An in vivo method for measuring the adsorption of plasma proteins to titanium in humans.

A novel method of collecting in vivo plasma proteins of humans from osteotomies prepared during insertion of an oral implant is described. A rod containing a collecting portion with a predetermined surface is introduced into the osteomy, removed, and transferred for enzyme-linked immunosorbent assay analysis. Two experiments were used to examine the feasibility of the method. In the first, titanium (Ti) rods with different roughness were exposed for 10 min to the blood. Blasted and acid-etched surfaces adsorbed four times more and acid-etched surfaces adosorbed two times more plasma proteins as compared to machined surfaces. In the second experiment, blasted and acid-etched rods were wetted for 10 s prior to the insertion. The adsorption for fibronectin, albumin, fibrinogen, and IgG was enhanced significantly compared with nonwetted rods. These results are discussed in the light of previous methods used in studies on adsorption. Thus, use of the collecting instrument enables aspects of human plasma-implant interface to be studied in a more realistic manner.

Genetic control of the antibody response. II. Further analysis of the specificity of determinant-specific control, and genetic analysis of the response to (H,G)-A--L in CBA and C57 mice.

CBA and C57 mice were tested for their ability to make an immune response to a related series of branched, multichain synthetic polypeptide antigens in which the antigenic determinants on the amino termini of the branched side chains were systematically varied. Neither strain responded to the polyglutamic acid determinant. Both strains responded well and equally to the poly(phenylalanine, glutamic acid) determinants. CBA mice responded poorly, and C57 mice responded well to two different antigens bearing poly(tyrosine, glutamic acid) determinants. CBA mice responded well, and CS7 mice responded poorly to two different antigens bearing poly(histidine, glutamic acid) determinants. The genetic control of the immune response to (H,G)-A--L appears to be dominant and polygenic, as it has been shown to be for (T,G)-A--L. The related antigens used in this study show extensive cross-reactions with antisera against other members of the related series.

Thymus independence of a collagen-like synthetic polypeptide and of collagen, and the need for thymus and bone marrow-cell cooperation in the immune response to gelatin.

Several inbred mouse strains were screened for their ability to respond to the ordered periodic collagen-like polymer (Pro-Gly-Pro)(n), to the random copolymer (Pro(66), Gly(34))(n), to the protein conjugate Pro-Gly-Pro-ovalbumin, to rat tail tendon collagen, rat tail tendon gelatin, and to Ascaris cuticle collagen. Differences were obtained in the magnitude of the antibody titers towards the above immunogens among the strains tested. The level of the response to the ordered polymer (Pro-Gly-Pro)(n) was not similar to that towards the random (Pro(66), Gly(34))(n), confirming differences in the antigenic determinants of the two immunogens. The role of the thymus in the immune response to (Pro-Gly-Pro)(n) and (Pro(66), Gly(34))(n) as well as to two collagens and gelatin, was studied in order to find out a possible correlation with the structural features of the immunogens. Heavily irradiated recipients were injected with syngeneic thymocytes, marrow cells, or a mixture of both cell populations and were immunized with the above-mentioned antigens. An efficient immune response to the ordered collagen-like (Pro-Gly-Pro)(n) was obtained in the absence of transferred thymocytes. The thymus independence of (Pro-Gly-Pro)(n) was confirmed when thymectomized irradiated mice were used as recipients. In contrast with these results, cooperation between thymus and marrow cells was necessary in order to elicit an immune response to (Pro(56), Gly(34))(n). Similarly, the immune response to the triple helical collagen was found to be independent of the thymus, whereas for an effective response to its denatured product, gelatin, thymus cells were required. These findings indicate that a unique three-dimensional structure of immunogens possessing repeating antigenic determinants plays an important role in determining the need for cell to cell interaction in order to elicit an antibody response.

Contribution of different cell types to the genetic control of immune responses as a function of the chemical nature of the polymeric side chains (poly-L-prolyl and poly-DL-alanyl) of synthetic immunogens.

Genetic regulation of immunological responsiveness was studied at the cellular level by comparing the limiting dilutions of immunocompetent cells from spleen, thymus, and bone marrow of high and low responders as a function of the poly-L-prolyl and poly-DL-alanyl side chains of two synthetic polypeptide immunogens. The spleens of immunized and unimmunized high responder DBA/1 mice were found to contain respectively, 18- and 7-fold more limiting precursor cells specific for (Phe, G)-A--L than the spleens of SJL low responder donors. These results, using a synthetic polypeptide built on multichain poly-DL-alanine, confirm the findings reported for polypeptides built on multichain poly-L-proline (1, 2), that there is a direct correlation between immune response potential and the relative number of immunocompetent precursors stimulated. Cell cooperation between thymocytes and bone marrow cells was demonstrated for both (T, G)-Pro--L and (Phe, G)-A--L. Limiting dilutions of thymus and bone marrow cells in the presence of an excess amount of the complementary cell type indicated an eightfold lower number of detected (T, G)-Pro--L-specific precursors in DBA/1 (low responder) marrow when compared with SJL (high responder) marrow. No differences were observed in the frequency of relevant high and low responder thymocytes for the (T, G)-Pro--L immunogen. These results are similar to those reported for the (Phe, G)-Pro--L (3). In contrast to the cellular studies reported for the Pro--L series of immunogens, the marrow and thymus cell dilution experiments for (Phe, G)-A--L revealed genetically associated differences in both the marrow and thymus populations of immunocytes from high (DBA/1) and low (SJL) responders. In addition to a fivefold difference in limiting marrow cell precursors (similar to that seen in the Pro--L studies), a striking difference was observed between the helper cell activity of high responder DBA/1 and low responder SJL thymocytes. This difference was indicated by the observation that low responder thymocyte dilutions followed the predictions of the Poisson model, whereas dilutions of high responder thymocytes did not conform to Poisson statistics. Transfers of allogeneic thymus and marrow cell mixtures from DBA/1 and SJL donors confirmed the syngeneic dilution studies showing that the genetic defect of immune responsiveness to (Phe, G)-A--L is expressed at both the thymus and marrow immunocompetent cell level. The parameters presently known for genetic control of immune responses specific for (Phe, G) (Ir-1 gene) and for Pro--L (Ir-3 gene) have been compared. The Ir-1 and Ir-3 genes are not only distinct by genetic linkage tests (to H-2) (5, 6, 9), but they are also seen to be different by cellular studies. Furthermore, expression of low responsiveness within a given cell population was shown to depend on the chemical structure of the whole immunogenic macromolecule.

Cellular basis of the genetic control of immune responses to synthetic polypeptides. I. Differences in frequency of splenic precursor cells specific for a synthetic polypeptide derived from multichain polyproline ((T,G)-Pro--L) in high and low responder inbred mouse strains.

SJL mice are high responders to the synthetic multichain polypeptide antigen (T,G)-Pro--L, whereas DBA/1 mice are low responders (10, 11). In order to determine whether the genetic control of immune response can be correlated with the number of antigen-sensitive precursor cells, spleen cell suspensions from normal and immunized SJL and DBA/1 donor mice were transplanted into lethally X-irradiated syngeneic recipients (incapable of immune response) along with (T, G)-Pro--L. Anti-(T, G)-Pro--L responses (donor-derived) were assayed in the sera of the hosts 12-16 days later. By transplanting graded and limiting numbers of spleen cells, inocula were found which contained one or a few antigen-sensitive precursors reactive with the immunogen. Using this method to estimate the relative numbers of such cells for the high responder SJL strain, one precursor was detected in approximately 1.3 x 10(6) and approximately 7.2 x 10(6) spleen cells from immunized and normal donors, respectively. In contrast, one precursor was detected in about 30 x 10(6) spleen cells from low responder DBA/1 mice, irrespective of whether the donors had been immunized. These results indicate that the genetic control of immunity to the synthetic polypeptide antigen investigated is directly correlated to the relative number of precursor cells reactive with the immunogen in high and low responder strains.

Cellular basis of the genetic control of immune responses to synthetic polypeptides. II. Frequency of immunocompetent precursors specific for two distinct regions within (Phe, G)-Pro--L, a synthetic polypeptide derived from multichain polyproline, in inbred mouse strains.

DBA/1 mice are high responders to the (Phe, G) determinant of the synthetic polypeptide (Phe, G)-Pro--L, whereas SJL mice respond well to the Pro--L region of this macromolecule (6). In order to determine whether the phenomenon described above is related to the number of antigen-sensitive units detected for both specificities, and whether responses to these determinants can be transferred independently, graded and limiting inocula of spleen cells from SJL, DBA/1, and F(1) donors were injected into X-irradiated, syngeneic, recipient mice with (Phe, G)-Pro--L. By this approach, one antigen-sensitive unit specific for (Phe, G) was detected in 1.7 x 10(6) and 8.5 x 10(6) spleen cells from immunized and nonimmunized DBA/1 donors, respectively. In contrast, one (Phe, G) relevant precursor was detected in 20 x 10(6) SJL spleen cells, irrespective of whether the donors had been immunized. On the other hand, for the Pro--L specificity, one limiting splenic precursor was found in 1.3 x 10(6) and in 3.4 x 10(6) cells for immunized and nonimmunized SJL donors, respectively; whereas one response unit was estimated for this determinant in 9.4 x 10(6) and in 38 x 10(6) spleen cells from immunized and nonimmunized DBA/1 mice. The findings reported here indicate that the phenotypic expression of the genetic control(s) for immune responsiveness to different immunopotent regions of (Phe, G)-Pro--L is directly correlated with the number of immunocompetent response units detected in two inbred mouse strains. In the spleens of immunized F(1) donors, similar frequencies of one limiting precursor in 3.0 x 10(6) and in 2.8 x 10(6) cells were detected for (Phe, G) and Pro--L, respectively. The results of a chi-square test for independence of (Phe, G) and Pro--L responses in F(1) animals is compatible with the hypothesis that the transferred spleen cells limiting the response to (Phe, G)-Pro--L are restricted to generate antibodies specific for only one of the two determinants of this macromolecule.

Studies on the competence of single cells to produce antibodies of two specificities.

Two series of experiments were performed, utilizing a modification of the hemolysin plaque technique which registers 19S antibody, in an attempt to determine the frequency of cells capable of simultaneously producing antibody to two non-cross-reacting antigens. Mice were immunized i.v. with rabbit and camel RBC and their spleens assayed for cells producing antibody against both antigens. 16,904 cells producing antibody of one or the other specificity, from 26 mice, were counted. Not one cell was detected which produced antibody of two specificities. Rabbits were immunized intradermally with HSA to which polyalanyl and p-azobenzenearsonate groups were chemically attached. The individual haptens, polyalanyl, and p-azobenzenearsonate groups were coupled to separate aliquots of SRBC, and the lymph nodes of immunized rabbits were assayed for cells releasing antibody against both haptens. In a study of 11 rabbits, after counting 27,845 cells producing antibody, we detected no "double" plaques.

Antibody response of inbred mouse strains to ordered tetrapeptides of tyrosine and glutamic acid attached to multichain polyalanine or polyproline. Tyr-Tyr-Glu-Glu is a major determinant of the random poly-(Tyr, Glu)-polyDLAla--polyLys.

Five inbred mouse strains which represent high and low responders to the random synthetic polypeptide poly(LTyr,LGlu)-polyDLAla--polyLLys, designated (T, G)-A--L, to which the immune response is controlled by an H-2-linked gene, were immunized with three ordered tetrapeptides composed of tyrosine and glutamic acid attached either to multichain poly-DL-alanine or to polyproline. Only one of the three antigenic determinants, namely tyrosyl-tyrosyl-glytamyl-glutamic acid (T-T-G-G), resembled the random peptide (T, G) in the pattern of immune responses elicited against it, and in the cross-reactivity of the specific antibodies with (T, G)-A--L. The immune response pattern to the other two ordered tetrapeptides, T-G-T-G and G-T-T-G, was different from that obtained with (T, G)-A--L, and no cross-reactivity was detected between the antibodies provoked with these peptides and (T, G)-A--L. Thus, it is suggested that T-T-G-G is a major determinant in the random (T, G)-A--L.

Genetic control of immune response. The dose of antigen given in aqueous solution is critical in determining which mouse strain is high responder to poly(LTyr, LGlu)-poly(LPro)--poly(LLys).

Antibody response to different doses of (T,G)-Pro--L, given in aqueous solution, was investigated in the high responder SJL and low responder DBA/1 strains by measuring hemolytic plaque-forming cells (PFC) in the spleens as well as hemagglutination titers in the sera. The gene responsible for the difference between the two strains in the response to this antigen, given in complete Freund's adjuvant, has been previously denoted Ir-3. This gene is not linked to the major histocompatibility locus. In the response to the optimal dose (1 mug) of antigen, no difference could be shown between the strains. The peak of the response and the numbers of direct and indirect PFC were similar in both strains in the primary and secondary response. After injection of higher doses (10-100 mug) of antigen, both the direct and indirect PFC responses were lower in the low responder than in the high responder strain. Moreover, the peak of the response occurred earlier in the high responder strain in the primary response to the 10 mu dose of antigen. After administration of a suboptimal dose (0.02 mug) of antigen, the low responder strain produced in the primary response 4-20 times more indirect plaques than the high responder strain. Also the number of direct plaques was higher in the low responder than in the high responder strain. The serum antibody responses to the optimal and higher doses of antigen were parallel to the PFC responses. From inhibition of PFC with free antigen, it was concluded that a similar proportion of cells was producing high and low affinity antibodies to (T,G)-Pro--L in both strains. High and low zone tolerance could be induced in the two strains with (T,G)-Pro--L, but no difference could be shown between the strains. It is suggested that the Ir-3 gene plays a role in the regulation of the balance stimulation and suppression according to the dose of antigen given.

Biological activity of the cleavage product of human immunoglobulin G with cyanogen bromide.

Treatment of human IgG with cyanogen bromide in 0.05 M HCl under specified conditions resulted in the cleavage of about half of its methionyl peptide bonds. A major fragment of about 5S was isolated from the reaction mixture by gel filtration in quantitative yield. The CNBr fragment reacted fully with goat antiserum against human light chain, but its reaction with anti-heavy chain was markedly decreased. The treatment with CNBr caused a drastic decrease in the following biological activities of IgG: complement fixing, skin binding, reaction with antiglobulin factors, and reaction with specific anti-Gm(12) serum. On the other hand, the reaction with serum of anti-Gm(1) or anti-Gm(4) specificity was not impaired and antibody activity, namely antistreptolysin and isohemagglutinin, was retained after the treatment with CNBr. It is concluded that the CNBr cleaves preferentially the methionyl bonds in the Fc portion of IgG, and that the major fragment obtained, denoted F(ab'')(2), has still the combining properties of a divalent antibody. The possible therapeutic uses of F(ab'')(2) are discussed.

Regulation of antibody response by cells expressing histamine receptors.

Spleen cells from immunized and unimmunized mice were either passed over histamine-rabbit serum albumin-Sepharose columns or rabbit serum albumin-Sepharose control columns. The immune response potential of 5 x 10(6) cells excluded from the two columns were compared with each other, and with an equal number of unfiltered cells by injection of the cell suspensions mixed with sheep erythrocytes into irradiated, syngeneic recipients. The direct and indirect anti-sheep erythrocyte plaque-forming cell responses generated by the cells passed over the histamine-bead column were significantly greater than the responses resulting from the inocula of unfiltered cells or cells passed over control columns. These results indicate the existence of a cell population expressing surface receptors for histamine, which functions to regulate antibody responses.

In vitro induction of a primary response to the dinitrophenyl determinant.

Primary antibody response against the dinitrophenyl group has been elicited in vitro after the stimulation of normal mouse spleen explants with 2,4-dinitrophenyl (DNP)-hemocyanin or alpha-DNP-poly-L-lysine (PLL). Antibodies were detected in the culture medium by the inactivation of DNP-T4 phage. The specificity of the reaction was manifested by the lack of the capacity of the medium to inactivate the unmodified bacteriophage and by the inhibition of the inactivation of DNP-T4 with DNP-lysine.

The nature of the antigenic determinant in a genetic control of the antibody response.

The response of inbred mouse strains to two polypeptides derived from multichain polyprolines, (T,G)-Pro--L and (Phe,G)-Pro--L, is different from the response of the same mouse strains to a similar series of polymers built on multi-poly-D,L-alanyl--poly-L-lysine, although the same short sequences of amino acids are attached to the side chains of the polypeptides in the two series. These results indicate that a portion of the side chain (e.g. polyalanine or polyproline) participates in the antigenic determinant. This was confirmed by studying the response of different mouse strains to two kinds of polypeptides: (T,G)-Pro-A--L 717 and 718 and (T,G)-A-Pro--L 719 and 721. Antibody assay of antisera to (Phe,G)-Pro--L with the cross-reacting antigens (T,G)-Pro--L and (Phe,G)-A-L indicates that different inbred mouse strains make antibodies specific for different parts of the same polypeptide. Thus, antibody from DBA/1 mice reacts almost exclusively with the (Phe,G) sequence, while SJL antisera bind only (T,G)-Pro--L and fail to bind (Phe,G)-A-L. The immune responses to the same amino acids on two different polypeptides (i.e. A--L and Pro--L) appear to be under separate genetic control.

The genetic control of antibody specificity.

The immune response to a synthetic polypeptide built on multichain polyproline, poly-L-(Tyr,Glu)-poly-L-Pro-poly-L-Lys [(T,G)-Pro--L], in the offspring of a cross between DBA/1 and SJL mice is under a genetic control superficially similar to the one operating for the immune response to a similar synthetic polypeptide built on multichain polyalanine, poly-L-(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys [(T,G)-A--L], in the offspring of a cross between CBA and C57 mice. In both cases, the genetic control is a quantitative trait in which the major gene(s) is (are) dominant and the trait is not linked to any of the known structural genes coding for mouse immunoglobulin heavy chains. However, the genetic control of response to (T, G)-Pro--L, designated immune response-3 (Ir-3), is qualitatively different from the one operating for (T,G)-A--L [immune response-1 (Ir-1)] in that it is not linked to the histocompatibility-2 (H-2) locus. A study of the immune response to a related polypeptide built on multichain polyproline, poly-L-(Phe,Glu)-poly-L-Pro-poly-L--Lys [(Phe, G)-Pro--L], in the DBA/1 x SJL cross has shown a genetic control of antibody specificity. F(1) x DBA/1 backcross anti-(Phe, G)-Pro--L sera segregate in their ability to bind (T,G)-Pro--L, and there is no linkage of anti-(T,G)-Pro--L binding capacity with the H-2(s) allele of the SJL grandparent. F(1) x SJL anti-(Phe, G)-Pro-L sera segregate in their capacity to bind poly-L-(Phe,Glu)-poly-D,L-Ala-poly-L-Lys [(Phe, G)-A--L] and the ability to bind (Phe, G)-A--L is clearly linked to the H-2(q) allele from the DBA/1 grandparent. Thus, in mice all responding well to a given antigen [(Phe, G)-Pro--L], the specificity of the antibodies produced [i.e., anti-(Phe,G) or anti-prolyl] is genetically determined. Cross-inhibition of binding m (DBA/1 x SJL)F(1) anti-(Phe,G)-Pro--L antisera indicates that the anti-(Phe,G) and anti-prolyl specificities are a function of two separate and largely non-crossreacting antibody populations.

The role of thymocytes and bone marrow cells in defining the response to the dinitrophenyl hapten attached to positively and negatively charged synthetic polypeptide carriers. Cell fractionation over charged columns.

An inverse relationship exists between the net electrical charge of immunogens and the antibodies they elicit (1). Results of an earlier study have demonstrated that the net charge phenomenon has a cellular basis, since the immune response potential of murine spleen cells to 2,4-dinitrophenyl (DNP) on a negatively charged synthetic polypeptide carrier was reduced by cell fractionation over negatively charged glass beads, whereas the response to the same hapten on a positively charged carrier was unaffected (14). To verify that the net charge correlation is expressed at the cellular level, spleen cells were fractionated over positively charged poly-L-lysine-coated glass bead columns, and their immunocompetence to DNP on positively and negatively charged carriers was tested by cell transfers in irradiated recipient mice. In this case, the fractionated cells showed reduced response potential to DNP on the positively charged carrier only. Thus, the cellular basis of the net charge phenomenon has been demonstrated for both positively and negatively charged immunogens (for the same specificity) by cell separation techniques over columns of opposite charge. In order to establish whether the cell population relevant for the charge properties of immunogens was of thymus or marrow origin, thymocytes and bone marrow cells were selectively passed over positively or negatively charged columns and mixed with unfractionated cells of the complementary type. Transfers of the filtered and unfiltered cell mixtures in irradiated recipient mice immunized with DNP on either a positive or a negative synthetic polypeptide carrier indicated that fractionation of thymocytes, but not of marrow cells, correlated with the spleen population. Thus, thymocytes fractionated over negatively charged columns and mixed with unfractionated marrow cells exhibited reduced response to DNP on the negative carrier, but normal responses to DNP on the positive carrier. The opposite result was obtained when thymocytes were passed over positively charged columns. No effect on the anti-DNP response was detected by filtration of bone marrow cells over columns of either charge. These findings indicate that it is possible to distinguish between thymocytes on the basis of their capacity to react with more acidic or more basic surfaces and that a population of thymus-derived cells may recognize immunogens on the basis of their overall electrical charge. No evidence was found by these techniques that marrow-derived cells contribute to the net charge phenomenon.

Inverse relationship between net electric charge on the antigen and that on the sensitized cell in cellular immune response: demonstration with basic encephalitogen of the brain.

An inverse relationship exists between the net-electrical charge of immunogens and the antibodies elicited (1). The cellular basis of the net charge phenomenon has been established for both positively and negatively charged immunogens, by cell separation techniques over columns of opposite charge (7, 8). To establish whether this phenomenon can be extended to include cell-mediated immunity, the response to basic encephalitogenic protein (BE) which induces experimental allergic encephalomyelitis (EAE) was now investigated. Lymph node cells from sensitized strain 13 guinea pigs were fractionated over positively and negatively charged columns and compared to unfractionated cell populations in two assay systems: (a) in vitro response to BE in terms of lymphocyte transformation and (b) the passive transfer of EAE to unsensitized syngeneic recipients. The response was found to be confined to the fraction of cells eluted from glass bead columns, namely, the more negative cells. Cells eluted from poly-L-lysine-coated glass bead columns (i.e., positive cells) were devoid of the capacity to respond to this antigen either in vivo or in vitro. It was previously established that thymocytes rather than bone marrow cells account for the inverse charge phenomenon as assayed by T-helper-cell function in in vivo antibody production (8). We have now extended the inverse charge effect to include cell-mediated immune response of the delayed hypersensitivity type.

Protein-bacteriophage conjugates: application in detection of antibodies and antigens.

Covalent attachment of proteins to bacteriophage yielded modified phage preparations with which it is possible to detect antibodies to proteins at concentrations as low as 0.5 to 2.0 nanograms per milliliter. Similarly, antibodies may be linked covalently to phage, and the resulting antibody-phage conjugate is useful in detecting proteins. An alternative method for quantitative determination of proteins is suggested, in which the inactivation of protein-phage by antibodies to protein is inhibited by the protein tested. With rabbit immunoglobulin G as the protein, as little as 0.3 nanogram per milliliter could he determind.

Stimulation in vitro with protein carrier of antibodies against a hapten.

Rabbits were immunized both with lysozyme and with dinitrophenylated bovine serum albumin. No antibodies against the dinitrophenyl hapten were produced when cell suspensions of their spleens were exposed in vitro to dinitro-phenyl-ovalbumin, whereas a positive response was obtained with dinitrophenyl-lysozyme. Moreover, when spleen cells from rabbits previously immunized with dinitrophenyl-bovine serum albumin were exposed in vitro to bovine serum albumin alone, they produced antibodies against the dinitrophenyl hapten.

Receptors for histamine can be detected on the surface of selected leukocytes.

Histamine insolubilized by chemical linkage via a protein or polypeptide carrier to Sepharose beads cannot penetrate cells. Even so, the resultant histamine-coated Sepharose binds leukocytes selectively. Despite the low molecular weight of histaminie, the binding is via preformed cell membrane receptors and can be specifically and competitively blocked or reversed by antihistamines.

Specific leukocyte receptors for small endogenous hormones. Detection by cell binding to insolubilized hormone preparations.

Receptors for small endogenous hormones on human leukocytes were studied by insolubilizing the hormones and incubating them with the cells. Histamine, norepinephrine, and prostaglandin E(2) (PGE(2)) were conjugated to either of two types of carrier: (bovine or rabbit) serum albumin or a random copolymer of DL-alanine and L-tyrosine. The conjugates were linked to agarose beads (Sepharose) and the resultant drug-conjugate-beads were incubated with leukocytes. Norepinephrine (when linked to its carrier via glutaraldehyde) and histamine preparations bound the majority of leukocytes. The binding appeared to be specific for the hormones tested. For example, the binding by histamine-rabbit serum albumin-Sepharose was prevented or reversed by high concentrations of histamine and histamine antagonists, but not by catecholamines or their pharmacologic antagonists. Similarly, binding of cells to the norepinephrine conjugate was inhibited by some catecholamines and propranolol, but not by histamine or histamine antagonists. Conjugates of norepinephrine linked via carbodiimide did not bind cells. The protein or copolymer carriers did not contribute to binding per se. The hormone-protein-conjugates bound more cells than the hormone-polymer conjugates. The former (unlike the free amines) failed to stimulate accumulation of cyclic AMP in leukocytes. The norepinephrine linked to polymer via glutaraldehyde, however, did stimulate leukocyte cyclic AMP accumulation, possibly because of the flexibility of the polymer. Columns of the various Sepharoses were used to determine the distribution of receptors to each hormone in mixed leukocyte populations. The majority of cells appeared to have receptors for both histamine and norepinephrine (bound through glutaraldehyde). Receptors to prostaglandins may have been detected by the column procedure, but their distribution could not be quantitated. The approach described provides a means to separate leukocytes on the basis of what are likely to be preformed receptors to small endogenous hormones, and to study the physiologic importance and function of the receptors.