RESUMEN
Saliva specimens stored for 18 months at -20 degrees C with or without glycerol and the anti-protease benzamidine-HCl, lost all antibody activity for S. mutans. IgA activity in processed whole saliva decreased significantly after one week when stored either at 4 degrees C or -20 degrees C with or without glycerol, although it was stable in parotid saliva for at least 40 days. Loss of activity prior to processing was significant in the first 24 h, and the addition of 50% glycerol and storage at -70 degrees after processing, prevented loss of antibody activity in both whole and parotid saliva. Diurnal variations in IgA, lactoferrin and the IgA secretion rate were insignificant in parotid saliva but showed some fluctuations in whole saliva. Albumin and lactoferrin levels exhibited the greatest fluctuation in whole saliva specimens although IgA and IgA antibody levels were still more characteristic of the patient than the time of sampling. Monthly variations in IgA, IgA antibody activity and other parameters were least in parotid saliva and e.g., values for parameters that were high in patients samples on the first month, remained high during the 4-month study period. Statistical analyses showed a high correlation between values obtained for most of the 15 parameters that were measured in parotid and whole saliva specimens collected from greater than 20 patients during 2 successive visits. Whole saliva values for albumin, lactoferrin and albumin levels in parotid saliva, were most variable but differences were not significant. Hence, patients with very low or very high values, even in whole saliva, can be identified within the population on the basis of specimens collected at a single time.
Asunto(s)
Caries Dental/inmunología , Raíz del Diente/inmunología , Anciano , Anticuerpos Antibacterianos/análisis , Formación de Anticuerpos , Ritmo Circadiano , Criopreservación , Humanos , Inmunoglobulina A Secretora/análisis , Saliva/inmunología , Proteínas y Péptidos Salivales/análisis , Manejo de Especímenes , Streptococcus mutans/inmunología , Temperatura , Raíz del Diente/patologíaRESUMEN
IgA, IgG and IgM antibody activity (ELISA Units/ml) to Streptococcus mutans, Actinomyces viscous and Escherichia coli CF8 in serum, parotid saliva and whole saliva was measured using the amplified ELISA (a-ELISA) while the concentration (microgram/ml) of each isotype of immunoglobulin as well as albumin and lactoferrin, was determined using sandwich ELISAs. Selection of suitable reagents from those commercially available was based on specificity tests using purified human immunoglobulin; most polyclonal reagents required further absorption to attain class specificity. Cross-absorption studies indicated the absence of patient antibodies that were cross-reactive among the bacteria studied, except for IgM in some cases. Expression of response in ELISA Units (E.U.) per microgram of immunoglobulin, i.e. specific activity, revealed that IgG specific activity was significantly higher in parotid saliva than in either whole saliva or serum for all bacteria studied; serum and whole saliva did not differ except for the higher specific activity in whole saliva to E. coli. The value of one E.U. was determined using the Comparative Antibody-immunoglobulin Capture Assay (CACA). Using this novel method, we estimated that about 0.05 percent of serum IgA was specific for Streptococcus mutans, 0.008 for Actinomyces viscosus and 0.004 for Escherichia coli CF8. The percentage of specific IgM antibodies was higher than for IgA and IgG. The concentration of IgA anti-Streptococcus mutans, Actinomyces viscosus and Escherichia coli levels are approximately 92 ng/ml, 25 ng/ml and 16 ng/ml in whole saliva and 46 ng/ml, 9.4 ng/ml and 6.3 ng/ml in parotid saliva.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Formación de Anticuerpos , Caries Dental/inmunología , Raíz del Diente/patología , Actinomyces/inmunología , Anciano , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Humanos , Inmunoquímica , Inmunoglobulina A/análisis , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/análisis , Saliva/inmunología , Proteínas y Péptidos Salivales/análisis , Streptococcus mutans/inmunología , Raíz del Diente/inmunologíaRESUMEN
Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.