Production of monoclonal antibodies against a new carcinoma-associated marker in view of developing a serological test.

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 kDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen.     

Detection of Lewis(a) antigen in sera of ovarian carcinoma patients by MOv2-MOv8 double-determinant radioimmunoassay

The monoclonal antibodies MOv2 and MOv8, raised against ovarian carcinoma, were found to be directed against two non-crossreacting epitopes expressed on the same molecule. Immunochemical analysis of the MOv8 recognized epitope showed that the Le(a) oligosaccharide, or commercial anti-Le(a) MAb, but not the anti-Le(b) MAb, prevented Ov8 binding to the reference target cell line (SW626), indicating that it is carried by the Le(a) antigen. Since we previously reported that MOv2 also recognises the Le(a) antigen, these data suggest that Mov8 and Mov2 were directed against different epitopes on the same oligosaccharide chain. Bearing in mind the knowledge of the biochemical nature of the monoclonal antibody recognized epitopes (CaMOv2 and CaMOv8), the presence of the circulating molecules recognized by them was analyzed by double determinant immunoradiometric assay (DDIRMA) in 103 sera from ovarian carcinoma patients. Patients with clinical evidence of the disease (ED) with MOv2 and MOv8 reactive and negative tumors had sera reactivity in 67% and 19% respectively. Also, 26% of the patients with no clinical evidence of disease (NED) had positive sera. When we investigated the relationship between MOv2-MOv8 DDIRMA sera positivity and red blood cells (RBC) Lewis phenotype, a strong correlation was found between the Le(a)+ phenotype and DDIRMA sera reactivity in healthy donors (6/6) and in ovarian carcinoma patients (9/10) whatever their clinical condition. No Le(a)- healthy donors gave evidence of MOv2-MOv8 reactive sera. In contrast, 33% and 57% of the sera from ED carcinoma patients with respectively Lea-b+ and Lea-b- phenotype were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivities of an anti-CEA peptide monoclonal antibody.

Synthetic peptides representing different areas of the CEA molecule were used as immunogens for the development of anti-CEA antibodies. Both polyclonal and monoclonal antibodies were generated using peptides composed of CEA amino acid positions 99-128 and 585-613, respectively. One MAb, designated CP4, generated using the CEA peptide 99-128, was chosen for a more detailed analysis of reactivity. MAb CP4 reacts in solid phase RIAs with CEA peptide 99-128 immunogen and purified native CEA. CP4 did not react with purified non-specific cross reacting antigen (NCA), even though there were two single amino acid differences between NCA and CEA in the 29 amino acid peptide. The affinity constants of CP4 for the CEA peptide 99-128 and native CEA are 4.07 x 10(9) M-1 and 5.75 x 10(8) M-1, respectively. When CP4 was reacted with purified CEA in Western blotting experiments, the Mr 180,000 glycoprotein characteristic of CEA was detected, but CP4 reacted to various size entities in tumor cell extracts. The results of liquid competition RIAs showed that the epitope that MAb CP4 recognized on native CEA is not available for binding when CEA is in solution. Physical (adsorption to a solid matrix) or chemical (deglycosylation or formalin-fixation) alteration of CEA is required for binding of CP4 to CEA. MAb CP4 reacted approximately 1,000-fold greater to deglycosylated CEA than native CEA. Immunohistochemical studies using formalin-fixed paraffin-embedded tissue sections demonstrated that, among carcinomas, CP4 reacts selectively with colorectal carcinomas, while normal colon is negative. Although stomach carcinoma is negative, dysplastic lesions and areas of intestinal metaplasia are reactive. Two of 7 normal stomach tissues showed focal cytoplasmic reactivity of the surface epithelium. CP4, therefore, appears to react with an epitope with highly restricted expression in colorectal carcinoma. These studies demonstrate the complexities in dealing with an anti-peptide MAb with reactivity to an epitope which is accessible only under certain conditions.     

Biochemistry and expression of myelomonocytic antigens

Six monoclonal antibodies (MAb) which react with myelomonocytic cells representing various stages of differentiation, and which precipitate six different cell surface molecules, were identified. A 50 to 55 kilodalton (Kd) glycoprotein, restricted in expression to mature cells of the monocyte lineage, was detected by immunoprecipitation with antibody MoS39. By using COS-7 cells transfected with a cDNA clone encoding the MoS39 antigen, various well-described anti-monocyte MAb, including Mo2, My4, Leu-M3 (MoP9), MoP15, MoS1, and 63D3, also bound to MoS39-expressing COS-7 cells, suggesting that this group of antibodies reacted with the same glycoprotein. Immature cells of the myelomonocytic lineage were shown to express two distinct molecules: one with an m.w. of 26 to 28 Kd identified by antibody SG133, and the second, a 130 to 140 Kd glycoprotein identified by MoU26. Mature granulocytes were found to express a 60 Kd molecule identified by antibody SG185 which was absent from other cells of this lineage. Two other molecules were shown to be present on both mature and immature cells of the granulocytic and monocytic lineages: a 130 to 140 Kd glycoprotein identified by antibody SG134, and a 160 to 170 Kd glycoprotein recognized by antibody MoU48.     

Cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human iris and ciliary body.

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.     

Complementation of expression of carcinoembryonic antigen and tumor associated glycoprotein-72 (TAG-72) in human colon adenocarcinomas.

Enzyme-labeled monoclonal antibodies (MAbs) were used in an immunohistochemical, dual-staining study of 10 colon adenocarcinomas. MAbs B72.3 and COL-4, reactive with the high molecular weight tumor-associated glycoprotein-72 (TAG-72) antigen and carcinoembryonic antigen (CEA), respectively, were labeled with horseradish peroxidase or alkaline phosphatase. Dual staining using the two MAbs on a single tissue section (formalin-fixed, paraffin-embedded) showed that greater numbers of carcinoma cells could be detected by using the combination of the two MAbs than could be detected by use of either MAb alone. In many tumors, some carcinoma cells reacted with MAb B72.3, some reacted with MAb COL-4, and some cells reacted with both MAbs. Only 1 of 10 carcinomas showed greater than 75% reactive cells when stained with each MAb individually. In 9 of 10 cases, however, greater than 75% of cells reacted when the combination of MAbs was used. Cell surface and cytoplasmic patterns of reactivity were observed with both MAbs while some pools of extracellular mucin were composed of both TAG-72 and CEA. This study supports the rationale for the use of a combination of anti-TAG-72 and anti-CEA MAbs for in vitro immunologic detection and potential in vivo immunodiagnostic and immunotherapeutic applications for these MAbs in colon adenocarcinoma patients.     

Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells

The reactivity of a panel of monoclonal antibodies (MAb) produced against non-T, non-B acute lymphoblastic leukemia cells was investigated by immunoperoxidase staining of sections of normal human kidney. The antigens of kidney reactive with the MAb were isolated by immunoaffinity chromatography and were purified further by immunoprecipitation. Two MAb, 44D7 and 44H9, reacted with determinants found exclusively on the basolateral membranes of proximal convoluted tubules. The 44D7 antigen isolated from kidney was biochemically similar to that isolated from leukemic cells. It was resolved as a multimeric complex with an apparent m.w. of 120,000 when analyzed by SDS-PAGE under nonreducing conditions. The 44H9 antigen has not yet been purified from kidney. MAb 50B4 reacted with components of the interstitium and with the mesangium of glomeruli. It immunoprecipitated a polypeptide chain of apparent m.w. 85,000, similar to that of the 50B4 antigen isolated from leukemic cells. MAb 44G4 also reacted with the mesangium of glomeruli and with the interstitium of the kidney. However, the endothelium of glomerular capillaries and of interstitial blood vessels has also reacted with MAb 44G4. The kidney antigen recognized by MAb 44G4 was characterized as a major polypeptide band, 95,000 m.w. (reduced) and 125,000 m.w. (nonreduced), a subunit structure analogous to the 44G4 antigen isolated from leukemic cells. MAb 44E3 reacted with all cellular elements of glomeruli, tubules, blood vessels, and interstitium. Two polypeptide chains of apparent m.w. 94,000 and 90,000 were immunoprecipitated from kidney by MAb 44E3, while a single polypeptide chain of 94,000 m.w. was precipitated from leukemic cells. Our results describe five new antigens with distinctive cellular distributions within kidney.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Immunohistochemical studies of colorectal carcinoma with a monoclonal antibody against carcinoembryonic antigen

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of colorectal carcinoma using a monoclonal antibody (MAb) against CEA. CEA has been demonstrated in 20 out of 22 rectum carcinomas (90.9%), in all of 23 colonic carcinomas, in none of 4 hyperplastic polyps and in 2 out of 6 adenomatous polyps (33.3%). CEA was found more often, and the intensity of the staining was stronger in well-differentiated carcinomas than in moderately and poorly differentiated carcinomas. No correlation was found between the presence of CEA in colorectal carcinoma and the stages of the disease. The mean values of serum CEA in patients with colorectal carcinoma and polyps with negative, weakly and strongly positive staining were 5.4 +/- 3.9 ng/ml, 28.3 +/- 23.8 ng/ml and 99.8 +/- 145.3 ng/ml respectively. Elevation of serum CEA occurred in 30 out of 39 (78.9%) cases with strongly positive CEA staining, in 4 out of 6 (66.7%) with weakly positive and in 1 out 9 (11.1%) with negative staining. A significant difference was found in serum CEA activity between the group with negative CEA staining and positive CEA staining (P less than 0.01). Our results suggest that the monoclonal antibody (MAb C27) can be used for the localization of CEA in conventionally prepared tissues of colorectal carcinomas by immunoperoxidase techniques for routine immunopathological diagnosis.     

S-antigen in rods and cones of the primate retina: different labeling patterns are revealed with antibodies directed against specific domains in the molecule.

S-antigen (arrestin) is a soluble 48 KD protein of the retinal photoreceptor cells. It has been found to have a function in regulation of the phototransduction cascade. Previous labeling experiments with anti-S-antigen (SAg) antibodies have yielded conflicting reports as to the presence of SAg in cone photoreceptor cells. In the present study we employed five monoclonal anti-SAg antibodies (MAb) directed against different known domains in the SAg molecule. MAb A9C6, D9F2, and C10C10 are directed against sequences in the carboxy half of the SAg molecule. MAb 5C6.47 and F4C1 are directed against the amino terminal. Immunoelectron microscopy was used in the localization of SAg in LR Gold-embedded baboon retinas. Green/red and blue cones were identified with MAb COS-1 and OS-2, respectively. MAb A9C6, D9F2, and C10C10 densely labeled rods and blue cones but not green/red cones. MAb 5C6.47 and F4C1 labeled rods and both blue and green/red cones. It appears that, in the baboon retina, different SAg molecules are present in the blue and green/red cones. Whereas the blue cone SAg shares common antigenic determinants with rods, both in the amino and carboxy terminals, the green/red cone SAg contains different antigenic determinants at the carboxy half of the molecule.     

Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: distinct mechanisms of action in vivo.

Monoclonal antibodies (MAb) reactive with the glycoprotein of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were used to protect mice against lethal infection. MAb which reacted with a number of distinct epitopes and which could neutralize the virus in vitro could also protect against infection in vivo. MAb which could not neutralize the virus in vitro but which were specific for the glycoprotein of a single serotype were also able to protect mice against lethal VSV challenge. Interestingly, a group of MAb which cross-reacted with the glycoproteins of VSV-Ind and VSV-NJ could passively protect against challenge with either serotype. It was shown that as early as 2 h after infection, neither neutralizing nor nonneutralizing MAb could protect. Nonneutralizing MAb were found to be less effective at in vivo protection than neutralizing MAb. Furthermore, nonneutralizing MAb demonstrated a much lower binding efficiency to intact virions than did neutralizing MAb. These observations, plus the fact that the nonneutralizing MAb could lyse virus-infected cells in the presence of complement, suggested that in vivo protection by these antibodies may involve cell-associated viral determinants. To compare the mechanisms by which neutralizing and nonneutralizing MAb protected in vivo, F(ab')2 fragments were used in protection experiments. Although the F(ab')2 of a neutralizing MAb was still able to protect animals lethal virus challenge, the F(ab')2 of a cross-reactive nonneutralizing MAb was unable to do so. The reactivity of nonneutralizing MAb with virions and the apparent necessity of an intact Fc portion for protection further distinguish these antibodies from those MAb that are able to neutralize VSV solely by binding to the glycoprotein.     

Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin

Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Purification and properties of Myxococcus xanthus cell surface antigen 1604.

A cell surface antigen complex from Zwittergent-solubilized Myxococcus xanthus has been purified by immunoaffinity chromatography with monoclonal antibody (MAb) 1604 and by subsequent gel filtration. We propose that the cell surface antigen (CSA) 1604 complex participates in intercellular interactions. The apparent total molecular mass of the CSA 1604 complex is 200 kilodaltons (kDa), as determined by gel filtration and by electrophoresis and Western immunoblot probing with MAb 1604. The antigen epitope recognized by MAb 1604 is on a 51-kDa polypeptide. The CSA complex also contains 14% neutral carbohydrate and a 23-kDa polypeptide that lacks the 1604 epitope. The carbohydrate is most likely part of a lipopolysaccharide (LPS) associated with the CSA, because an MAb recognizing an O antigen epitope from the LPS of M. xanthus also reacted with CSA 1604 on Western immunoblots. Thus, the 200-kDa CSA complex consists of 97 +/- 6 kDa of protein and many associated LPS molecules. The LPS evidently produces the multiplicity of bands observed on Western immunoblots between 100 and 200 kDa. The association with LPS may contribute to the negative charge of the CSA 1604 complex, which has a pI of 4.3. The CSA was clustered on the surface of intact M. xanthus cells after labeling with MAb 1604 and immunogold. Furthermore, fractionation studies indicated that cells grown on a plastic surface had 50% of their total CSA 1604 in the cytosol, 39% in the membrane fraction, and 8% in the periplasm. Saturable binding studies with 125I-MAb 1604 indicated that there were 2,400 CSA 1604 sites per cell. The Kd for MAb 1604 binding to the cell was 9 nM.     

Production and Characterization of Monoclonal Antibodies Specific to Closterovirus-like Particles Associated with Grapevine Leafroll Disease

Three hybridoma lines secreting monoclonal antibodies to a closterovirus-like particle (GLRV 3) associated with grapevine leafroll disease were produced. One of the antibody (MAbt) reacted with one extremity of the filamentous virus particle whereas the other two (MAb2 and 3) reacted with the entire surface of the virus particle. Using MAb2 in ELISA it is possible to detect 46 of the 50 GLRV-3 isolates. In order to detect all the 50 isolates, it is necessary to use MAb1 in ELISA in conjunction with polyclonal antibodies. In immunoblotting, the three MAbs recognized a 43 Kd viral protein.     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Immunohistochemical study of carcinoembryonic antigen (CEA) in normal and embryonic human tissues using a CEA-specific oncoprecipitin, krustacin

Using immunoperoxidase technique, antibodies to CEA (Ab) were compared to a glycoprotein krustacin (Kr) extracted from Pagurus prideauxii, which has an ability to precipitate specifically CEA. It was found that Kr and Ab reacted in a similar manner with embryonic and normal gastrointestinal tissues, revealing practically identical localization of the antigen in the tissues and cells. It was possible, however, to note some quantitative and qualitative differences in the distribution of antigen, which showed that Kr and Ab reacted with different determinants of the CEA molecule.