Adsorption Of Sera Research Articles

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed Enterococcus faecalis strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant Enterococcus faecium strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous E. faecalis strain and on a vancomycin-resistant E. faecium strain. The capsular polysaccharides from E. faecalis 12030 and E. faecium 838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-alpha-D-glucose-1-2 glycerol-3-PO4 with substitution on carbon 2 of the glucose with an alpha-2-1-D-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15 E. faecalis strains and 7 vancomycin-resistant E. faecium strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.

Evaluation of methods for detecting alloantibodies underlying warm autoantibodies.

In pretransfusion testing of patients whose sera contain autoantibodies reacting optimally at 37 degrees C, it must be determined whether alloantibodies are also present. Two approaches, testing a 1-in-5 dilution of patients' sera and the adsorption of sera in the presence of polyethylene glycol (PEG), have been proposed as alternatives to the time-consuming approach of adsorbing sera with ficin- or ZZAP-treated red cells (RBCs). The three approaches were compared. Patients' sera containing warm autoantibodies, with and without alloantibodies, were retested 1) after dilution (1-in-5) and 2) after adsorption with allogeneic RBCs in the presence of PEG. Results were compared to those after adsorption with ZZAP-treated allogeneic RBCs. Dilution (1-in-5): Twenty-seven of 119 sera (7/26 [27%] with and 20/93 [22%] without alloantibodies) did not react; one example each of alloanti-D, -E, -e, -Fy(a), and -Jk(a), and two examples of anti-Jk(b) were not detected at a dilution of 1 in 5. Alloantibodies were identified in 5 (19%) of 26 1-in-5 diluted sera containing alloantibodies; 87 (73%) of 119 sera still reacted with all cells and would have required further workup. PEG adsorption: Thirty-nine sera were tested after parallel PEG and ZZAP adsorptions. The PEG adsorptions required a total of 55 aliquots of adsorbing cells and 13.75 hours, whereas ZZAP adsorptions required 61 aliquots and 30.5 hours. All alloantibodies (anti-D [3], -C [2], -c [1], -E [4], -K [2], -Fy(a) [1], -Jk(a) [2], -Jk(b) [1]) reacted in the adsorbed serum-PEG mixtures at a strength equal to or greater than that in the ZZAP-adsorbed sera. Although the 1-in-5 dilution approach is convenient, only 22 percent of warm autoantibodies without alloantibodies were nonreactive, and 27 percent of alloantibodies of potential clinical significance were not detected. PEG adsorption appears to give similar results to those of ZZAP adsorption, but it has the advantages of eliminating the cost and time of prior treatment of the allogeneic adsorbing cells and of a reduction of at least a 50 percent in adsorption time.

Serological cross-reactions between Bartonella and Chlamydia species: implications for diagnosis.

Diagnosis of Chlamydia or Bartonella infections continues to rely mainly on serology. However, serological cross-reactions between members of these genera have recently been described. Sera from eight patients originally diagnosed as having Chlamydia pneumoniae endocarditis reacted with both Chlamydia sp. and Bartonella quintana antigens (microimmunofluorescence technique). Adsorption of sera with B. quintana or C. pneumoniae antigens removed anti-C. pneumoniae antibodies, whereas adsorption with C. pneumoniae antigens did not change antibody titers to B. quintana. Western blot analysis confirmed the presence of cross-reacting antigens and showed antibody patterns in all sera to be compatible with a Bartonella infection. These patients were therefore probably suffering from Bartonella-induced rather than Chlamydia-induced endocarditis. In contrast, sera from 10 patients presumed to be suffering from C. pneumoniae pneumonia did not display anti-B. quintana antibodies, although cross-reacting antigens were revealed by Western blotting. This work highlights the possibility that cases of infective Bartonella endocarditis are erroneously diagnosed as chlamydial infections.

Easy-to-perform modified Elek test to identify Shiga-like toxin-producing diarrhoeogenic Escherichia coli

We determined whether Shiga-like toxin I (SLT-I) -producing diarrhoeogenic Escherichia coli could be detected by a modified Elek test. The test (SLT Elek test) is based on the principle of the Elek test and the Ouchterlony double-gel diffusion. The development of the SLT Elek test was preceded by a preliminary study; the purpose of the latter was to establish whether a simplified purification procedure of SLT-I (involving bacterial sonic extract, “Affi-Gel Blue” chromatography and anion- and cation-exchange liquid chromatography) could be employed in the preparation of rabbit antisera to SLT-I. SLT-I-specific antisera were obtained after adsorption of sera with bacterial sonic extract from non-toxigenic E. coli. A total of 135 strains of E. coli were tested by the SLT Elek test (100 SLT-I-negative and 35 SLT-I-positive). The results of the SLT Elek test and the Vero cell test correlated well: 30 strains gave positive results and 100 strains gave negative results in both tests. Only 5 strains gave discrepant results: they were weakly positive in the Vero cell assay, whereas 3 gave borderline reactions and 2 were negative in the SLT Elek test. Positive predictive value was 1, negative predictive value was 0.98; the SLT Elek test was 91 % sensitive and 100 % specific.

Diagnosis of systemic candidiasis by enzyme immunoassay detection of specific antibodies to mycelial phase cell wall and cytoplasmic candidal antigens.

Diagnosis of systemic Candida infections was attempted by the use of an enzyme-linked immunosorbent assay (EIA) to detect IgG antibodies towards cell wall-bound and cytoplasmic candidal antigens. Cell wall antigens were sequentially solubilized by treatment of germinated blastoconidia of Candida albicans (ATCC 26555 strain) with beta-mercaptoethanol (beta ME extract) and digestion with Zymolyase 20T, a beta-glucanase preparation (Zymolyase extract). Protoplasts obtained after treatment with Zymolyase were osmotically lysed (cytoplasmic antigens). Sera were obtained from patients with systemic (n = 28) and superficial (n = 46) candidiasis. Control sera were obtained from normal healthy individuals (n = 31) and from hospitalized patients at low (n = 36) and at high (n = 13) risk of developing systemic candidiasis yet showing no symptoms of candidal infection. Detection of antibodies in crude sera samples by EIA using all of these antigenic extracts was highly specific (98-100%), but sensitivity of the method was low (3.5-17.8%). However, adsorption of sera with latex microspheres coated with purified Candida mannan in order to selectively remove anti-mannan antibodies prior to EIA improved the diagnostic efficiency of this test. Improvement was particularly noticeable when the beta ME extract was used as antigenic preparation, yielding a sensitivity of 89.2% and a specificity of 98.6%.

Intracellular location of the autolytic N-acetylmuramyl-L-alanine amidase in Bacillus subtilis 168 and in an autolysis-deficient mutant by immunoelectron microscopy.

Antisera against purified autolytic N-acetylmuramyl-L-alanine amidase from Bacillus subtilis 168 were prepared in rabbits. They neutralized the enzymatic action of the purified amidase acting on isolated sodium dodecyl sulfate (SDS)-treated walls from the same organism. They also inhibited the lysis of native walls, but only after the walls lysed partially. Amidase adsorbed to insoluble walls still combined with antibody. Antisera did not stop the lysis of whole cells. Lowicryl HM20 sections of both strain 168 and its autolytic mutant strain FJ6 were prepared by the progressive-lowering-of-temperature technique, immunolabeled with the antisera, and visualized with colloidal gold particles as markers. The highest concentration of gold particles seemed to be in the septa of dividing cells, followed by the side walls. There was some labeling of the cytoplasm. Adsorption of sera with SDS-treated walls reduced the overall labeling of sections considerably but did not alter the relative intracellular distribution of particles. The results for strains 168 and FJ6 were similar. Labeling of SDS-treated walls unexpectedly revealed the presence of a wall-bound amidase fraction.

IgG Oligoclonal Bands in Sera of HIV-1 Infected Patients Are Mainly Directed Against HIV-1 Determinants

In a series of 60 HIV-1-infected individuals, serum electrofocusing analysis disclosed clonally restricted IgG patterns in 9 patients (15%), most with limited disease progression (stages WR1-WR3). These oligoclonal bands had a very heterogeneous light chain pattern, and most showed specificity for HIV-1 in affinity-driven transfer studies; virus specificity was more clear-cut following adsorption of sera with the relevant antigen. These findings further stress the profound B-cell function derangement in HIV-1 infection; their possible relevance to AIDS-associated lymphoma development is discussed.

Immune inhibition of virus release from herpes simplex virus-infected cells by human sera.

Human sera contain antibody (IVR antibody) which will inhibit the release of herpes simplex virus type 1 from virus-infected cells. This antibody activity was removed by adsorption of sera with virus-infected cell extract. There was a positive correlation between IVR and neutralizing antibody activity, particularly when measured by augmented neutralization test; measurement of IVR antibody was equally as sensitive as measurement of neutralizing antibody by augmented neutralization test. IVR antibody levels provided indication of a history of recurrent herpes labialis, the pattern of antibody response following primary herpetic infection, and indication of response to Skinner herpes vaccine in human subjects. It is suggested that consideration should be given to measurement of IVR antibody in both clinical and epidemiological studies of herpes and other virus infections.

Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection.

Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from H. pleuropneumoniae serotypes 1 and 7; however, several OMPs and the lipopolysaccharide or polysaccharide determinants of these serotypes appeared to be type specific.

Anti-Fab antibodies in humans. Predominance of minor immunoglobulin G subclasses in rheumatoid arthritis.

Isoelectric focusing analyses of sera from patients with rheumatoid arthritis (RA) demonstrate two populations of antibodies directed against the Fab portion of pooled human IgG. One population is composed of polyclonal alkaline anti-Fab antibodies (alpha FABA) and the other, acidic alpha FABA which are more clonally restricted. In this study we have identified the immunoglobulin classes and subclasses of these antibodies in RA sera. Enzyme-linked immunosorbent assays (ELISA) demonstrated alpha FABA in RA sera to be predominantly IgG. A large portion of IgG alpha FABA existed as immune complexes, inasmuch as dialysis of RA sera against 6 M urea before ELISA analysis was necessary for maximal detection of alpha FABA activity. Chromatofocusing of RA sera isolated alpha FABA of different charges and revealed the acidic clonally restricted alpha FABA to be IgG4 and IgG3, whereas the polyclonal alkaline group contained IgG1, IgG2, and IgG3. Overall, acidic IgG3 and IgG4 comprised 70% of IgG alpha FABA, and high levels of IgG4 were seen in most RA sera. When alpha FABA were elevated in normal sera, they were primarily of the IgG4 subclass, and also existed as immune complexes. Serum anti-Fab activity was removed by adsorption of sera with Fab fragments. Anti-Fab antibodies of both kappa and lambda light-chain types were present in RA sera, and F(ab')2 fragments of RA serum immunoglobulin were found to possess anti-Fab activity. These studies indicate that alpha FABA in RA sera are limited to the IgG class, and that most of these antibodies exist as immune complexes and display clonal and minor IgG subclass restriction.

Study of agglutinins to Brucella abortus, B canis and Actinobacillus equuli in horses.

Horses at a veterinary teaching hospital and a slaughterhouse were surveyed for antibodies to Brucella abortus, B canis and Actinobacillus equuli. Four of the 141 hospitalised horses and none of the 73 slaughtered horses had titres of 1:100 or greater to B abortus. Six horses of both populations reacted to the card test. One was culture positive. A card test using B canis antigen was positive in 38 per cent of the sera from hospitalised horses and all of the slaughtered horses. Twenty (27.4 per cent) of the latter group had high tires in a tube agglutination test. High titres could not be reduced by 2-mercaptoethanol serum treatment. The titres appeared to be associated with advanced age but not to sex. Adsorption of sera with B canis did not affect titres to A equuli but the reverse was true.

Immune response to stage-specific surface antigens of the parasitic nematode Trichinella spiralis.

Rats were infected with the nematode Trichinella spiralis and the primary serum antibody response to antigenic surface proteins of infective larvae, intestinal worms, and newborn larvae was studies. 1 wk after infection, the sera contained antibodies to surface antigens of both infective larvae and intestinal worms. These early sera, however, failed to react with newborn larvae surface antigens. In addition, adsorption of sera with living intestinal worms or infective larvae removed antibodies to surface antigens of the homologous stage only. Finally, the time-course of appearance of antibodies that mediate eosinophil adherence to the surface of each stage of the parasite. We concluded that in a primary infection in rats, the surface proteins of T. spiralis used in this study are antigenically stage specific. Furthermore, they could be targets for the stage-specific, antibody-dependent eosinophil-mediated destruction of this parasite, known to occur in vitro.

Circulating immune complexes in sera from patients receiving procainamide

Sera from patients receiving procainamide (PrA) therapy were investigated for their ability to inhibit an antibody-dependent cell-mediated cytotoxicity system (ADCC). Before the start of therapy 1 out of 20 sera from patients were inhibitory. Two months following the onset of therapy, 21 out of 29 sera (72%) inhibited ADCC. Sera from 13 out of 29 (44%) patients receiving PrA for periods greater than 6 months still displayed inhibitory factors. The inhibitory factors were found by sucrose-gradient ultracentrifugation to be located mainly in fractions 7 S to 19 S. Adsorption of sera with anti-human IgG linked to Sepharose removed inhibitory factors whereas similar treatment with anti-human IgM Sepharose did not, indicating that IgG was present in the inhibitory material. These results are consistent with the hypothesis that PrA induces IgG-containing immune complexes.

Cytomegalovirus Specific IgM and IgG Response in Humans Studied by Radioimmunoassay

An indirect solid phase micro-radioimmunoassay (RIA) was adapted for the measurement of anti-cytomegalovirus class-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). Cytomegalovirus (CMV) antigen (Ag) was added to the wells of microtiter plates and desiccated onto the bottom surface of the wells. Serial dilution of human CMV antisera were added and allowed to react with the Ag. The amount of viral antibody (Ab) was determined by measuring the specific binding of 125I-labeled goat anti-human IgG (125I-AHIgG) and/or IgM (125I-AHIgM). The multiple factors which influence the test were determined. Serial samples of sera from CMV-positive patients showed progressive increases in Ab titers on the basis of specific binding of 125I-AHIgG. The titers of IgM class anti-CMV Ab were also determined with the same sera, and enhancement of the titers was demonstrated when the incubation periods of the first Ag-Ab reaction were extended from 1 to 3 hr. The competition between the different Ig classes of Ab for CMV Ag and the involvement of rheumatoid factor (RF) were investigated. The anti-CMV IgG that may compete with the IgM determination was removed by adsorption of sera with Staphylococcus aureus (Cowan 1). The RF that would cause false anti-CMV IgM results was adsorbed with glutaraldehyde-insolubilized IgG. Our results indicate that this RIA is a practical, specific, and reproducible technique for detection of specific IgG and IgM Ab to CMV.

Studies on Organ Specificity

Summary The interactions between human sera and human and monkey thyroid antigens were studied by the fluorescent antibody method, complement fixation, and tanned cell hemagglutination. Three types of antibodies which stain human thyroids were demonstrated in human sera by the fluorescent antibody method. The antigens are characterized by staining of: a) thyroid colloid, b) cytoplasm of thyroid epithelium, and c) thyroid nuclei. Antibodies to thyroid colloid and to the cytoplasm of thyroid epithelium frequently occur together. Some sera appear to contain only antibodies to the cytoplasm of the thyroid epithelium. Antibodies to thyroid colloid and to the cytoplasm of thyroid epithelium were further differentiated by: a) differences in the chemical stability of the two antigens, b) selective removal of antibodies which stain thyroid colloid by adsorption with thyroglobulin. Sera which stained the cytoplasm of thyroid epithelial cells by the fluorescent antibody method also fixed complement with human thyroid microsome, to a lesser extent with human thyroid mitochondrial fraction and not at all with the nuclear fraction. Adsorption of sera, even with well washed microsomes, removed both the cell and the colloid staining antibodies. Immunofluorescent staining of the cytoplasm of thyroid epithelium occurred with the patient's own thyroid sections in three cases thus confirming the autoantibody nature of the reaction. All sera which stained thyroid colloid by the fluorescent antibody method also gave strong hemagglutination reactions. The converse was not always true. Hemagglutination occurred with the patient's own thyroid extracts in two cases thus confirming the autoantibody nature of this type of reaction. One of 17 sera studied stained all three thyroid consituents, the colloid, the cytoplasm of the epithelial cells, and the thyroid nuclei. Nuclear staining and colloid staining antibodies were removed by selective adsorption. Sera containing nuclear antibodies (mostly from patients with lupus erythematosus) stained no other component of thryoid sections but stained nuclei of all organs. Pathologic diagnosis and serologic findings were compared in 46 thyroidectomized patients. Thyroglobulin autoantibodies occurred in all chronic nonspecific thyroiditis cases. No other generalization could be made.

Hemagglutination and Hemagglutination-Inhibition Studies with Echo Viruses

Summary A study of several factors which may affect hemagglutinin (HA) production revealed that a dilute inoculum combined with a relatively late harvest time favored maximum HA titers of ECHO 6 viruses grown in monkey kidney tissue culture. Infectivity to HA ratios of 6 to 7 logs were obtained with viruses which attain high infectivity titers (B phase), while the ratios obtained with the low titer viruses (S phase) were approximately 4 to 5 logs. A survey of ECHO 6 strains revealed that 9 of 24 tested produced HA. HA production could not be correlated with infectivity titers, phase, or number of passages in tissue culture. Cross hemagglutination-inhibition (HI) tests with hyperimmune rabbit antisera revealed a high degree of specificity among the serotypes tested. Differences were noted among several ECHO 6 HA antigens in their ability to detect HI antibody. Nonspecific inhibition of ECHO virus hemagglutination was demonstrated. Adsorption of sera with 0.1% bentonite resulted in almost complete removal of nonspecific inhibitors, whereas this procedure had no apparent effect on specific HI antibody.