Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell-associated antigen.

A mouse hybridoma cell line, FluIgM-1, which secretes IgM specific for the hapten fluorescein (FLU) was developed to allow detailed analysis of the effector-cell blockade (ECB) phenomenon, in which contact of antibody-forming cells (AFC) with specific antigen results in marked reduction of antibody secretion. Treatment of hybridoma cells with highly substituted FLU conjugates (e.g., Flu20gelatin) resulted in inhibition of plaque formation. The data indicated close parallels with the ECB of normal spleen AFC, both in speed of onset and the dose of antigen required. The inhibition of antibody secretion was confirmed with a biosynthetic-labeling procedure which demonstrated that this was a result of reduced Ig synthesis. The inhibitory effect appeared to be confined to antibody synthesis, in the total protein synthesis, DNA synthesis, and cell-doubling times were unaffected. The association of FLU conjugates with the cells during and following ECB was studied directly using fluorescence microscopy and the fluorescence-activated cell sorter. These experiments showed that FLU conjugates capable of causing blockade aggregated on the cell surface, that the clearance of cell-associated antigen correlated with recovery from ECB, and that at all times when cell associated antigen was detectable, a portion remained bound to the cell surface and was susceptible to enzymatic removal. The latter observations supported previous findings suggesting that ECB was mediated by extracellular antigen. The direct observation of aggregates of antigen on the surface of blockaded cells is consistent with a mechanism involving cross-linking of Ig receptors. Finally, Fc receptors were not present on hybridoma cells, excluding their involvement in induction of ECB.

Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies.

We have examined the cells involved in the development of contact sensitivity to FITC in CBA mice. After skin painting with antigen, the number of dendritic cells (DC) in the draining lymph nodes increased by 30 min, was maximal at 48 h, and returned to normal by 6 d. Derivation of some DC from Langerhans' cells of the skin was indicated from the presence of Birbeck granules observed in some DC isolated 24 h after skin painting. The DC acquired FITC and by 8 h there were two populations, one highly fluorescent and the other less fluorescent. The highly fluorescent cells were present between 8 h and 3 d after sensitization, and during this period the DC were potent at initiating primary proliferative responses of normal syngeneic T lymphocytes in vitro. Between days 3 and 5 the numbers of lymphocytes in the draining lymph node increased. During this period purified T lymphocytes did not express detectable levels of antigen, but enriched B cell populations expressed antigen transiently on day 1, 2, or 3 after exposure to antigen. The results showed that, during a 3-d period after exposure to antigen, DC expressed antigen and stimulated T cell proliferation. We speculate that low amounts of FITC binding selectively to veiled cells or lymph node DC in the first hours after exposure to antigen are not immunogenic but that Langerhans' cells acquire high levels of antigen, enter the nodes, and initiate immune responses.

The modulation of lymphocyte functions by molecules secreted by macrophages. I. Description and partial biochemical analysis.

Culture fluids of peritoneal exudate cells rich in macrophages stimulated DNA synthesis of thymocytes and, to lesser extent, of spleen cells. We also investigated the effects of culture fluids from macrophages on the in vitro response to a hapten-carrier protein (fluorescein-menocyanin) using spleen cells from immune mice. Macrophage culture fluids contained an activity that increased the plaque-forming cell response of both IgG and IgM class. This increase was observed in the absence of any added hapten protein to the culture. The helper function of T lymphocytes (as evidenced by challenging with the hapten on the homologous carrier) was also increased by the macrophage culture fluid. However, this enhancement was best observed in conditions of relatively low T-cell activity. Also, the macrophage fluid allowed spleen cells of nude athymic mice to make a plaque-forming cell response to sheep red blood cells of both the IgM and IgG class. The macrophage was the cell source of the stimulatory molecule since it was generated only in cultures of macrophages devoid of significant number of lymphocytes. Stimulatory activity was not found in cultures of lymphocytes, mouse embryo cells, or 3T3 cells. The thymocyte stimulatory molecule did not contain H-2 antigens, was resistant to diisopropylfluorophosphate treatment, eluted from Sephadex with a size ranging from 15,000 to 21,000 daltons, and was sensitive to chymotrypsin and pepsin.

Induction of immunological tolerance requires that the B cells can respond to the polyclonal B-cell-activating properties of the thymus-independent antigens.

Mice were rendered specifically tolerant to the fluorescein isothiocyanatedextran (FITC) epitope by injection of FITC-dextran B512. Their spleen cells were removed at various times and cultivated in vitro with different polyclonal B-cell activators, such as lipopolysaccharide (LPS), purified protein derivative of tuberculin, and native dextran. LPS caused the appearance of high affinity anti-FITC plaque-forming cells to an equal extent with cells from untreated and tolerant animals, whereas native dextran failed to activate cells from tolerant mice, although it was a potent activator of normal cells. It was concluded that tolerance induction only affects those B cells that could respond to the polyclonal B-cell-activating properties of the tolerogen, but not other B cells having an identical set of Ig receptors directed against the tolerogen.

Intracellular fusion of sequentially formed endocytic compartments.

A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.

Location and dynamics of a membrane-bound fluorescent hapten. A spectroscopic study.

In the preceding paper (Petrossian, A. and Owicki, J.C. (1984), Biochim. Biophys. Acta 776, 217-227), we describe the binding of a monoclonal anti-fluorescein antibody to a membrane bound fluorescein-lipid hapten. Those results suggest that some of the hapten fluorescein moiety is extended away from the membrane surface and is available for antibody binding, while some of the hapten is sequestered and not immediately available for antibody binding. In this paper, we carry out a spectroscopic study of the membrane-bound hapten and show that there is more than one physically distinct fluorophore environment, with the sequestered hapten associated with the phospholipid headgroup region. The amount of membrane-associated fluorophore depends upon the membrane lipid composition: most of the fluorophore is associated when the lipid is unsaturated or branched-chain phosphatidylcholines (PC), whereas the hapten is largely extended for PC/cholesterol mixtures. The effect of cholesterol on the availability of membrane-bound hapten to antibody binding is not unique to this system. The conversion between sequestered and extended hapten is slow (minutes).

Interaction of antibodies with liposomes bearing fluorescent haptens.

Kinetic and equilibrium aspects of the recognition of antigenic model membranes by antibodies have been studied. Monoclonal anti-fluorescein IgG and its monovalent Fab fragment were allowed to interact with a fluorescein-lipid hapten that was incorporated into phospholipid vesicles. The binding was assayed in the nanomolar hapten concentration range by monitoring the quenching of hapten fluorescence by antibody. The rate and strength of the binding depended on the lipid composition of the vesicles; cholesterol enhanced both. The biphasic binding kinetics observed at high antibody concentrations for some compositions, plus additional spectroscopic evidence, led us to hypothesize that the hapten existed in a composition-dependent equilibrium between at least two conformations: (1) extended away from the membrane surface, available for binding, and (2) sequestered at or in the surface, unavailable for binding. The rate and strength of IgG binding were always greater than those of Fab, indicating bivalent binding by the IgG. This binding was intra-vesicular, since no agglutination of the vesicles was detected.

Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA.

We found in preliminary studies using 125I-labelled antibodies that an antibody bound to a solid-phase antigen was recognized more efficiently than an antibody adsorbed directly to the solid phase. The present study was designed therefore to quantitate the differential recognition of an antibody adsorbed directly to the solid phase and an antibody bound to antigen on the solid phase using the amplified enzyme-linked immunosorbent assay (a-ELISA), and to compare results with the amounts of specific antibody determined by quantitative immunoprecipitation. The degree of differential recognition was quantitated for rabbit IgG and SIgA anti-ovalbumin (anti-OA) and anti-fluorescein, and was found to be dependent upon the isotype of the antibody and not its specificity. The ratio describing the differential recognition of SIgA antibodies (1.8) was much less than for IgG antibodies (greater than 30) and remained constant over the titration range analyzed while the ratios obtained for IgG varied substantially (25-60) over the same range. These ratios of differential recognition were used to estimate rabbit IgG antibody levels to OA, bovine serum albumin, ferritin and alpha-lactalbumin. The estimates obtained were consistently much less than total antibody levels measured by quantitative precipitation. The use of glutaraldehyde-aggregated OA in the ELISA, however, increased the amount of IgG anti-OA and SIgA anti-OA capable of recognizing OA adsorbed on plastic from 12 to 50 and from 30 to 80%, respectively.

Linkage of low and high affinity anti-fluorescein idiotype families.

Data presented in this study describes the isolation and characterization of two anti-fluorescein (F1) hybridoma proteins 3-24 and 12-40, both IgG1, kappa with a Ka = 2.8 and 3.4 X 10(6) M-1, respectively, at 37 degrees C. These clones inhibited (6.8 +/- 2.8 -20.8 +/- 0.6% at 1 microgram/well) the idiotype-anti-idiotype interactions (IAII) of anti-F1 clones 3-13 and 3-17, which define a previously described low affinity idiotype family. Antibodies 3-24 and 12-40 also inhibited (45.0 +/- 3.0 and 61.3 +/- 5.6%, respectively, at 1 microgram/well) an IAII defined by a high affinity (Ka = 5.2 +/- 1.5 X 10(9) M-1 at 37 degrees C) anti-F1 clone, 4-4. Hybridoma proteins 3-13 and 3-17 possess similar affinities for F1 (Ka = 3.8 +/- 5.1 and 5.9 +/- 4.0 X 10(4) M-1) and are known to be idiotypically unrelated to clone 4-4. While 3-24 and 12-40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Qmax and lambda max) of bound F1. All IAII (3-13, 3-17, 9-40 and 4-4) were inhibited greater than 80% by the presence of 10(-4) M F1 or F1-BSA. In addition, four intermediate affinity (6.0 X 10(6) less than or equal to Ka less than or equal to 5.3 X 10(8) M-1) anti-F1 clones, comprising a second previously described idiotype family (designated the 9-40 family) were further analyzed. Inhibition of the 9-40 IAII by all heterologous proteins in the 9-40 family (except clone 5-27), and clones 3-24, 12-40 and 4-4 ranged from 87.7 +/- 1.3 to 95.4 +/- 1.0% at 1 microgram/well. Titration of the 9-40 IAII inhibition by antibodies 9-40, 3-24, 12-40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3-24, 12-40, 9-40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9-40 family, 3-24 and 12-40 were more closely related to intermediate affinity clone 9-40 than high affinity clone 4-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyclonal antibodies specific for liganded active site (metatype) of a high affinity anti-hapten monoclonal antibody.

Syngeneic polyclonal antibodies were elicited to an affinity labeled high affinity (2-3 X 10(10) M-1) anti-fluorescein murine IgG2a monoclonal antibody. Hyperimmune ascites fluid was tested for reactivity with homologous liganded, affinity labeled and non-liganded Fab fragments derived from the high affinity antibody. Binding results demonstrated antibody specificity for the liganded or affinity labeled site, but no reactivity with either the non-liganded form or the fluorescyl ligand. Kinetic analysis showed that the rate of dissociation of the fluorescein ligand was slowed down significantly upon binding of the anti-affinity labeled reagent to the liganded antibody. Antibodies specific for the affinity labeled prototype were not reactive with the liganded form of an IgM monoclonal anti-fluorescyl antibody of the same affinity but idiotypically unrelated. Results of the immunological studies suggested that the antibody active site stabilized by bound ligand differed from the idiotype of the antibody. The term "metatype" was proposed for the immunological definition of the liganded active site to distinguish it from idiotype (non-liganded). The general nature of metatopes is discussed in terms of conformational or sequential epitopes.

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.

Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family.

The T-dependent BALB/c murine immune response to fluorescein (F1) is characterized by structural heterogeneity at the protein level exemplified in part by a significantly wide range of affinities (Ka), and apparent lack of dominant idiotypes. In order to generate an idiotype family, xenogenic anti-idiotype (anti-ID) antibodies raised against anti-F1 monoclonal antibody (MCA) 4-4 (Ka = 1.7 X 10(10) M-1) were used in a solid-phase radioimmunoassay (SPRIA) to screen 68 anti-F1 hybridomas generated from multiple cell fusions for idiotypically related immunoglobulins. Four affinity-purified MCAs (designated 9-40, 10-25, 5-14 and 5-27) bearing 4-4 idiotypic determinants (ID 4-4) exhibited discrete isoelectric focusing spectrotypes (pI range = 6.8-7.7), significantly different fluorescence quenching values (38-95%) of bound ligand, binding affinities ranging from 3.3 X 10(7) to 5.3 X 10(8) M-1, similar active site inaccessibility to iodide, and closely related fine-specificity patterns for fluorescyl analogues. Idiotypic relatedness of each MCA to prototype 4-4 was quantitated by SPRIA, the results demonstrating that: each 125I-labeled MCA bound significantly to solid-phase anti-ID 4-4, and the concns of heterologous MCAs 9-40, 10-25 and 5-14 required for 50% inhibition of 125I-4-4/anti-ID 4-4 binding were comparable to homologous Ig protein. The finding that ID 4-4 bearing anti-F1 MCAs exhibit various binding properties and affinities is consistent with variable-region somatic diversification in anti-F1 affinity maturation.

C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits.

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.

Cryoprecipitation properties of a high-affinity monoclonal IgM anti-fluorescyl antibody.

A high affinity (Ka = 2-3 x 10(10) M-1) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temp. insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temp. (4 degrees C) had been shown to be reversible at higher temp. and in the presence of fluorescyl ligand, indicating antigen binding site involvement. The primary objectives were to isolate and identify structural component(s) responsible for insolubility at low temp. Procedures developed for production and isolation of the monomeric subunit (IgMs) involved thiol reduction and gel filtration separation in the presence of the zwitterionic detergent, CHAPS. Fab and (Fc)5 fragments generated by papain digestion were purified sequentially by gel filtration (Sephadex G-200) and affinity chromatography (fluorescein-Sepharose). A protocol for covalent labeling of Fab fragments with the fluorescent chromophore 2,5 DNS-Cl was developed in order to measure steady-state fluorescence polarization. In kinetic and equilibrium cryoprecipitation assays, nonliganded 18-2-3 IgM and IgMs demonstrated insolubility. Bound fluorescyl ligand, increased ionic strength (0.5 M NaCl) or basic pH (greater than 8.0) abrogated cryoprecipitation. Isolated Fab or (Fc)5 fragments did not exhibit low temp. insolubility. Decreased cryoprecipitation occurred when (Fc)5 fragments were added to nonliganded 18-2-3 IgM. Fluorescence polarization of 2,5 DNS Fab 18-2-3 fragments indicated lack of Fab-Fab aggregation. Results suggested that electrostatic interactions involving 18-2-3 antibody combining sites with interactive sites in the Fc region of homologous IgM were responsible for the phenomenon of cryoprecipitation.

Charge transfer between fluorescein and tryptophan as a possible interaction in the binding of fluorescein to anti-fluorescein antibody.

Steady-state and time-resolved fluorescence studies of fluorescein (Fl) and 9-hydroxyphenylfluoron (HPF) bound to high-affinity rabbit anti-Fl IgG antibody (anti-Fl IgG) have been performed. The heterogeneity in the fluorescence properties observed for Fl bound to anti-Fl IgG is reduced for HPF bound to anti-Fl IgG. A charge transfer between a tryptophyl residue in the binding site and the hapten was considered as a possible binding interaction. Fl was observed to form complexes in solution with the amino acids tryptophan, tyrosine and methionine, probably due to charge transfer. Also, the fluorescence of tryptophyl residues of the protein is quenched on binding. While such charge transfer complexes may be present, there is no direct evidence that charge transfer complexes between Fl and tryptophan are necessarily present for Fl bound to all high-affinity anti-Fl IgG molecules.

Induction and suppression of contact sensitivity to fluorescein isothiocyanate (FITC).

Although both fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP) covalently couple primarily to the epsilon-amino group of lysine residues of surface glycoprotein, FITC is structurally different from TNP. In this paper, we investigated the immune regulatory mechanisms in contact sensitivity (CS) to FITC by the administration of FITC and FITC-conjugated epidermal cells (FITC-EC) via various routes. Mice injected with FITC via i.p. or i.v. route did not induce CS but induced hyporesponsiveness to the following sensitization with FITC painting. Mice injected with FITC via s.c. route induced neither CS nor hyporesponsiveness to the following FITC painting. Administration of FITC via i.v. route was demonstrated to induce hapten-specific suppressor T cells. Inoculation of FITC-EC via s.c. or i.p. route induced CS, whereas injection of FITC-EC via i.v. route did not induce CS but induced hyporesponsiveness to the following FITC painting. The results are compared with the previous data obtained by trinitrobenzene sulfonate and trinitrophenyl-conjugated epidermal cells.

Modulation of the primary and secondary antifluoresceyl antibody response in rats by 17 beta-estradiol.

Estradiol (E2) enhances humoral immunity, but the quantitative and temporal relationship between serum E2 and antibody titers is unclear. The present study was therefore designed to determine whether the sex of the animal, ovariectomy (OVX), and replacement with various tonic levels of serum E2 influenced the primary and secondary antifluorescein antibody titers of rats. Two experiments were performed. In Exp I, intact male and intact female Lewis inbred rats were given primary and booster (secondary) immunizations with fluorescein-keyhole limpet hemocyanin. Primary and secondary bleedings for antifluorescein antisera were obtained. In Exp II female Lewis inbred rats were randomly distributed among the following treatment groups: sham OVX, sham implanted; OVX, sham implanted; and three groups of OVX and implanted with E2-filled Silastic capsules of 0.5, 1.5, or 5.0 cm lengths. Immunization with fluorescein-keyhole limpet hemocyanin and bleedings for antifluorescein antisera followed the same schedule as in Exp I. In Exp I, females had significantly higher antifluorescein antibody titers than males during the primary and secondary response. In Exp II, OVX significantly depressed the primary and secondary antifluorescein antibody titers relative to sham OVX controls. During the primary antifluorescein antibody response, a significant quadratic relationship was seen between antibody titers and serum E2 when measurements included the pharmacological range of the serum E2 concentrations. A significant linear relationship existed between serum E2 and antifluorescein antibody titers when the same data were examined over a range of 15-130 pg E2/ml. This linear relationship is more significant during the primary response than the secondary response. It is concluded that females generate higher antifluorescein antibody titers than males; OVX significantly depresses the antifluorescein antibody response; E2 is immunoenhancing in the physiological range and immunosuppressive in the pharmacological range; and the presence of E2 during the primary response may be more critical to immunoenhancement.

Probing the nucleotide-binding site of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase with anti-fluorescein antibodies.

Antibodies raised against fluorescein were unable to bind to the fluorophore when bound at the nucleotide-binding site of native (Ca2+-Mg2+)-ATPase, as judged by fluorescence quenching assays or competitive ELISAs, but were able to bind when the ATPase was denatured. Indirect ELISAs, in which native and denatured FITC-ATPase were used to coat ELISA plates, were unable to detect the difference in accessibility of the fluorescein bound to the native and denatured ATPase. These results indicate that the nucleotide-binding site is relatively inaccessible in the native structure, even though fluorescence energy transfer studies [(1987) Biochim. Biophys. Acta 897, 207-216] indicate that this site must be close to the surface of the ATPase. In addition the results suggest that the indirect ELISA method may be of limited value in probing the accessibility of epitopes using antibodies.

Fractionation of human lymphocyte subpopulations on immunoglobulin coated Petri dishes.

This report describes a simple solid-phase technique for the positive selection of lymphocytes labeled with fluoresceinated antibodies. B lymphocytes were labeled with fluoresceinated anti-human Ig or monoclonal anti-human Ia (L243), and then were bound to plastic culture dishes coated with affinity purified goat anti-fluorescein specific antibody. Bound cells were eluted at 37 degrees C with 1 mM fluorescein-L-lysine phosphate-buffered saline. Functionally the eluted Ig positive cells responded to pokeweed mitogen (PWM) by in vitro secretion of IgM, as measured by radioimmunoassay of culture supernatants. The secretion of IgM was dependent on the addition of T lymphocytes. Moreover, the isolated B cells were functionally receptive to 'help' and 'suppression' by T cells with and without Fc receptors for IgG respectively. T cell subsets were fractionated on plastic culture dishes coated with heat aggregated rabbit or human IgG. the non-bound cells (enriched T(gamma-)) provided collaborative 'help' in the PWM induced IgM secretion response by human B lymphocytes. The bound cells (enriched T(gamma+)) eluted with 0.01 M EDTA in phosphate-buffered saline, suppressed IgM secretion. This method can be adapted to fractionate subsets of lymphocytes for which a fluoresceinated antibody is available. For routine functional studies, the isolation of cell types with conventional or monoclonal antibodies does not require the use of a fluorescence activated cell sorter, but only an antifluorescein labeled Petri dish. In conclusion, a rapid solid-phase technique enables us to prepare enriched populations of functionally active lymphocytes.