Diagnostic and clinical immunology最新文献

Weekly blood samples from five healthy volunteers were obtained for 13 weeks to assess the variations in the immunological phenotypes of peripheral blood lymphocytes and monocytes. Monoclonal antibodies to eleven lymphocyte and two monocyte antigens were evaluated using two-color flow cytometry. Average week-to-week differences were less than 5% for all monoclonal antibodies and combinations except for I2+, NKH-1+, MO1+, and MO2+ cells. The observed variations presumably reflected both technical variables and responses of the immune system to daily environmental insults.

Several observations indicate that smooth (S) and rough (R) Salmonella strains display the capacity to spontaneously adhere to lymphoid cell membrane. Such a phenomenon is confined to T lymphocytes and affects both CD4+ and CD8+ cells. As far as receptor structures on lymphocytes surface are concerned, the lipopolysaccharides (LPS) of the bacterial cell wall play a key role in human and murine cytoadherence. In addition, evidence has been provided that LPS of gut flora induce bacterial binding as assessed by the evaluation of cyto-adherence at different anatomical sites. Interestingly, cells mediating nonspecific immune responses are not involved in the bacterial binding, since the unbound fraction is highly enriched for cytotoxic and T helper cells. The in vivo occurrence of binding in typhoid fever patients suggests that this activity may represent an earlier event during the course of infection. These findings are also supported by the demonstration that chemotherapeutic treatment abolished bacterial binding in both vitro and in vivo systems. Finally, the production of lymphokines following bacterial stimulation points out the importance of bacterial/immune system interaction in the development of immune response during gram-negative sepsis.

A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody.

An H9-HTLV-III-infected cell line was used as a target in a short-term (3-hr) Cr release assay to assess its sensitivity to lysis by peripheral blood lymphocytes (PBL) from normal donors. The single cell cytotoxicity assay (SCCA) on poly-L-lysine-coated coverslips was used to investigate further the mechanism of binding and killing. Uninfected H9 and K562 cell lines were studied as controls. Our results argue in favour of a natural killer (NK) mechanism being operative on an H9-HTLV-III-infected cell line owing to the following findings: (1) the cell line is sensitive to lysis in a short-term assay; (2) its sensitivity is significantly higher than K562; and (3) the kinetics of lysis, as assessed by SCCA, is similar to that of K562, with a more efficient killing being detectable against H9-HTLV-III. Furthermore, a phenotypic analysis of effector cells suggests that CD4+ lymphocytes are also involved in the lysis of this target. Our data provide evidence for an immune mechanism that may be operative in HTLV-III infection. We then studied, by this method, five groups of patients: one (n = 20) affected by acquired immunodeficiency syndrome (AIDS), one (n = 20) by AIDS-related complex (ARC), one (n = 20) by lymphadenopathy syndrome (LAS), one group (n = 40) of HTLV-III seropositive, apparently healthy people, and one (n = 40) of healthy HTLV-III seronegatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Multimarker studies were conducted on 195 lymph node, 59 bone marrow, 44 peripheral blood, eight body fluid, and eight internal organ specimens. The markers were identified by fluorochrome-labeled antibodies quantified with flow cytometry. T-cell receptor gene rearrangements were used for the determination of T-cell clonality. These studies confirmed that CD 19 (B4, Leu 12) is highly sensitive for B-lymphoblastic leukemia, CD 7 (Leu 9) is highly sensitive for T-lymphoblastic leukemia, and CD 5 (Leu 1) is highly sensitive for chronic lymphocytic leukemia. When these markers were compared to antigens of the same cell lineage (e.g., CD 19 to CD 20 [Leu 16] or to surface immunoglobulin, CD 7 to CD 3 [Leu 4], and CD 5 to CD 3), a marked discrepancy between them was diagnostic of the corresponding tumor. T-cell marker discrepancy (CD3 vs. CD 7) was demonstrated in T-cell lymphomas, but it was also shown occasionally in polyclonal T-cell populations. On the other hand, a marked discrepancy between the percentages of a B-lineage (CD 19 or CD 20)-positive and a surface-immunoglobulin-positive population is a reliable phenotype for the diagnosis of a surface-immunoglobulin-negative B-cell lymphoma.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to tetanus in dried blood samples spotted on filter paper discs has been developed. A good correlation was found between the two methods, filter paper disc assay and direct blood assay (r = 0.91). The specificity of the test was determined by a competitive inhibition assay using goat anti-tetanus-toxoid antibodies coupled to horseradish peroxidase. The interassay coefficient of variation is 10-12%. The antibodies were measurable until 4 months of storage of filter paper discs at room temperature (25 degrees C). This procedure is useful for mass screening of anti-tetanus antibodies using dried blood samples on filter paper discs.

Ribonucleoprotein (RNP) antigen-antibody precipitin bands obtained by two-dimensional double immunodiffusion were systematically analyzed and compared using a microcomputer and a SPSS-PC statistical analysis package. Precipitins rated in respective agarose concentrations ranging from 0.3% to 1.5% were obtained from twofold dilutions of a calf thymus nuclear extract (CTNE) containing RNP antigen. These dilutions were reacted against the same dilutions of plasma containing anti-RNP antibody. A Mean Quality Index Score was calculated at each agarose concentration from the sum of ratings assigned for the intensity (thickness) of precipitin bands times the sum of ratings assigned for the clarity (clearness and sharpness) of the precipitin bands. This product was divided by the total number of possible precipitin reactions. The Mean Quality Index Score for the 0.4% agarose was significantly higher than the other concentrations (p less than or equal to 0.05). This method or similar approaches may allow a more systematic way of comparing precipitins developed in gel immunodiffusion.

Circulating immune complexes isolated from clinical filarial patients' sera by 3% polyethylene glycol precipitation were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on vertical slab gel. Silver staining of the gel after electrophoresis revealed 20 and 30 protein bands without and with reduction of immune complexes, respectively, in the molecular weight range of 148-18K. Filarial antigen in protein eluates from gel slices was detected by sandwich enzyme-linked immunosorbent assay. The eluates from four gel slices, viz., IC-2, IC-4, IC-7, and IC-9, showed filarial antigenic activity. These antigen fractions were characterized and explored for their diagnostic use. IC-7 and IC-9 fractions and microfilariae excretory-secretory (mf ES) antigen share common antigenic determinants as revealed by the fact that saturation of immobilized antibodies with IC-7 or IC-9 inhibited the binding of mf ES antigen coupled to penicillinase. IC-9 fraction appears to be useful in serological differentiation of Wuchereria bancrofti infected persons from those with disease manifestations. Biochemical characterization of the IC-9 fraction revealed the protein nature of the antigen. Comparison of the electrophoretic profiles of immune complexes and W. bancrofti mf ES antigen revealed several common protein bands. The 55 K protein band with antigenic activity was observed in both preparations.

Monoclonal antibodies (MAbs) against HLA antigens can give better information about the serology and biochemistry of the human major histocompatibility complex (MHC) than HLA antisera obtained from pregnant women. To increase the very limited panel of MAbs against HLA we immunized mice with human cultured cell lines and fused their spleen cells with the Ag8-653 myeloma. We produced MAbs against HLA-A1, A2/w69, A2/28, A2/11/25/26/28/29/30/31/33/34, A25/32, B7/22, and B13. The best dilution medium to store the MAbs in Terasaki plates was RPMI 1640 supplemented with 7% bovine serum albumin. The MAbs are also excellent reagents to investigate public HLA antigens and to stain HLA antigens in cryosections of transplanted organs.