls (31.0 +/- 0.3 mg/100 ml, P less than 0.001). An elevation of plasma fibrinogen degradation products in 42 per cent of our patients, and a positive linear relationship between platelet counts and At III levels ( r = 0.29, P less than 0.01), provided additional evidence for chronic disseminated intravascular clotting in diabetes mellitus. Diabetic retinal complications were more frequent in patient with low plasma At III levels (50.6 per cent of cases) than in those exhibiting At III concentrations within a normal range: 32.4 per cent of cases (X2 = 6.09, P less than 0.02). It is postulated that the low levels of At III encountered in diabetes result from excessive consumption, and that the deficiency may be responsible for the onset and/or aggravation of intravascular clotting. At III deficiency may therefore contribute to vascular degenerative complications, particularly those leading to diabetic retinopathy.

Immunochemical Measurement of the Vitamin D-Binding Protein in Rat Serum*

The vitamin D-binding protein (DBP) was measured in rat serum using a single radial immunodiffusion technique. Normal serum levels at birth (74 +/- 11 mg/liter, mean +/- SD) were lower than during the last day of fetal life (130 +/- 14 mg/liter) and much lower than in adult rats. A marked sex difference in DBP occurred after puberty: male values (656 +/- 52 mg/liter) were significantly higher than female values (472 +/- 46 mg/liter). The sex difference could be abolished by either adult gonadectomy or transpharyngeal hypophysectomy. Implantation of a pituitary gland under the renal capsule in hypophysectomized male rats further decreased the DBP concentration, suggesting that PRL suppresses the DBP level. A similar decrease was also observed at the end of pregnancy and during lactation. Administration of androgens to either normal female or gonadectomized male rats increased their DBP concentration to the normal adult male level. The serum levels of total 25-hydroxyvitamin D did not fluctuate according to the concentration of DBP, indicating that the concentration of "free 25-hydroxyvitamin D" is not regulated at a constant level.

Distinct Glycosylation of Serum Proteins in Patients With Cancer: Brief Communication 2

Concanavalin A and wheat germ agglutinin, lectins that interact with serum glycoprotein in a manner similar to the antigen--antibody reaction, were used as "antibodies" in a single radial immunodiffusion technique to test a coded serum panel (from the National Cancer Institute, Bethesda, Md., and the Mayo Clinic, Rochester, Minn.) containing a) 99 serum samples from patients with different types of malignant neoplasms of the gastrointestinal tract, prostate gland, and lung, b) 50 samples from patients with benign diseases of the same organs as those affected in the cancer patients, and c) 50 samples from apparently healthy smokers. The resulting precipitation rings were not correlated to serum protein concentration, and the differences (demonstrated by Student's t-test and with a generalization of the one-sided two-sample Kolmogorov-Smirnov statistic for evaluating diagnostic tests) established that serum glycoproteins are glycosylated differently in cancer patients than in people without cancer.

Studies on Epstein-Barr virus-related antigens. I. Indirect single radial immunodiffusion as a useful method for detection and assay of soluble antigen.

A useful method for the detection and assay of Epstein-Barr virus (EBV)-related soluble antigen has been developed by the application of the indirect single radial immunodiffusion technique which is frequently used for quantitative measurements of immunoglobulins and other soluble proteins. When the extracts of EBV-determined nuclear antigen (EBNA)-positive non-producer cells (Raji and NC-37) were applied to agar plates containing seropositive human serum, followed by overlay with anti-human IgG serum, ring-shaped precipitates with high specificity were clearly evident. The size of such precipitin rings was proportional to the amount of the antigen. This method is simple and applicable for a quantitative assay of a particular EBV-related soluble antigen and antibody and the sensitivity is equivalent to that seen with the complement fixation test.

Phenytoin‐Induced Depression of Salivary IgA and Gingival Hyperplasia

IgA concentrations were determined in saliva from epileptic patients taking phenytoin and in saliva from healthy controls, by single radial immunodiffusion technique. Mean salivary IgA in epileptic children was 7.23 mg/ml; in healthy children, 16.44 mg/ml. Corresponding values for adults were 13.53 and 19.48 mg/ml, respectively. In 14 out of 84 samples, salivary IgA levels were too low for quantitative analysis. Salivary IgA levels were normal in untreated patients and fell during treatment with phenytoin. Gingival inflammation was commonly accompanied by an increase of salivary IgG and serum-derived IgA, whereas compensatory increase of IgM was infrequent. Phenytoin-induced deficiency of salivary IgA can result in increased susceptibility to gingival inflammation, which is considered one of the predisposing factors for subsequent development of gingival hyperplasia.

Specific protein assays in protein-calorie malnutrition

Assessment of serum protein concentrations in protein-calorie malnutrition was carried out by two specific immunochemical methods, the Single Radial Immunodiffusion technique and the Automated Immunonephelometric procedure. The correlation coefficient ( r) between the two methods was above 0.85 for all the serum proteins assayed. The automated nephelometric assay procedure has the advantages that it is rapid and that a large number of samples can be readily processed. The system in its present form could not be readily used to quantitate proteins such as prealbumin whose serum concentration was less than 50 mg/100 ml. The single radial immunodiffusion technique gave more consistent results for IgA estimation than the automated technique. With the exception of C4 and the immunoglobulins, all the serum proteins assayed were lower in children with protein-calorie malnutrition than in control children. During refeeding in children with protein-calorie malnutrition the first serum protein to show a significant rise was C3. This was followed by transferrin, then prealbumin and lastly albumin. C4 did not show any significant change. In experimental protein-calorie malnutrition in rats, the first serum protein to show a significant reduction was C3. This was followed by transferrin and then albumin. On the rats these serum proteins returned to normal values in the same order.

Estimations of active and inactive carbonic anhydrase isozyme B in human red cells

Human erythrocyte carbonic anhydrase isozyme B was measured by a specific and immunological method. The levels of carbonic anhydrase B were determined in normal subjects, patients with hyperthyroidism, and patients with chronic obstructive lung disease and patients with epilepsy under treatment with acetazolamide, using the rapid assay method of single radial immunodiffusion. The levels of carbonic anhydrase B showed a decrease in hyperthyroidism, and increased in chronic obstructive lung diseases and epilepsies. Closely negative correlations were observed between carbonic anhydrase B and T 3 resin sponge uptake or protein bound iodine (PBI) levels in hyperthyroidism. Simultaneously, carbonic anhydrase B-dependent esterase activity (active carbonic anhydrase B enzyme) was determined kinetically using the immunoadsorbent method in the above-mentioned patients. The results were compared with the total enzyme protein (active and inactive carbonic anhydrase B enzyme), estimated by the single radial immunodiffusion technique. In patients treated with acetazolamide, the “true” specific activity of the carbonic anhydrase B (carbonic anhydrase B-dependent esterase activity/total carbonic anhydrase B protein) decreased remarkably. In chronic obstructive lung disease, no remarkable changes were observed. On the other hand the specific activity was elevated in hyperthyroidism to 2 times higher than that of normal subjects. The clinical significance of these active enzymes is discussed.

Identification and estimation of plasma proteins in human dentine

The total non-collagenous matrix (NCM) of human dentine was isolated by EDTA extraction followed by collagenase digestion, both procedures being carried out within a dialysis sac. The presence of serum albumin, transferrin and the immunoglobulins (IgG, IgA and IgM) was demonstrated by double diffusion on Ouchterlony plates. Quantitation of the amounts of these serum proteins in human dentine was achieved by the single radial immunodiffusion technique. One gram of dentine NCM was found to contain 20.1 mg of albumin, 2.1 mg of transferrin and 4.0 mg of IgG; IgA and IgM each occurred at a level of 0.24 mg/g.

Carbonic anhydrase isozymes, hemoglobin-F and glutathione levels in lead-exposed workers

Red cell carbonic anhydrase B and C isozymes and hemoglobin F were studied by the single radial immunodiffusion technique in lead workers exposed to low concentrations of lead. The lead contents, δ-aminolevulinic acid dehydrase and glutathione levels were simultaneously determined. Carbonic anhydrase B, glutathione and δ-aminolevulinic acid dehydrase activity in lead-exposed workers showed relatively low values compared to those of normal subjects not exposed to occupational lead exposure in the past or present. Subjects with blood lead contents of 30 μg per 100 ml or higher showed a significant increase in hemoglobin-F and a significant decrease in carbonic anhydrase B and glutathione levels. These findings indicate that the syntheses of these red cell components are affected by lead, and hence in addition to δ-aminolevulinic acid dehydrase activity, these components were also found to be of clinical value for the diagnosis of subclinical lead poisoning.

Protein A production in different strains of Staphylococcus aureus under varied growth conditions.

The protein A content of extracts of staphylococcal cells was estimated by the single radial immunodiffusion technique (Mancini technique) and by a haemagglutination test. The grouping of S. aureus strains according to protein A production using these methods was essentially similar to that based on their different affinity to FITC‐labelled immunoglobulins. High yields of protein A were obtained from staphylococcal cells which were harvested after growth on solid medium and extracted in 0.07 M phosphate buffer at pH 5.9 for 10 days at room temperature. The presence of sodium chloride (7.5 per cent) in the medium suppressed the formation of protein A. The immunogenic properties of both protein A producing strains and protein A were dependent on the presence or absence of serum (IgG) in the medium. Due to the complex formation between protein A and IgG, even highly purified protein A induced antibodies which reacted specifically with immunoglobulins when serum or blood had been present in the medium.

Quantitative Distribution of Brucella Antibody amongst Immunoglobulin Classes in Vaccinated and Infected Cattle

Summary The quantity of specific Brucella antibody present in the serum of vaccinated cows and calves and the serum and whey of naturally infected cattle was determined using the single radial immunodiffusion technique. In vaccinated cattle the great majority of antibody produced following vaccination was found in the IgG1 and IgM classes with similar quantities present in the serum of both cows and calves. At the peak of the response 23% of IgG1 and 37% of IgM was specific Brucella antibody. In cattle which were positive to the agglutination test most of the serum antibody was IgG1 with smaller amounts of IgM antibody present. On the other hand cattle classified as inconclusive had much less antibody in both the IgG1 and IgM classes than those classified as positive. In colostrum and milk whey positive to the agglutination test 24-54% of the IgA and 36-46% of the IgG1 present was specific Brucella antibody.

Polymer Enhancement of Automated Immunological Nephelometric Analysis, as Illustrated by Determination of Urinary Albumin

Abstract We have developed a new automated immunonephelometric method for determination of albumin in urine, by exploiting the polymer-enhancing effect on the immunological reaction. The original continuous-flow manifold has been simplified and the reaction time shortened to about 3 min. The antiserum was diluted 100-fold in a solution (100 g/liter) of polyethylene glycol (av mol wt, 6000) and mixed with the prediluted sample in the continuous-flow system. The method is highly sensitive and is unaffected by high blank and (or) high absorbance values of the specimens. Day-to-day variation was 3.8-4.3% (CV). Accuracy, as estimated from recovery experiments, was also good. A comparative study of 176 urines with the single radial immunodiffusion technique showed a correlation coefficient of 0.994. We therefore suggest the new method for routine use for determination of urinary albumin, because it is fast, accurate, precise, and sensitive, and requires only small amounts of antiserum.

Analysis of Albumin in Meconium for Early Detection of Cystic Fibrosis

Albumin in meconium was quantitated by means of a single radial immunodiffusion technique, which is described in detail. The method shows good precision and accuracy for this material, when certain conditions associated with collection, preparation, and analysis are observed. The method can discriminate between healthy newborns and newborns with cystic fibrosis.

Immunoglobulin levels as related to age in nonhuman primates in captivity. II. Rhesus monkeys.

Immunoglobulin levels in relation to age were studied in the sera of 154 rhesus monkeys. The quantitation of immunoglobulins was performed by a modified single radial immunodiffusion technique with class-specific antisera against rhesus monkey IgG, IgA, and IgM. The development of IgG was found to be gradual from the second year of life up to young adult age. The IgA and the IgM developmental curves showed an increase in the first three years. A slight drop in the concentrations at the age of four was followed by a rapid rise up to the normal adult values already at the age of five. The presence of IgD was detected by cross-reaction with an antihuman IgD serum in only two of 154 rhesus monkey sera.

Evaluation of a specific protein analyser

A specific protein analyser (Technicon Autoanalyser II for Immunoprecipitin Techniques, Technicon Instruments Corp., Tarrytown, N.Y. 10591, U.S.A.) has been assessed for the estimation of immunoglobulins A and G (IgA and IgG), haptoglobin, and α 1-antitrypsin (α-AT). Values determined by the analyser procedure correlate well with values obtained by the single radial immunodiffusion technique. The effect of haemolysis has been studied and shown to have no effect on the quantitation of IgA, IgG and α 1-AT by this method. Reference sera and antisera to IgA and IgG from two sources have been investigated, and good correlations obtained. Normal ranges for IgA and IgG have been determined. The estimation of immunoglobulin M (IgM) has also been investigated and as no satisfactory results have been obtained using the recommended method, further studies are continuing.

Discrepant equations derived from the single radial immunodiffusion technique

Discrepant equations derived from the single radial immunodiffusion technique

Discrepant equations derived from the single radial immunodiffusion technique

Discrepant equations derived from the single radial immunodiffusion technique

PCB-induced suppression of the humoral and cell-mediated immunity in guinea pigs

The effect of PCB feeding at levels of 0, 10, 50 and 250 ppm, on the humoral and cell-mediated immune response is described. Three experiments were carried out with guinea pigs. A suppression of the humoral immunity, after stimulation with one dose of tetanus toxoid (alum-adsorbed) was found at the 50 ppm level.The antitoxin titres were statistically significantly decreased as determined with the passive haemagglutination test. Also, the number of tetanus antitoxin-producing cells in the stimulated popliteal lymph nodes were reduced, as was shown with the indirect fluorescent antibody technique. In a second experiment, animals were immunized with two doses of tetanus toxoid. A decreased immunological response was observed again at the 50 ppm level using single radial immuno-diffusion and fluorescent anti-body techniques. In addition lymphopenia was found. In the third experiment the cell-mediated immunity was stimulated with Freund's Complete Adjuvant. The skin reactions after tuberculination, as a parameter of the cell-mediated immunity, were statistically significantly reduced at the 50 ppm level. In all 3 experiments, organ weights were recorded and histological studies were performed. Stress was not considered responsible for the reduced immunological responses. A high mortality occurred at the 250 ppm level. Cachexia and depletion of the lymphoid system and liver damage were the most important findings in these animals. PCB contents of pooled liver samples are given.

Intravaginal Immunization of Humans with Candida Albicans

Abstract Ten women were immunized intravaginally with a Candida albicans vaccine. Antibody was measured by a radioactive single radial immunodiffusion technique, and antibody activity was expressed as the area of the precipitin ring. The mean rise in serum antibody was 41 to 66 mm2. The mean rise in cervicovaginal secretion antibody was from <12 mm2 to 74 mm2. The cervicovaginal secretion antibody was removed by absorption with anti-IgA, but not appreciably by anti-IgG or anti-IgM. These results suggest that cervicovaginal secretion IgA antibody is locally produced and can be stimulated by local application of antigen.