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PubMed1971

Serological diagnosis of human influenza infections by immunoprecipitin techniques.

In immunodiffusion tests employing disrupted influenza virus (A2/Hong King/68, X-31 strain) as antigen, precipitin lines corresponding to three virus antigens can be distinguished-namely, the ribonucleoprotein, the haemagglutinin, and the neuraminidase. In the present study a comparison was made of such immunodiffusion tests and conventional diagnostic methods (complement-fixation and haemagglutination-inhibition tests) for serological diagnosis of A2/Hong Kong/68 infections in man. Precipitin tests in which the acquisition or reinforcement of precipitins for A2/Hong Kong/68 virus were detected, were found to be as sensitive as conventional methods for the serological diagnosis of influenza. In convalescent human sera precipitin lines corresponding to influenza A ribonucleoprotein were frequently detected, lines corresponding to A2 neuraminidase were less frequent, and those corresponding to haemagglutinin were least frequent. Precipitin tests had considerable advantages over other methods of serological diagnosis of influenza. They were rapid and simple to perform and were not susceptible to the effects of nonspecific reactions. In addition the antibody response to each of three antigens of the influenza virus could be detected in a single test system.

Complement Fixation Tests↗
PubMed1977

Pseudomonas aeruginosa infection in cystic fibrosis. Relative prevalence of antibodies in serum against cathodic and anodic migrating P. aeruginosa antigens determined by immunoelectrophoretic methods.

Sera from 119 patients with cystic fibrosis were examined for precipitating antibodies against anodic migrating P. aeruginosa antigens by means of crossed immunoelectrophoresis. The presence of precipitins against cathodic migrating P. aeruginosa antigens was investigated by means of classical immunoelectrophoresis. Thirty-six per cent of the sera contained precipitins against both anodic and cathodic antigens, 20% contained only precipitins against anodic antigens, none contained only precipitins against cathodic antigens, and 44% had no demonstrable P. aeurginosa precipitins. The amount of extra information obtained by combining the results of classical immunoelectrophoresis with the results of crossed immunoelectrophoresis was small.

Adolescent↗
PubMed1984

Immunochemical studies on the binding properties of Brucella abortus lipopolysaccharides to bovine precipitating antibodies.

The binding properties of Brucella abortus smooth lipopolysaccharides (LPS) to the precipitating antibodies in the serum of a B. abortus S2308 infected cow were studied by quantitative precipitin and precipitin-inhibition assays. Crude B. abortus lipopolysaccharides (cLPS) and chemical modification products of strains 1119.3, 19 and 2308 (virulent) were used for this study. In the quantitative precipitin assay, 13 micrograms of cLPS precipitated about 4.0-5.2 micrograms nitrogen of precipitating antibodies in 10 microliter of serum. After alkaline treatment (0.125 N NaOH at 56 degrees C for 1 hr), the alkali-treated lipopolysaccharides (aLPS) from S19 and S1119.3 maintained the maximum precipitability, but the precipitation profile was changed from a sigmoidal to a bell-shaped curve, and aLPS from S2308 was reduced to about 40% of its reactivity. These results indicate that some of the antigenic determinants blocked by other molecules were reshielded by alkaline treatment, and possibly that structural differences exist between virulent (S2308) and nonvirulent (S19 and S1119.3) strains. Among the monosaccharides and glycosides tested for inhibition of precipitation, methyl alpha-D-mannose was most active and about 3.3 times as active as D-mannopyranose (DMan) and only 1/200 as active as acid hapten or the carbohydrate moiety of cLPS prepared from S1119.3 (expressed as a monosaccharide of mol. wt 200) implying that the carbohydrate determinants of this antigen are probably related to a DMan alpha 1----linked compound. The precipitability of the antibodies in this serum was greatly reduced after absorption with cLPS and the precipitin reaction was completely inhibited by the carbohydrate moiety of LPS (O-antigen) S1119.3 demonstrating that the common antigenic determinants (hapten) of B. abortus LPS reacting with these precipitating antibodies are in the region of the carbohydrate moiety of the LPS.

Animals↗
PubMed1983

Further evaluation of factors affecting a rapid ELISA procedure for detection of IgG antibodies to Candida albicans.

A rapid enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG antibodies against a cytoplasmic antigen of Candida albicans. The optimum conditions and time required for each step were investigated. Pre-equilibration at 37 degrees C and constant agitation of immunoreactants resulted in a total test time of 1 h for examination of single serum dilutions. Initial binding of the antigen at 37 degrees C to the solid phase occurred within 4-6 min and was dependent on concentration. A serum dilution of 100-fold resulted in adequate discrimination between precipitin test-positive and -negative sera. Interaction of antibody and the bound antigen was maximal after 6 min incubation at 37 degrees C and was dependent upon the precipitin titre of the serum. Ten minutes was selected as the optimal incubation time for each of the stages, by which time maximal binding had occurred, irrespective of antibody affinity for antigen. The ELISA was completed by incubation at 37 degrees C for 10 min with alkaline phosphatase-conjugated anti-human IgG. Discrimination between positive and negative test sera was achieved by incorporating in each test run precipitin test-positive and -negative reference sera from groups of persons with or without C. albicans colonization or infection. Closest agreement between sera positive for C. albicans precipitins by counter-immunoelectrophoresis and the rapid ELISA was seen when reference negative sera were selected from non-colonized individuals.

Antibodies, Fungal↗
PubMed1996

Precipitating Pseudomonas aeruginosa antibodies and antimicrobial therapy in cystic fibrosis patients.

Forty patients with cystic fibrosis were studied bacteriologically and serologically. Precipitating Pseudomonas aeruginosa antibodies were monitored by crossed-immunoelectrophoresis (CIE) in order to evaluate the possibility of preventing chronic colonization by Pseudomonas aeruginosa by cycles of antimicrobial therapy. Sputum or pharyngeal aspirate and serum samples from all patients were analyzed by means of spread on selective media and CIE, respectively. Significant differences in the number of precipitins were obtained: noncolonized and intermittently colonized patients had no precipitins, whereas the number of precipitins in the chronically colonized patients varied from 11 to 44. An increase in the number of precipitins could be a good marker for initiation of therapy with antimicrobial agents that are either active against Pseudomonas aeruginosa or able to inhibit the release of virulence factors.

Adolescent↗
PubMed1986

Diagnosis of systemic candidiasis: development of co-counterimmunoelectrophoresis.

To improve the results of counterimmunoelectrophoresis for serological diagnosis of candidiasis, a new method was devised to characterize human sera precipitins: co-counterimmunoelectrophoresis. Cellulose acetate was chosen as the support in order to observe identity reactions between precipitin lines from serum systems run conjointly (thus, co-counterimmunoelectrophoresis). Different experimental antisera (anti-somatic and anti-cytoplasmic antigens and anti-whole fixed germ tube antisera) were tested for reactions of identity with sera from selected patients with systemic candidiasis caused by Candida albicans. The reaction of patient sera and anti-germ tube antiserum with a selected somatic antigen always resulted in an identity reaction as proven by the continuity of major precipitin lines. The precipitating system, named the co-specific precipitin line, involved polysaccharide antigens; it was not present in human control sera.

Adult↗
PubMed1985

Precipitating antibodies against Micropolyspora faeni in equines in north-western India.

Prevalence of serum precipitins against Micropolyspora faeni, Thermoactinomyces vulgaris and Aspergillus fumigatus, employing the counterimmunoelectrophoresis (COIE) and Ouchterlony's double diffusion (DD) techniques, is reported in 162 of the equines stationed at two military installations in north-western India. M. faeni specific precipitins were demonstrable in 58 of 112 mules from site I in the mountainous region whereas the results were negative for all of the 50 horses examined from site II located in the plains. Of the 58 M. faeni positive mules, 45 (78%) had signs and symptoms suggestive of chronic obstructive pulmonary disease (COPD) while the remaining 13 (22%) were apparently free from any respiratory disorder. The more frequent occurrence of M. faeni precipitins in the symptomatic than in the asymptomatic group of mules was found to be statistically significant (P less than 0.01). Precipitins against A. fumigatus were concomitantly demonstrated in 5 of the mules afflicted with COPD and found to be positive for M. faeni.

Animals↗
PubMed1982

Immunochemical characterization of binding sites of hybridoma antibodies specific for alpha (1 leads to 6) linked dextran.

The combining sites of seven BALB/c IgM, four BALB/c IgA and one C57BL/6 IgA hybridoma antibodies specific for alpha (1 leads to 6) linked dextran were probed by precipitin and precipitin inhibition assays. The 12 antibodies are able to bind to linear determinants in the interior of the dextran molecule; some have sites complementary to six alpha (1 leads to 6) linked glucose residues and other have sites complementary to seven alpha (1 leads to 6) linked glucose residues. From the analysis of the precipitins and precipitin inhibitions, it is concluded that no two hybridoma proteins have identical binding sites.

Animals↗
PubMed1967

Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum.

1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an alpha-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-(14)C]Leucine or -valine and d-[1-(14)C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea-pig serum or fraction I, immunological identity being apparent with some lines. The microsome-bound substances thus represent serum glycoproteins or precursors of them. 8. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [(14)C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.

Animals↗
PubMed1984

Antibody to Aspergillus fumigatus antigens in normal sera: influence on positive-negative discrimination in ELISA.

The response of precipitin-negative sera from non-selected asthmatic patients against a range of somatic and culture filtrate antigens of Aspergillus fumigatus in ELISA for anti-A. fumigatus IgG is described. Antibody to the various antigens was widely distributed but the precise distribution was dependent upon antigen type. To determine how the selection of negative reference sera from precipitin-negative sera can influence the discrimination between precipitin-positive and -negative sera in ELISA, panels of 10 sera were chosen from the extremes of the frequency distribution and by random selection. The closest agreement between precipitin testing and ELISA was seen when randomly-selected negative reference sera were incorporated in the assay, although the degree of correlation was dependent upon the detector antigen. These findings demonstrate the requirement for careful selection of sera included as negative reference sera in ELISA for anti-A. fumigatus IgG.

Antibodies, Fungal↗
PubMed1971

New approaches for the laboratory recognition of M types of group A streptococci.

The successful classification of Group A streptococci by the capillary precipitin technique requires a complete series of M type antisera which are sufficiently potent and specific to give unequivocal type-specific reactions with all the serotypes. Specific antisera for this purpose have been prepared by absorption with heterologous streptococci. Unabsorbed antisera have been employed here in the Ouchterlony double-diffusion agar-gel test to identify the M type of streptococci. Techniques have been developed for making this method of M typing fully reliable. The results reported here confirm and amplify the original findings of Michael and Massell (3). With crude HCl extracts and unabsorbed M type antisera, a precipitin line due to the M protein and another to the group-specific carbohydrate are the two major reactions observed. These reactions, however, are usually readily distinguishable. There was a surprising lack of cross-reactive precipitin lines due to non-type-specific protein antigens in the extracts. Although many of the unabsorbed M type antisera can be employed in the double-diffusion tests, the group-specific antibody must be removed from some of the unabsorbed antisera to avoid confusing cross-reactions. Absorption of these antibodies has been achieved by means of a specific immunoabsorbent column prepared from para-aminophenyl-beta-N-acetylglucosamine and cyanogen bromide-activated Sepharose. Excellent agreement was observed between the M typing results obtained on 117 field strains by the conventional capillary precipitin method and the Ouchterlony double-diffusion method.

Absorption↗
PubMed1983

Inability of counterimmunoelectrophoresis to detect echovirus in cerebrospinal fluid.

Methods for rapid detection of viral antigens in cerebrospinal fluid (CSF) are needed to aid in the differentiation of viral from bacterial meningitis. The formation of precipitin bands in patients with suspect viral meningitis utilizing viral antisera in a counterimmunoelectrophoresis (CIE) system has been described. To investigate further the possible value of CIE in the diagnosis of viral meningitis, the specificity of the CSF precipitin bands was studied. Precipitin bands were formed between commercially available type-specific antisera and cell culture supernatant fluids. Precipitin bands were also formed when control CSF was used as an antigen. Using type-specific antisera produced against purified virus, enteroviral antigens were not detected in CSF from patients from whom CSF viruses had been isolated. CIE lacks sufficient sensitivity for the detection of echovirus 11 antigens in CSF.

Antigens, Viral↗
PubMed1979

Genetic variation in the M antigen of group A streptococci: reassortment of type-specific markers and possible antigenic drift.

The phylogenetic relationships of 73 strains of group A Streptococcus isolated over a six-year period from a population with a high prevalence of streptococcal infections were analyzed with use of four serotype-specific markers: the serum opacity factor (OF), the T antigen, the determinant of the M antigen that precipitates with antibody, and the M antiphagocytic determinant. OF inhibition tests divided the strains into five subtypes: a, b, c, d, and e. Strains within subtypes a, b, and c shared a previously unidentified M precipitin. The identification of this new common M precipitin was based on absorption studies, double agar immunodiffusion, and immunoelectrophoresis analysis. Antisera directed against representative strains from subtypes a, b, and c contained cross-reactive bactericidal antibody, and absorption studies indicated that determinants of the M precipitins were identical, whereas determinants of resistance to phagocytosis were closely related but not identical. The association of a common M precipitin with three different OF antigens, two different T antigens, and three different, yet similar, antiphagocytic determinants is discussed in light of the possibility that the genes that code for these antigens have reassorted or recombined. Moreover, the possibility that differences in the antiphagocytic determinants of these strains have resulted from antigenic drift in a common gene is considered.

Absorption↗
PubMed1978

Some immunological investigations on antithrombin III 'Budapest'.

Immunoelectrophoretic studies were carried out on plasma from a patient with an abnormal antithrombin III molecule (antithrombin III 'Budapest'). One-dimensional immunoelectrophoresis using a commercial antibody to antithrombin III (antibody to alpha2-AtIII) showed two precipitin peaks of alpha2-AtIII antigen. Two-dimensional immunoelectrophoresis showed two precipitin peaks, one with normal electrophoretic mobility, the other with increased electrophoretic mobility. It was shown that alpha2-AtIII antigen with normal mobility was identical to normal alpha2-AtIII, and that the alpha2-AtIII antigen with increased electrophoretic mobility was antigenically deficient, and appeared to be present in higher concentration than the normal alpha2-AtIII antigen. Although one-dimensional immunoelectrophoresis on plasma from the patient's son showed only one peak, two-dimensional immunoelectrophoresis revealed that the son also had two populations of alpha2-AtIII, one with normal mobility, the other with increased mobility. However, the alpha2-AtIII antigen with normal mobility appeared to be present in higher concentration than the alpha2-AtIII antigen with increased mobility. One- and two-dimensional immunoelectrophoresis using a different commercial antibody to alpha2-AtIII showed only one precipitin peak using the patient's plasma. The precipitin peak observed following two-deminsional immunoelectrophoresis was asymmetric and showed increased mobility.

Adult↗
PubMed1984

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against Micropolyspora faeni and Thermoactinomyces vulgaris.

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37 C and vigorous agitation during incubation enabled the total test time required for the procedure to be reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive-negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.

Antibodies, Anti-Idiotypic↗
PubMed1975

Chicken antichromatin antibodies: specificity to different chromatin fractions.

Specific complement-fixing and precipitin antibodies were induced in chicken to chromatin from WI-38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement-fixation reaction. These antibodies are species specific and they react at the complement-fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.

Animals↗
PubMed1977

The immunochemistry of peptidoglycan. Antibodies against a synthetic immunogen cross-reacting with an interpeptide bridge of peptidoglycan.

An albumin-peptide conjugate was synthesized, which carries pentaglycine peptides with C-terminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein-peptide-conjugates, immunoaffinity chromatography of the antisera on Sepharose-(Gly-Gly-Gly-Gly-Gly)n and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C-terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L-serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the corresponding peptidoglycans. For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanograms of peptidoglycan could still be detected.

Amino Acids↗
PubMed1983

Milk antibodies in pulmonary inhalation.

To determine the association between inhalation and increased circulating milk antibodies, the presence of milk precipitins and haemagglutinating titres of antibody to casein and lactalbumin were determined in a series of 100 children studied by radionuclide 'milk scan' and/or barium swallow for possible milk inhalation. Sixty-five were investigated because of reflux and/or inhalation (REFLUX), while 35 were evaluated after near-miss sudden infant death (SID). Inhalation was demonstrated in 23/65 Reflux and 9/35 Sid patients. The incidence of milk precipitins was 22% (5/23) of the REFLUX with demonstrable inhalation, 12% (5/42) of the REFLUX without demonstrable inhalation, 0% (0/23) SID and 2% (21/1005) of an unselected series of hospital patient sera. The incidence of milk precipitins was increased in cases with demonstrated inhalation and lower respiratory tract symptoms. Determination of antibody titres to casein and lactalbumin did not provide additional benefit in the diagnosis of milk inhalation. Detection of milk precipitins can provide additional support to the diagnosis of milk inhalation.

Animals↗