Serum antibody levels in smoker and non-smoker saudi subjects with chronic periodontitis.

BACKGROUND: Cigarette smoking is a significant risk factor for the initiation and progression of periodontal disease. Studies have shown altered serum and gingival crevicular fluid inflammatory cytokine profiles, immune cell function, and altered proteolytic regulation in smokers. The observations are not consistent, and to date, there is no clear mechanism to explain how smoking may affect periodontal disease. Hence, the present study was undertaken to assess the alterations of serum immunoglobulin levels in smokers with periodontitis and its potential role as a risk indicator of the disease process. METHODS: In this study, 30 patients who smoked and 30 patients who did not smoke with chronic periodontitis and 30 healthy subjects were enrolled. Serum immunoglobulin (Ig) G, IgA, and IgM levels were estimated with immunoturbidimetric assay. The IgG subclass (IgG1, IgG2, IgG3, and IgG4) levels were performed using single radial immunodiffusion assay. RESULTS: Levels of serum IgG and IgA were significantly lower in smokers compared to non-smokers and healthy controls (P <0.001). Although IgM levels were low in smokers, it was not significant. Of the four subclasses of IgG studied, the IgG2 was found to be significantly lower among smokers with periodontitis. CONCLUSIONS: Current observations indicate that cigarette smoking may be associated with the suppression of B-cell function and immunoglobulin production. The alteration of antibody levels further explains the potential mechanism by which smoking exacerbates periodontal disease. Further studies at the molecular level may highlight the specific mechanism by which tobacco can interact with cells of the immune system and its impact on periodontal disease process.

Immunochemical determination of the serum protein reacting with antibody against human urinary trypsin inhibitor by single radial immunodiffusion: use of polyethylene glycol.

A single radial immunodiffusion method is described for determining the serum protein which reacts with antibody against the urinary trypsin inhibitor, but does not react with anti-inter-alpha-trypsin inhibitor antibody. Since the amount of this protein could not be determined with an agar plate containing antibody alone, we first prepared an agar plate containing 4% polyethylene glycol 6 000 and 1 mg of anti-urinary trypsin inhibitor gamma-globulin and confirmed that the amount of this protein could be measured accurately from the sizes of precipitin rings after 72 h incubation at room temperature. Levels of this serum protein, alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were measured in normal human serum by the single radial immunodiffusion method, and it was confirmed that the level of this serum protein did not correlate with the levels of these 3 other proteins.

Diffusion-in-gel thin layer immunoassay (DIG-TIA): optimal conditions for quantitation of antibodies.

A new technique for the quantitation of antigen-antibody reactions, diffusion-in-gel thin layer immunoassay (DIG-TIA), has been developed. The principle of DIG-TIA is that antibodies are allowed to form a concentration gradient by radial diffusion in an agar gel poured on top of an antigen-coated plastic surface. After removal of the gel and visualisation by means of condensation of water vapour on the plastic surface, the antigen-antibody reactions appear as zones of increased wettability. The concentration of antigen used for coating, pH of the agar, incorporation of Tween 20 in the agar, size of diffusion basins, time of diffusion, and reinforcement by anti-immunoglobulin have been studied with regard to their influence on sensitivity and precision in the detection of antibodies with DIG-TIA. A photographic technique for permanent recording of wettability patterns is also described.

A modification of the automated immune precipitin method for quantitation of human serum immunoglobulins.

The use of the automated immune precipitin method to measure human serum immunoglobulins has been reported by several groups. The authors encountered difficulties in evaluating the technic even when they incorporated such recent modifications as the use of polyethylene glycol to enhance the reaction. By altering the flow pattern and prediluting each specimen, they were able to increase sampling rate, decrease processing time, and decrease the frequency of line obstructions while obtaining reproducible standard curves without using polyethylene glycol. There was good correlation between the results obtained by their modification of the technic and those obtained by the Fahey radial immunodiffusion method. As with other automated immune precipitin systems, abnormally high values gave characteristic notched peaks and lipemic specimens were centrifuged in order to obtain the infranatant for quantitation. The present modification of the automated immune precipitin system provides a rapid, simple and efficient method to quantitate human serum immunoglobulins.

Procerain, a stable cysteine protease from the latex of Calotropis procera.

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.

A simple lysoplate method of lysozyme determination with samples dried on filter paper.

A simple method for quantifying lysozyme in serum and urine has been established by modifying the procedures of sample application in the lysoplate method reported by Osserman and Lawlor [1]. A strip of filter paper is partially immersed in a liquid sample, dried at room temperature and cut into discs which are later placed on the surface of an agarose gel containing Micrococcus lysodeikticus. The follow-ng procedures for determination are carried out as described in the lysoplate method. There was no statistically significant loss of enzyme activity during the sample preparation. Lysozyme dried on filter paper is so stable that it can be stored at room temperature for at least 13 weeks and can be mailed. The sensitivity of the method is increased by pretreatment of the filter paper with Triton X-100 and consequently corresponds to that of the lysoplate method. The reproducibility of our method is practically good since the variation coefficient of the diameters of lytic zones within assays is around 1%. There is a very close correlation between lysozyme levels determined by this method and by the lysoplate method with serum samples obtained from patients with various hematological diseases (r = +0.994). The method can be utilized for routine clinical determinations of lysozyme.

Differential measurement of monomeric and polymeric IgA by one-directional immunoelectrodiffusion.

A technique to specifically quantify monomeric IgA and total IgA in colostrum has been developed using a modified one-dimensional immunoelectrophoretic assay. This method employed electrophoresis in antibody-containing polyacrylamide-agarose gel in the presence of a gel barrier which blocks polymeric IgA. The addition of PEG (polyethylene-glycol 6000) to the anodic gel increased the sharpness of the peaks, the height of which was proportional to the antigen concentration. This method proved to be sufficiently simple, precise, reproducible (CV less than 3%) and linear (from 20-300 mg/l) to measure the monomeric IgA: total IgA ratio rapidly (14 +/- 4.5% for 20 samples in duplicate). Immunoelectrodiffusion studies confirmed that human colostral and serum IgA standards could be used to determine directly monomeric IgA, total IgA and polymeric IgA levels (by difference) rather than to apply correction factors to estimate these IgA levels.

Application of polyethylene glycol turbidity assay to detection of circulating immune complexes in cancer patients.

A rapid, reproducible immune complex screening assay was used to quantitate levels of circulating immune complexes in the sera of normal subjects, patients with documented increases in immune complexes from rheumatoid arthritis, patients with clinically or microbiologically documented infections, and patients with cancer. Although wide variations in individual values within the groups were noted and the concurrent elevation of polyethylene glycol-circulating immune complex levels by infection was documented as expected, significant differences were found in the values in patients with cancer compared with those in normal subjects. The overall clinical application of polyethylene glycol-circulating immune complex screening is discussed and current application of screening of serial sera samples from individual patients for correlation with measurable tumor volume is proposed.

Discrepancy of serum IgA levels determined by single radial immunodiffusion and laser nephelometry in patients with IgA nephropathy.

The levels of IgA in sera were quantitated by single radial immunodiffusion (SRID) and laser nephelometer (LN) to evaluate a precise amount of serum IgA in patients with IgA nephropathy, other glomerular diseases and healthy adults. Thirty patients with IgA nephropathy, twenty patients with other glomerular diseases and eighteen healthy adults were examined. It was shown that the levels of IgA in sera measured by LN were significantly higher than those in sera measured by SRID in patients with IgA nephropathy associated with increased levels of serum IgA. This suggests that the levels of serum IgA measured by SRID in patients with IgA nephropathy were affected by the physicochemical characteristics of circulating IgA in patients with IgA nephropathy. It is postulated that an increase of serum IgA in patients with IgA nephropathy may be mainly due to an increase of polymers rather than a monomer of IgA.

A comparison of three isolation methods for obtaining immunoglobulin A from turkey bile.

Turkey IgA was isolated from bile by three methods: ammonium sulfate precipitation, polyethylene glycol (PEG) extraction, and lambda-carrageenan extraction. The isolated immunoglobulin fractions were compared using double diffusion, immunoelectrophoresis (IE), and enzyme-linked immunosorbent assay (ELISA). Results indicated that all three methods of isolation are sufficient for the initial isolation step for purification of the immunoglobulin fraction in turkey bile. Because of contaminating IgG, IgM, and other high-molecular-weight proteins, further purification by column chromatography is needed to isolate pure IgA. The lambda-carrageenan extraction method appears to be the method of choice for precipitating the immunoglobulin fraction in bile, because of the high antibody activity after extraction. Like ammonium sulfate precipitation, lambda-carrageenan and PEG extraction are not sufficient as single-step purification methods and should be used as the initial step in the purification of IgA.

The relation between salivary IgA and caries in renal transplant patients.

OBJECTIVE: The purpose of this study was to evaluate the effect of immunosuppressive drugs on the level of salivary immunoglobulin A (IgA) in patients who have received kidney transplants and the relation between the levels of salivary IgA and dental caries incidence. STUDY DESIGN: Patients who had undergone renal transplantation (n = 28, aged 18-54) were divided into 3 groups according to postsurgical period (0-6 months [G(1)], 6-12 months [G(2)], and >12 months [G(3)]). A healthy control group (n = 10, aged 17-49) was also included in this study. Saliva samples were collected from all patients by the spitting method. After collection, the samples were frozen immediately at -40 degrees C until analysis by the single radial immunodiffusion method. All fissure caries were examined clinically, and proximal caries were examined clinically and radiographically; caries status was determined according to the decay surface index. The findings were evaluated statistically by means of correlation analysis, the Kolmogorov-Smirnov test, and the 1-way Kruskal Wallis analysis of variance method. RESULTS: Salivary IgA levels of the patients who had undergone renal transplantation were found to be significantly lower than those of the control patients (G(1) = 6.76 mg/dL, G(2) = 6.80 mg/dL, G(3) = 7.84 mg/dL, and control group = 10.84 mg/dL, P <.001). However, the caries status of the patients who had undergone renal transplantation was not different from that of the control subjects for the first year after the transplant operation. The salivary IgA values of the 3 groups of patients who had undergone transplantation were not significantly different from each other. Thus, it was observed that a decrease in the level of salivary IgA does not result in an increase in caries incidence within 12 months after renal transplantation. The caries rate in the third group of patients who had undergone renal transplantation was found to be significantly different from those in the first and second groups. CONCLUSION: Low salivary IgA levels caused by immunosuppression are not correlated or associated with higher levels of dental caries within the first 12 months after renal transplantation. However, the incidence of dental caries was higher for patients who had undergone renal transplantation than for control subjects 12 months after renal transplantation. Because of the diagnostic processes used, dental caries may not become evident until after 12 months.

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.

Development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin G.

OBJECTIVE: To develop an automated turbidimetric immunoassay (TIA) for measurement of bovine IgG. SAMPLE POPULATION: 24 bovine serum samples. PROCEDURE: IgG concentration was determined by use of the TIA, and results were compared with those of the radial immunodiffusion (RID) method. Variables were determined, using commercially available reagents and a clinical biochemical analyzer. For the TIA, polyclonal goat anti-bovine IgG (Fc specific) serum, bovine IgG calibrator serum, and polyethylene glycol reaction buffer were used. Sample concentrations were determined by the instrument, using the linear regression method of least squares. The accuracy of this assay was validated by referencing to a purified bovine IgG standard and by recovery of control standards. Parallelism was documented by assay linearity and serial sample dilution linearity. Interference resulting from hemolyzed samples was examined. RESULTS: The TIA method correlated positively (r = 0.9957) and significantly (P < 0.05) with the RID method, yielding a regression equation with slope of 0.78708 and y-intercept of 1.02102. Bias attributable to hemolysis was not observed. CONCLUSIONS: The TIA method is automated, accurate, and precise for bovine serum IgG quantification. CLINICAL RELEVANCE: This assay provides sample results in approximately 10 minutes and may be used as an alternative to the manual RID method.

Sensitivity and specificity of a modified agar gel precipitation test and its application to the diagnosis of enzootic bovine leukosis.

Sensitivity and specificity of a modified agar gel immuno-diffusion (AGID) test for the diagnosis of enzootic bovine leukosis was compared in 609 sera using the standard method, the modified test, and ELISA. The modifications concerned the composition of agar gel (1.8% Bacto-agar Difco), addition of polyethylene glycol (PEG 6000, 4%) and enlarging the diameters of the wells from 6 to 10 mm for sera. The change in well diameters and the addition of PEG 6000 increased the sensitivity of the test by about 60-100%, favourably influenced the proper interpretation of the results, especially with weak positive sera. ELISA proved to be more sensitive either than the modified (by 4.1%) or standard AGID (by 5.25%) tests. The modified AGID test may find wide application in laboratory practice, especially when a more sensitive technique is not available.

[Protein assay by immunoprecipitation with Centrifichem centrifugal analyser]. / Dosage des protéines par immunoprécipitation sur analyseur centrifuge Centrifichem.

The authors present an automatic technic of estimation of ten serum proteins (albumin, alpha-antitrypsin, orosomucoid, alpha-1-macroglobulin, haptoglobin, C'3 complement, transferrin, IgM, IgG, IgA) by immunoopacimetry on a Centrifichem centrifugal analyser. This technic is derived from an immunonephelometric method, with the following differences: --the use of two concentrations of polyethylene glycol permitting one to obtain for all proteins, a constant levelling-out of the reaction within 3 minutes; --by a first reading at 3 seconds avoiding the prior determination of blank reagents and sera; --by the use of two dilutions of serum permitting calculation of all concentrations. The results are reproducible and are satisfactory: 95% of the coefficients of variation are less than +/- 5% in the case of all the proteins. The correlation with IDR is good. The coefficients vary from 0.91 to 0.99.

Serum transcortin levels in patients with chronic hemodialysis.

Patients with renal insufficiency who are hemodialyzed 3 times a week either through a cuprophane membrane or through a polyacrylonitrile (PAN) membrane have the same transcortin and cortisol levels as patients with renal insufficiency who are not dialyzed. Moreover, hemodialysis did not influence acutely the latter transcortin levels.

[Determination of an intrinsic value of diffusion coefficient of active principle in polymeric matrix for the formulation of a controlled release system]. / Détermination d'une valeur intrinsèque du coefficient de diffusion d'un principe actif dans une matrice polymérique en vue de la formulation d'un système à libération contrôlée.

The study of diffusion in a polymeric system often poses difficult experimental problems, notably when the rate of release is determined in a liquid receptor phase which can interact with the system. In this publication, we propose two experimental methods to determine the diffusion coefficient by a gel-gel kinetic release study, in which the receptor is a same gel unloaded initially. The first method is based on the use of radioactive tracers and a multi-channel radioactivity counter to obtain, at different times, the concentration profiles in the diffusion medium. The theoretical model is given for a gel donor with the concentration C0 of diffusing molecules is below or above the solubility Cs. The second method is based on the measurement of the displacement of solubility front during the diffusion within the system in the case C0 > Cs. The theoretical model shows that this displacement is a function of square root of t. For illustration, two experimental determinations of testosterone diffusion coefficient on the one hand in a silisic acid gels and on the other hand in an acrylic acid gels are given.

[The humoral immune response to the influenza virus: the characteristics of the enriched virus-neutralizing fraction of hyperimmune mouse serum]. / Gumoral'nyi immunnyi otvet na virus grippa: kharakteristika obogashchennoi virus-neitralizuiushchei fraktsii giperimmunnoi syvorotki myshei.

Certain characteristics of immunosorbents prepared on the basis of aminocellulose and oxycellulose were determined. The affinity chromatography of sera from influenza-virus-immunized mice was shown to yield with the former of the immunosorbents a fraction of antibody in which the relative portion of antibodies detectable by neutralization test was higher than that in the original hyperimmune serum. The preparation of pure antibodies obtained with the oxycellulose-based immunosorbent contained antibodies detectable by NT and HI test in a ratio similar to that in the unfractionated hyperimmune serum. It is concluded that aminocellulose and oxycellulose differ in the nature of chemical interaction with influenza virion proteins owing to which differences in the pattern of interaction of immunosorbents prepared on their basis with virus-specific antibodies are observed. The process of influenza virion neutralization by antibodies was also shown to be a multi-hit reaction, the ratio of NT- and HI-detectable antibodies in a preparation affecting the kinetics of the virus-neutralizing effect of the substance. An increase in the preparation of a portion of NT-detectable antibodies reduced the time in which the number of antibody molecules sufficient for virus particle neutralization is adsorbed to it.