Red blood cells imaging and antigen-antibody interaction measurement

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.     

Kinetics of specific and nonspecific adhesion of red blood cells on glass.

Fixed spherical human red blood cells suspended in 17% sucrose were allowed to adhere on either clean glass surfaces or glass surfaces preincubated with antibodies specific to a certain blood group antigen. The adhesion experiments were performed in an impinging jet apparatus, in which the cells are subjected to stagnation point flow. The objective of this study was to compare the efficiencies of nonspecific and specific (antigen-antibody mediated) adhesion of red blood cells on glass surfaces. The efficiency was defined as the ratio of the experimental adhesion rate to that calculated based on numerical solutions of the mass transfer equation, taking into account hydrodynamic interactions as well as colloidal forces. The efficiency for nonspecific adhesion was nearly unity at flow rates lower than 85 microliter/s (corresponding to a wall shear rate, Gw, of 30 s-1 at a radial distance of 110 microns from the stagnation point). The values of efficiency dropped at higher flow rates, due to an increase in the tangential force. The critical deposition concentration is found to occur at 120-150 mM NaCl, which is consistent with the theoretically predicted values. At low salt concentrations, the experimental values are higher than the theoretical ones. Similar discrepancies have been found in many colloidal systems. Introducing steric repulsion by adsorbing a layer of albumin molecules on the glass completely prevents nonspecific adhesion at flow rates below 60 microliter/s (Gw congruent to 15 s-1). The efficiency of specific adhesion depends both on the concentration of antibody molecules on the surface and the flow rate. Normal red cells adhere more readily through antigen-antibody bonds than fixed cells. Fixed spherical cells have a higher adhesion efficiency than fixed biconcave ones.     

Kinetic Studies of a Doubly Bound Red Cell Antigen-Antibody System

The Polybrene method for detection of red cell antibodies which utilizes continuous flow equipment was modified so that kinetic studies could be performed on red cell antibodies doubly bound between adjacent red cells. In the anti-Rh_o-Rh_o erythrocyte system, deaggregation by temperature was studied over an antibody concentration range of from approximately 1 to 500 antibody molecules per erythrocyte, a residence time range of approximately eightfold, and a temperature range of from 10 to 55°C. The rate of dissociation of antigen-antibody complex, as determined from deaggregation of antibody-dependent red cell aggregates, was found to be of apparent zero order. The apparent activation energy for the antigen-antibody reaction under the experimental conditions was determined and found to be higher than would be expected for singly bound antigen-antibody systems. Possible explanations are considered for these findings in terms of an antigen-antibody bond-breaking model.     

Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. II. Protein-protein bond.

In an extension of the previous paper, we describe the force dependence of break-up of doublets of latex spheres cross-linked by protein G-IgG bonds via the Fc region of the antibody. The receptor, the monoclonal Bear-1 antibody, was either covalently linked to 4.75-microns aldehyde/sulfate (A/S) latex spheres in a one-step reaction, or physically adsorbed to the 4.63-microns carboxyl-modified latex spheres used in Part I of this paper. The spheres were suspended in 19% buffered Dextran 40 containing the ligand, the bivalent recombinant protein G (Gamma-Bind G), and observed in the counter-rotating cone and plate Rheoscope. Break-up of doublets, tracked individually under the microscope, as well as in populations of 50-150 particles, was studied over a range of normal force from 20 to 260 pN. In individual particle studies, the fraction of doublets of spheres with covalently linked IgG breaking up in the first 10 rotations, increased from 16% in the low-force to 63% in the high-force range. In population studies, the fraction broken up increased with duration and magnitude of the applied force, and decreased with increasing ligand concentration. Moreover, doublets of physically adsorbed IgG spheres required significantly lower force than doublets of covalently linked IgG spheres for the same degree of break-up, possibly because of surface detachment of IgG molecules rather than rupture of receptor-ligand bonds. Computer simulation, using the Bell stochastic model of break-up and a Poisson distribution for the number of bonds, described in Part I, showed that the parameters of the protein-protein bond differed significantly from those of the carbohydrate-protein bond studied in Part I of this paper, the former being much more responsive to force than the latter.     

Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up.

We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-microns-diameter tubes, and the hydrodynamic normal force at break-up, Fn, is computed from the measured doublet velocity and radial position. Previous results showed a large range of Fn, the mean of which increased with [antiserum], and an absence of clustering at discrete values of Fn. Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of Fn. The results showed that the data are as scattered as ever, with Fn varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in Fn could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (Fn from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91% survived into the constant force region; 16% of these broke up after time intervals, tP, of 2-30s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, tc, from < 1 to 10 s. Thus, doublets do indeed break up under a constant shear stress, if given time. The average time to break-up decreased significantly with increasing force, while the fraction of doublets broken up increased. At a given Fn, the fraction of break-ups decreased with increasing [IgM], suggesting that the average number of bonds had also increased.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

To date, nanoscale imaging of the morphological changes and adhesion force of CD4⁺ T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4⁺ T cells. The AFM images revealed that the volume of activated CD4⁺ T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4⁺ T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.     

A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

Functional differentiation of B lymphocytes in congenital agammaglobulinemia. II. Immunochemical analysis of the in vitro primary immune response.

Cultures of peripheral blood lymphocytes (PBL) in which specific hemolytic plaque-forming cells (HcPFC) had been induced were labeled with 14C-amino acids. Antigen-specific products in the culture supernatants were characterized by using indirect immune precipitation in conjunction with specific immunoabsorbents and/or gel filtration followed by SDS-polyacrylamide gel electrophoresis. After 5 days of culture with antigen (sheep red blood cells or ovalbumin) newly synthesized IgM and specific IgM antibody were demonstrated in culture supernatants from normal donors and from four out of five patients with congenital agammaglobulinemia (cAgamma). Secreted products bound specifically to antigen and pretreatment of labeled supernatants with anti-mu and anti-L chain antisera, but not with anti-gamma antiserum, prevented binding. Typical mu- and L chains constituted only a proportion of the anigen-binding peptides recognized by the anti-mu reagents. Induction of IgM antibody synthesis was dependent on the presence of antigen and was correlated with the generation of HcPFC. No major differences between the antigen-induced products of cAgamma and normal PBL were observed. These findings suggest that in the absence of terminal B cell differentiation in vivo, certain patients with cAgamma possess precursor cells that can respond to antigen in vitro with the synthesis of specific humoral products, including IgM antibody.     

Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells

Summary The binding and internalization of endogenous growth hormone in Chang hepatoma cells were localized on the cell surface and in the Golgi-endoplasmic reticulum-lysosome (GERL) area by various indirect immunocytochemical labeling techniques, namely, peroxidase or colloidal gold conjugated to secondary antibody, and avidin-biotin complex methods. Rabbit antiserum and monoclonal antibodies raised against HPLC-purified porcine growth hormone were used in this study. In fixed material, antigen-antibody complexes were found to be homogeneously distributed along the cell membrane. Control groups showed negative binding on the cell surface. Trypsin treatment before immunolabeling removed antibody binding completely, but hyaluronidase was ineffective. Pretreatment of lectins did not block the recognition of primary antibody to antigen molecules on cell surface. Internalization of the antigen-antibody peroxidase or gold complexes was demonstrated in the cells, which were immunolabeled at 4°C, and then reincubated for 0–30 min at 37°C before fixation. After reincubation, the internalized ligand complexes were found in vesicles near the cell surface or in the GERL area near the Golgi apparatus which, however, did not label for peroxidase. These findings suggest that the trypsin-sensitive growth hormone, specifically bound and internalized into Chang hepatoma cells, is localized in the GERL instead of the Golgi apparatus and might be involved in the mechanism of tumor cell growth.     

Isolation of suppressor T lymphocytes adherent to histamine conjugated albumin and their role in suppression of IgG response to SRBC

Antigen-specific immunosuppression to sheep red blood cells (SRBC) in BALB/c mice was induced by repetitive injection of high doses of soluble components derived from a hypotonic lysis of SRBC. This form of unresponsiveness induced both IgM and IgG suppression. The IgG, but not the IgM suppression, was stable and persisted in an adoptive transfer. The transferred IgG unresposiveness could be terminated by the removal of a cell population that bound to histamine-rabbit serum albumin-Sepharose (HRS). Such a population could actively suppress normal immunocytes as well as immunocytes in which suppression was eliminated by removal of cells adherent to HRS. The suppressor population shows immunospecificity typical of T cells and its suppressive activity is completely abrogated by anti-θ antibody-induced lysis. Furthermore, we demonstrated that the cell, by virtue of separation on insolubilized histamine columns, has surface characteristics different from the helper T memory cell. Functional B and T immunocytes were found in the SRBC-suppressed mice. We therefore conclude that this type of antigen-induced immunosuppression is maintained solely through an active T cell-mediated suppression.     

Impedimetric immunosensors based on the conjugated polymer PPV

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.     

B 细胞膜CD20 抗原的分布与单分子力谱探测

CD20抗原分子在B细胞上表达下降是慢性B淋巴细胞白血病 (B-CLL) 的标志性特征。采用激光扫描共聚焦显微镜 (LSCM) 和量子点标记相结合的方法对正常和B-CLL外周血CD20+B淋巴细胞膜表面CD20抗原分子的表达及分布进行了荧光成像。同时,采用原子力显微镜 (AFM) 对CD20+B细胞的形貌及超微结构特征进行了表征,并且将AFM针尖用生物素化的单克隆抗体进行修饰,对CD20+B细胞表面的CD20抗原-抗体之间的单分子力谱进行了探测。LSCM荧光图像显示,B-CLL CD20+B淋巴细胞上CD20分子的表达量比正常CD20+B淋巴细胞显著降低。AFM结果显示,B-CLL CD20+B淋巴细胞超微结构比正常的粗糙。力谱结果显示,CD20抗原-抗体的相互作用力大约是非特异性黏附力的5倍,CD20分子在正常CD20+B淋巴细胞膜上分布比较均匀,小部分有聚集现象,反之,在B-CLL CD20+B淋巴细胞膜表面分布稀疏。利用以上两种方法能进一步观察到B-CLL外周血B淋巴细胞的异常,并在一定程度上解释临床上B-CLL病人对利妥昔的低反应现象,为针对抗原CD20的治疗用药选择提供参考。     

Properties of the cell surface Fc-receptor induced by herpes simplex virus.

Detection of peroxidase-antiperoxidase soluble complexes (PAP) bound to the surface of herpes simplex virus-infected cells has been used to demonstrate virus-induced Fc receptors and to study their distribution. The PAP method is more sensitive than hemadsorption with immunoglobulin-coated sheep red blood cells, and can be used to study localization by light and electron microscopy. Our results indicate that capping takes place after the receptor is engaged by antigen-antibody complexes and that at least a portion of the bound ligand is internalized.     

Functional properties of T and B cells isolated by affinity chromatography from rat thoracic duct lymph.

Rat thoracic duct lymphocytes (TDL) were separated into two fractions by passing the cells through a column of rabbit anti-rat F (ab′)₂ antibody coupled to Sephadex G-200. Cells with readily detectable surface immunoglobulin (Ig) were retained on the gel, whereas those without surface Ig were recovered in the effluent. Adherent cells were retrieved by eluting the column with rat Ig. Both dividing and nondividing lymphocytes were separated by this procedure. The adherent and non-adherent fractions contained functionally active lymphocytes as judged by a thymidine incorporation technique and the immunological performance of the cells after transfer to normal recipients. Antibody forming cells and B memory cells were concentrated in the adherent fraction. The non-adherent fraction contained antigen-sensitive T cells which initiate graft versus host reaction and specifically sensitized lymphocytes of the kind which transfer resistance to L. monocytogenes.     

Non-histone chromatin proteins of mouse spleen cells during the primary immune response to sheep red blood cells and aggregated human gamma-globulin.

1. The protein/DNA ratio in chromatin of spleen cells increased during immunization; the ratio was the highest at the time of the maximum antibody synthesis, then decreased to the control values. 2. In the spleen cells of mice immunized with sheep red blood cells or aggregated human gamma-globulin, several characteristic fractions of non-histone chromatin proteins were preferentially synthesized: two antigen-specific fractions were observed in the phase of IgM and IgG synthesis, respectively, and the third, unspecific fraction was detected when the antibody synthesis ceased.