Inhibition of specific antibody binding to adult male Schistosoma mansoni by adsorbed host serum components

The ability of anti-schistosome antibody to bind to adult male Schistosoma mansoni was studied, using fluoresceinated Staphylococcus aureus to detect specific antigen-antibody interaction at the parasite's surface. Both freshly perfused parasites and parasites which had had their adsorbed host antigens removed by elution were employed in a series of experimental manipulations to ascertain under what conditions specific antibody binding occurs and what conditions or factors are necessary for the parasite to reconstitute its surface so that specific binding is precluded. Neither normal mouse serum nor normal mouse IgG bound in a specific manner to either fresh or eluted worms. Slight binding was noted with immune mouse serum on both fresh and eluted worms, while immune IgG produced weak binding on fresh worms, but strong binding to eluted worms. This strong binding was reduced to the level seen on fresh worms by pre-incubation of the eluted worm in normal IgG prior to incubation in immune IgG and binding was completely negated by pre-incubation in either normal mouse serum or normal mouse serum minus IgG. The binding of immune IgG to eluted worms was not diminished by pre-incubation in mouse albumin, bovine albumin, or fetal bovine serum. These studies demonstrate that adsorbed host serum components can inhibit specific antigen-antibody interaction at the parasite's surface and suggest that a degree of specificity exists in what the parasite adsorbs from the host. These data further suggest that the protective serum factor or factors may include, but are not limited to, host IgG.     

Use of an immunoaffinity column for tetrachlorodibenzo-p-dioxin serum sample cleanup

Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provided antigens that generated antibodies in chickens. Competitive ELISA analysis demonstrated that the antibodies isolated from egg yolk (IgY) bound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The antibodies were linked to CNBr-Sepharose to generate an immunoaffinity column. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retained by the column and could be eluted by increasing the Tween 20 concentration. The binding efficiency for 10.7 ng per ml gel matrix ranged from 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind ¹⁴C-1,3,7,8-TCDD from serum samples. Diluting the serum 1:20 with 0.05% Tween 20 increased the binding efficiency. Alternately, ethanol–hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropriate preaffinity column cleanup for serum samples. After this preaffinity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%.     

Cross-reactivity of anti-human immunodeficiency virus type 1 gp41 antibodies with human astrocytes and astrocytoma cell lines.

An antigen expressed by astrocytes in human brain tissue and by various human astrocytoma cell lines was shown to cross-react with a monoclonal antibody generated against amino acids (aa) 584 to 609 of the transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1). This region is an immunodominant segment of gp41, and high levels of antibodies against this epitope have been detected in both serum and cerebrospinal fluid of HIV-infected individuals at all stages of HIV infection. Immunohistochemistry with this monoclonal antibody demonstrated the presence of a cross-reactive antigen in human brain tissue, with an increased frequency and intensity of staining in HIV-positive individuals when compared with HIV-negative controls. By using a panel of HIV-positive and -negative sera, we show that antibodies in HIV-positive serum specifically bound to the surfaces of human astrocytoma cells. HIV-positive sera depleted of antibodies recognizing gp41 aa 584 to 609 showed a significant diminution in cell surface binding. Conversely, the serum antibodies that bound to and were eluted from the aa 584 to 609 peptide also bound to the astrocyte cell surface. To identify the target antigen, the immunoreactivity of three astrocytoma cell lines was examined. By immunoprecipitation of metabolically labeled cell lysates and Western blot (immunoblot) analysis, we identified a protein of approximately 100 kDa as the target antigen. Cross-reactive antibodies between HIV proteins and astrocyte epitopes, such as this 100-kDa protein and others previously reported, suggests that an autoimmune response against these target antigens may disrupt the normal functions of astrocytes.     

Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

Isolation and characterisation of two sperm membrane proteins recognised by sperm-associated antibodies in infertile men

Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP). Using different immunoassays, these 3 preparations were tested for their ability to bind fish antibody. As determined by ELISA, the specific antibody binding activity was higher in SAP than in CSP. As determined by dot-blot immunoassay, the specific antigen binding activity versus SAP was higher in bile than in plasma antibodies. A different number of antigenic components of SAP and ESP were identified by immunoblotting performed with plasma or bile antibodies. These results led to the conclusion that T. bernacchii parasitism by nematodes involves plasma and bile anti-parasite antibodies. Furthermore bile antibodies were found to be more reactive and more heterogeneous than plasma.     

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

Sphere-linked immunodiagnostic assay (SLIDA): an electron microscopic method for detecting specific antibodies

We have developed a sensitive method, sphere-linked immunodiagnostic assay, using specific antigens covalently bonded to microspheres for the detection of antibodies in serum. In this method, specific antigens, such as the capsid proteins of tobacco mosaic virus and tobacco etch virus, were independently, covalently bonded to plastic micropheres of 0.5 microns or 0.9 microns in diameter. The antigen-linked spheres were then exposed to normal serum or serum containing specific antibody, followed by treatment with gold-labeled secondary antibodies. The binding of the gold-labeled secondary antibodies to the specific primary antibodies on the spheres acted as an indication of the presence of the specific primary antibodies. The spheres were then examined and photographed by transmission electron microscopy. The number of gold particles bound to the spheres was counted manually using the photographs. The gold labeling was found to be specific and sensitive, enabling detection of antibodies present in highly diluted antisera. The efficiency and sensitivity of the technique for detection of antibodies were compared with those of the enzyme-linked immunosorbent assay and found to be highly sensitive. The technique was also used for testing for the presence of antibodies to herpes simplex virus as well as antibodies to Staphylococcus enterotoxin using microspheres coated with the respective antigens. We believe that this technique could be applied clinically when needed for detection of antibodies to other viruses, such as the Human Immunodeficiency Virus.     

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.     

Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA.

The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.     

Identification and partial characterization of a parasite antigen in sera from humans infected with Wuchereria bancrofti

The purpose of this study was to identify and characterize soluble parasite antigens present in sera from humans infected with the filarial nematode Wuchereria bancrofti. Affinity chromatography and immunoblot methods were used to demonstrate a 200,000 m.w. circulating parasite antigen in sera from infected humans which corresponded to an antigen released by adult W. bancrofti during in vitro culture. Two monoclonal antibodies were produced to this antigen by immunizing mice with antigens from Dirofilaria immitis, a filarial parasite that is closely related to W. bancrofti, and screening cell fusion supernatants by enzyme immunoassay and counterimmunoelectrophoresis inhibition. The antibodies bound to a single repeated epitope (not phosphorylcholine) that was resistant to heat, acid, and protease treatments but sensitive to periodate oxidation. Immunoperoxidase studies showed that the epitope was concentrated in the cuticle and reproductive organs in D. immitis, and it was released in relatively large amounts by adult female D. immitis in vitro. The epitope is also present in antigens of other species of filarial and nonfilarial nematodes, but on the basis of preliminary studies, its presence in human serum appears to be specific for W. bancrofti infection.     

Analysis of antibody cross-reactivity in experimental American trypanosomiasis.

Susceptible C3H/He mice were immunized with the avirulent Corpus Christi strain of Trypanosoma cruzi and subsequently infected with virulent Brazil stain organisms. Seventy days after infection sera were isolated and analyzed on western blots of electrophoretically separated T. cruzi antigens prepared from culture-form parasites (primarily epimastigotes). More than 25 bands were identified. The antibodies were fractionated by elution from various regions of western blots corresponding to average molecular weights of approximately greater than 130, 77, 70, 60, 48, or 38 kDa. Each of these antibody preparations was then incubated with strips of nitrocellulose containing all of the electrophoretically separated T. cruzi, and cross-reactivity was determined. Antibodies isolated from the 130-, 77-, and 70-kDa regions all cross-reacted with each other. Antibodies eluted from the 60-kDa region bound antigens in the 60-, 70-, and the 77-kDa regions. More importantly, antibodies eluted from every region bound antigens in the 70-kDa region. Conversely, antibodies eluted from this region bound to antigens in all of the other regions. These data indicate the presence of (a) common antigenic epitope(s) in T. cruzi infections in these mice that is predominantly found in the 70-kDa antigen-antibody complex on western blots.     

The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Characterization of epitopes of human secretory component on free secretory component, secretory IgA, and membrane-associated secretory component

We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.     

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime — bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound protein was recovered by incubating the adsorbent with 2-mercaptoethanol. Crude antibody and uterine cytosol was prepared for affinity chromatography in buffer containing 10^?3 to 10^?2M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S ag SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200–300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.