Indirect Staining Method Research Articles

627. Differences in C. difficile Toxin A Binding in Humans: Adults vs. Infants

BackgroundChildren less than 1 year of age are commonly colonized with toxin-producing C. difficile, but appear to be immune to the associated colitis. Animal studies suggest that young infants lack receptors for C. difficile toxin, though this has never been documented in humans.MethodsTissue from infants (<6 months) and adults > 21 years were studied. Toxin A binding was assessed using an indirect staining method, which included incubation with toxin A (List Labs) and detection with a rabbit polyclonal anti-sera (Lee Labs). A trained pediatric pathologist assessed the extent of staining in a blinded fashion. In other studies, toxin A was labeled with rhenium-188 and incubated with albumin-blocked tissue sections (four-infant and six-adult) for 1 hour. After washing, gamma counts were measured and the average percentage of retained radiolabeled toxin A calculated. Fisher exact tests and ANOVA were used for analyses. All studies were done in compliance with our institutional IRB.ResultsSix of 13 (46%) adult specimens were found to have reactivity on both the apical epithelial surface as well as crypt staining. Another six had reactivity localized only to the basal and lateral surface of the crypts. One specimen demonstrated no reactivity at all. For neonates (n = 15), no specimens were found to have reactivity localized to the apical epithelial surface, though four specimens had reactivity at the basal epithelial surface (P value for comparison of apical staining 0.0046) (see figure). Average percentage of retained counts for control (no tissue), infant and adult colon sections were, 0.318 ± 0.147, 0.305 ± 0.079 and 0.48 ± 0.114, respectively (P = 0.051). ConclusionImmunohistochemistry and radiolabelling studies indicate that neonatal colon section binds C. difficile toxin A less strongly and in a different distribution pattern (i.e., without apical staining) when compared with adult colon sections. These findings are consistent with previous animal studies and support the paradigm that a lack of toxin receptors in the infant colon contributes to immunity against C. difficile colitis. Additional studies are needed to define the presence of specific receptors and determine if a similar phenomenon applies to toxin B binding.Disclosures All authors: No reported disclosures.

Validating The Prognostic Significance Of FCRL2 In Predicting IGHV Mutation Status, Clinical Disease Progression, and Survival In CLL

B cell chronic lymphocytic leukemia (CLL) can be stratified into indolent and aggressive subtypes according to the degree of somatic hypermutation evident in the immunoglobulin heavy chain variable region gene (IGHV). Despite some discordance with IGHV mutation status, both CD38 and ZAP-70 are routinely analyzed in clinical practice and are considered useful prognostic factors for segregating patients with aggressive-unmutated CLL (U-CLL). Members of the Fc receptor-like (FCRL1-5) family have immunoregulatory function and are preferentially expressed by B cells. Their distinct distribution pattern among subsets in healthy donors suggests that the FCRLs may also have useful diagnostic and/or therapeutic potential in B lineage malignancies. Previous work employing a receptor-specific monoclonal antibody (mAb-7F2) and an indirect staining method demonstrated that FCRL2 is particularly upregulated on the surface of indolent-mutated CLL (M-CLL) cells. Among 107 CLL samples, 52 U-CLL and 55 M-CLL, FCRL2 overlapped with IGHV status with 94.4% concordance, relative to 76.6% for CD38 (cutoff of 30%), and 80.4% for ZAP-70 according to the T/B ratio (cutoff of 4.0) or 74.8% by the % method (cutoff of 20%). Because several years have passed since our initial analysis using this reagent, we updated the clinical information to reassess the relationship among these prognostic parameters with FCRL2 staining and to further determine the usefulness of this biomarker in CLL. Similar to IGHV status [Hazard ratio (HR)=5.03], FCRL2 could also, but more powerfully, predict survival (HR=8.58). Both these features were superior to CD38 (HR=1.29) and ZAP-70 by either the T/B ratio (HR=1.42) or % (HR=1.04) approaches. The median survival for FCRL2 positive cases was 26.2 years and for FCRL2 negative donors was 12.7 years. With regard to disease progression, FCRL2 positive cases had a median treatment free interval of 13.5 years, whereas for FCRL2 negative donors it was only 3.2 years. Given these promising results, we pursued optimizing FCRL2 detection and assayability by flow cytometry to validate its clinical utility for diagnostic and prognostic purposes in CLL. A screen of FCRL2-specific mAbs led to the identification of a novel subclone termed 3E11, which by epitope mapping reacted with a site discrete from that of 7F2. The binding kinetics of these two mAbs also differed. According to surface plasmon resonance studies using an FCRL2-Fc recombinant protein, 3E11 possessed ∼2.5X better nanomolar affinity and a slower dissociation rate than 7F2. The superiority of 3E11 was also evidenced by its relatively stronger surface staining reactivity in flow cytometry analyses. To achieve a one-step staining strategy, 3E11 was directly conjugated with PE. Use of this PE-labeled reagent demonstrated brighter and more selective FCRL2 reactivity on M-CLL versus U-CLL (N=30) cells using either the post-Ficoll or whole blood-lysis preparation methods. Importantly, its reactivity was also stable over time (Concordance Correlation Coefficient of 0.9). As a promising CLL biomarker capable of selectively detecting the MT-CLL subtype and predicting disease progression and survival, the current findings demonstrate the practicability and reliability of a newfound mAb for validating the prognostic value of FCRL2 in CLL. Disclosures:LI:University of Alabama at Birmingham: Filed Patent, Filed Patent Patents & Royalties. Davis:NIH an University of Alabama at Birmingham: Patent Filed, Patent Filed Patents & Royalties, Research Funding.

Microwave antigen retrieval in formaldehyde-fixed human brain tissue.

AbstractThe use of formaldehyde as a reagent for fixing tissue was first identified over a century ago (1), and because of its efficacy in a broad range of situations, at varying concentrations and with virtually all tissues, it has remained one of the most widely used fixatives. Formaldehyde is a reactive electrophilic species (2,3) that reacts readily with primary amines, amide groups, aromatic rings, and alcoholic hydroxyl groups, crosslinking various biological macromolecules. For histological purposes, this crosslinking is highly desirable in that it allows considerable morphological detail to be maintained, but such fixation invariably results in a corresponding time-dependent loss of antigenicity (4). Consequently, early immunocytochemical protocols used fresh/frozen or alcohol-fixed tissue only (5), retaining tissue antigenicity, but at the expense of morphology. Despite the effects of formalin fixation, however, the loss of antigenicity is neither complete nor irreversible. Indeed, formaldehydefixed tissue has been successfully used for the detection of many different tissue antigens, such as neurotransmitters (6), neuropeptides (7), cell surface markers (8), and enzymes (9). The suitability of formalin-fixed tissue for immunocytochemistry has been further enhanced with the development of sensitive indirect staining methods, such as PAP (peroxidase antiperoxidase) (10) and, more recently, avidin-biotin complex (ABC) methods (11).KeywordsCell Surface MarkerPretreatment MethodMicrowave PretreatmentHeat PretreatmentMicrowave Antigen RetrievalThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Blood Island is a Site of Formation of the Primary Embryo Thrombocyte in the Chick Blastoderm: (anti-thrombocyte antibody/embryo thromboblast/site of production/blood island/chick embryo).

Using the immunohistological technique we inquired at what developmental stage and in which site of chick blastoderm does the embryo thrombocyte (ET) begin to differentiate. An anti-ET antibody was raised against rabbits by injecting ETs isolated from blood of 10 day chick embryos. By applying the indirect staining method to smear preparations of blood collected from developing embryos it was confirmed that cytoplasm of the ET showed more intense staining than that of the erythroid cell and that the ET population could be distinguished from the erythrocyte population by this antibody. Cells showing the intense staining could be detected first in blood islands of the area opaca vasculosa of stage 9+ blastoderms. These embryo thromboblasts were found singly or in groups of a small number at dorsal periphery of cell clusters in the blood island. The electron microscopy revealed that embryo thromboblasts appeared in the same position in the stage 9+ blastoderm. At stage 10+ or later embryo thromboblasts were also present adhering to the vascular endothelium or free in the vessel lumen. We conclude that ETs start differentiating from primitive mesenchymal cells localized in the blood island of the area opaca vasculosa at stage 9 or earlier, migrate thereafter to vessel lumen, and enter the blood stream.

An investigation of optimal gold particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy.

We investigated the optimal gold particle size for use with polarized incident light (epi polarization) microscopy with immunogold immunohistological preparation in both immunogold indirect (IGS) and silver-enhanced immunogold-silver staining (IGSS) techniques. A range of gold particle sizes from 5 nm-40 nm was used along with tissue of known immunoreactivity with a well-characterized primary monoclonal antibody. Checkerboard titrations were carried out for each technique and for each particle size. The preparations were viewed using a standard polarized incident light microscope and assessed in a semi-quantitative manner. Adequate visualization of gold particles was achieved using the indirect staining method only with a particle size of 40 nm. With silver enhancement (IGSS), particles of all sizes were clearly seen. However, 5-nm particles were considered optimal for this method because of reduced background staining, high titration of antisera possible, and crisp localization of the visual signal.

Direct and indirect immunofluorescence analysis of bacterial populations by flow cytometry

Bacillus anthracis spores and Escherichia coli were stained with fluorescein-conjugated antibody using direct and indirect methods, then analyzed by means of a commercial flow cytometer. To reduce the cytometer's fluorescence component resulting from unreacted conjugate, reaction mixtures were either dilluted or were centrifuged through a sucrose solution using a moving zone technique. Evidence is produced that the fluorescence statistics for centrifuged samples closely represent the fluorescence distribution of stained single bacteria in the reaction mixture at the end of incubation; in particular, centrifugation did not cause aggregation of bacteria. Centrifugation is proposed as more effective than mere dilution for use with a wide range of bacterial concentrations, and the moving zone technique is to be preferred to conventional centrifugation in which bacteria tend to aggregate in the pellet. In indirect assays, it was shown that the washing step after reaction with antibacterial antibody may be omitted. The performance of direct and indirect staining methods was compared, including the use of either Staphylococcus aureus protein A or polyclonal sheep anti-rabbit antibody as the indirect reagent. When the bacterial concentration in reaction mixtures was increased the median fluorescence intensity fell, indicating that specific antibody had become limiting at low concentrations of the polyclonal antibody preparations. The implications of this for the design of flow cytometry assays of bacteria are discussed.

39]Immunoperoxidase staining of S-100 and calmodulin in tissue sections and cultured cells

Publisher Summary This chapter describes the methods for direct and indirect immunoperoxidase staining of S-100 proteins and calmodulin in tissue sections and cultured cells. Preparation of peroxidase-labeled antibodies is now widely used for light and electron microscopic immunocytochemistry, and is also applicable to stain a specific antigen on nitrocellulose sheets transferred from polyacrylamide gels. The immunoperoxidase method exhibits the advantage of the stability of the enzyme reaction product, the easiness in the counter staining by the conventional staining methods, and usefulness in the immunoelectron microscopic study. The immunofluorescent method often provides a higher resolution at the light microscopic level. For visualization of antibody molecules bound to antigen by the immunoperoxidase method, direct staining (using peroxidase directly conjugated to specific antibody) or indirect staining (in which tissue or cells are reacted with the specific antibody followed by reaction with peroxidaselabeled second 3,-globulin) is employed. There are some modifications of the indirect staining method, such as the peroxidase-anti-peroxidase (PAP) method, in which PAP complex (rabbit anti-peroxidase and free peroxidase) is linked with the primary rabbit antigen-antibody complex by the addition of goat-anti-rabbit IgG antibody.

Inconsistencies detected by flow cytometry following immunofluorescence staining with anti-Thy 1 antibodies

A number of inconsistencies have been observed when anti-Thy 1 reagents are used for immunofluorescence. (1) When indirect staining procedures are used, the differences in staining between high and low Thy 1 populations may be obscured, (2) with both direct and indirect staining methods, variable peaks of ‘low Thy 1’ cells can be seen, apparently derived from the high Thy 1 population by loss of antigen and/or antibody during the staining procedure. This disparity is quite variable, appears to occur at random and is independent of a large number of technical variables tested. Similar inconsistencies are not apparent with other lymphocyte markers (e.g., Lyt 1, Lyt 2, TLa, peanut agglutinin, H2, Ig).

Use of Immunofluorescence and Viability Stains in Quality Control

Immunofluorescence has been evaluated as a rapid quality control technique for the detection of low levels of wild yeasts in culture yeast or other brewery samples. Antisera have been prepared against antigenic groups A to F, pooled and absorbed with culture yeast. Using the indirect staining method and fluorescein isothiocyanate or rhodamine as fluorochrome, levels as low as 10 wild yeasts per million cells can be detected in three hours. Combined immunofluorescence and viability staining allows the differentiation of live and dead, culture and wild yeast cells, on the same slide by alternating the light sources. The preferred method uses fluorescein isothiocyanate, excited by incident blue light, for wild yeast detection, combined with methylene blue, viewed by transmitted light, for viability differentiation. Fluorescein diacetate is a useful viability stain for yeasts although agreement between results comparing it with methylene blue and slide culture viabilities is not exact for heat-stressed cells. Some nonbrewing yeasts require heating for two minutes at 50° C before consistent results are obtained with fluorescein diacetate. An examination of the mechanism of the action of methylene blue as a viability stain has suggested that the action is one of permeability, rather than permeability followed by enzymatic reduction.

Wall structure of the Neurospora hyphal apex: immunofluorescent localization of wall surface antigens.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965), i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behing the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III, and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener. Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be alpha-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.

Histochemical lectin affinity technique by means of FITC-labeled serum protein fractions.

A two-step affinity technique is described for light microscopic demonstration of the Concanavalin A, Agaricus bisporus lectin and Ricinus communis lectin binding sites by means of various FITC-labeled human and rabbit serum protein fractions. Experiments for the visualization of the Lens culinaris lectin and the Pisum sativum lectin binding sites gaves negative results. The technique consist of two reaction steps which involve the incubation of tissue sections in the lectins followed by the visualization of receptor-bound lectins with FITC-labeled serum protein fractions basing on their carbohydrate content. The specificity of the technique could be demonstrated by the addition of the hapten or by incubation in the FITC-labeled serum protein fractions only. In contrast to the direct or indirect staining methods only very small amounts of purified lectins are necessary.

Purification of mouse alpha1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization.

Mouse alpha1-fetoprotein (AFP) was isolated from amniotic fluid by immunoadsorbent columns and preparative electrophoresis. Specific antibodies were isolated from monospecific hyperimmunsera by use of immunoadsorbents, and subsequently coupled with horseradish peroxidase. At the light microscopical level, purified antibody-peroxidase conjugates were used for the cellular localization of AFP in fetal liver by direct and indirect staining methods. Fixatives containing ethanol or aldehydes were tried for antigen staining. Prior to immunocytological reactions, endogenous peroxidases were inhibited by hydrogen peroxide.

Indirect fluorescent-antibody method for the identification of Corynebacterium vaginale.

The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale.

Indirect Fluorescent-Antibody Method for the Identification of Corynebacterium vaginale

The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale.

Immunofluorescence Studies of Chick Lens FISC and α-Crystallin Antigens during Lens Morphogenesis and Development

Indirect staining method of Weller and Coons [23] was applied to detect the presence of FISC and α-crystallin lens antigens in the embryonic and post-embryonic chick lenses. Specific antiserum against

IMMUNOLOGIC STUDIES OF TWO CASES OF FATAL MYASTHENIA GRAVIS.

The presence of antibodies to skeletal muscle in the sera of some patients with myasthenia gravis was first demonstrated by direct and complement immunofluorescent staining by Strauss et al.1Subsequently, we found that these antibodies may be demonstrated by the indirect staining method as well, that they combine also with heart muscle (thus they are referred to as SH antibodies), and that they behave as autoantibodies,2ie, the patient's serum reacted with his own muscle tissue in the in vitro test system. A second antibody which is less commonly found in sera of patients with myasthenia gravis reacts only with skeletal muscle. This so-called S antibody was also demonstrated to be an autoantibody by a test-tube complement-fixation-test method. More recently a highly sensitive tanned-cell agglutination test for antibodies to skeletal and heart muscle has been elaborated in this laboratory.3Again the SH antibodies were found to combine