Influence of lipid environment on insulin binding in cultured hepatoma cells

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.     

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys)

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.     

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein

A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains.     

Apolipoprotein D and cross-reacting human plasma apolipoproteins identified using monoclonal antibodies

We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.     

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.     

Tangier disease apolipoprotein A-I compared with normal plasma A-I using monoclonal antibodies

The molecular defect in Tangier disease is unknown. We have compared the electrophoretic and immunoreactive properties of Tangier disease and normal apolipoprotein A-I using four monoclonal antibodies. We verified that the molecular weight, pI and CNBr-cleaved fragments of Tangier disease and normal apolipoprotein A-I were not different, excluding the possibility that dimers, aggregates or fragments of apolipoprotein A-I could be responsible for its rapid catabolism in this disease.     

Detection of immunological heterogeneity of an isolated, purified protein (vervet apolipoprotein A-I)

Using a single goat antiserum, we have identified immunological heterogeneity of purified apolipoprotein A-I from high density lipoprotein of vervet monkeys. We examined whether the apparent heterogeneity was due to separate antigenic sites within the polypeptide sequence or rather on the different isoproteins, which result in charge heterogeneity of this protein. The apolipoprotein A-I was cleaved with cyanogen bromide and the resulting three fragments were purified and characterized. By using immunodiffusion, each of the fragments was found to show a characteristic and different reaction to the antiserum. By contrast, apparent identity was found by immunodiffusion among the separate isoprotein forms of apolipoprotein A-I. We have concluded that the immunological heterogeneity of apolipoprotein A-I was due to different antigenic sites within the primary sequence of apolipoprotein A-I.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.     

Characterization of antigenic determinants on human solubilized apolipoprotein B. Conformational requirements for lipids

The expression of low density lipoprotein (LDL) antigenic determinants in the delipidated and solubilized apolipoprotein B (apo-B) free of sodium dodecyl sulfate (SDS) has been studied. Of the six distinct determinants which react with previously characterized monoclonal antibodies against LDL (Milne, R.W., Theolis , R., Jr., Verdery , R.B., and Marcel , Y.L. (1983) Arteriosclerosis 3, 23-30), only one, that recognized by antibody 1D1 , was expressed on the soluble apo-B, indicating that soluble apo-B may be partly denatured. The average immunoreactivity of apo-B with antibody 1D1 was similar to or lower than that of intact LDL (mean 36%, range 93-20%). Therefore, delipidation and solubilization did not expose on apo-B any additional site reactive with 1D1 . When apo-B was equilibrated with either SDS micelles or with cholesterol-lecithin liposomes, the immunoreactivity of the determinant recognized by antibody 2D8 was partially regenerated, but not that of the others. In contrast, incubation of apo-B with microemulsions containing a hydrophobic core of cholesteryl esters also restored the antigenicity of the determinants reacting with antibodies 3F5 , 4G3 , and 5E11 . However, the regeneration of these antigenic determinants could only be achieved when solubilized apo-B was treated with SDS prior to equilibration with microemulsion preparations. In conclusion, three types of antigenic determinants have been identified on apo-B. The first type, such as that recognized by antibody 1D1 , is expressed both on LDL and on apo-B and is constituted by the primary and secondary structure of apo-B. The second type, an example being that recognized by 2D8 , is a conformational determinant which requires the presence of amphipathic lipids such as lecithin and cholesterol or SDS micelles. The third type, which reacts with antibodies 3F5 , 4G3 , and 5E11 , represents different conformational determinants which require the association of apo-B with lipid structures having a cholesteryl ester hydrophobic core. It may be significant that the latter determinants are those close to the LDL receptor-binding site on apo-B and that this domain of apo-B has a complex tertiary and quaternary structure as evidenced by the conformational requirements of the antigenic determinants.     

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

Monoclonal antibodies recognizing different epitopes of the 27-kDa gap junction protein from rat liver

Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.     

Distribution of Epitopes of Trypanosoma cruzi Amastigotes During the Intracellular Life Cycle within Mammalian Cells

ABSTRACT. In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intraceullar life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein—Ssp-4 defined by MAB 2C2 [5]: MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellared forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intraceullular proliferation of parasites, and processed for immjno-electron microscopy and confocal immunoflurescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrance-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.     

Identification of two antigenic epitopes on SARS-CoV spike protein

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1. The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. A number of monoclonal antibodies were developed against the fusion protein. In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively. Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively. Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.     

Variable cross-reactivity of Pseudomonas aeruginosa lipopolysaccharide-code-specific monoclonal antibodies and its possible relationship with serotype.

The core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) was analysed by four LPS-core-specific human monoclonal antibodies (mAbs; FK-2E7, MH-4H7, OM-1D6 and NM-3G8). Reactivity of these mAbs to about 180 P. aeruginosa strains was tested. FK-2E7 bound to strains of Homma serotype E and I at a frequency of about 90%, to strains of serotype M at about 50%, and to strains of serotype A and G at about 30%. MH-4H7 bound to P. aeruginosa strains of serotype A, F, G, H, K and M at a high frequency (45-87%), but did not bind to any strains of serotype B, C, E and I. OM-1D6 and NM-3G8 bound to P. aeruginosa strains in a nearly serotype-specific manner. OM-1D6 reacted with all strains of serotype G so far tested, and a few strains of serotype M. Furthermore, L-rhamnose in the LPS core of serotype G was an immunodominant sugar recognized by OM-1D6 as an epitope. NM-3G8 bound to only a few strains of serotype B and M. The variable reactivity of these mAbs suggests that antigenic heterogeneity of the LPS core is somewhat related with (O-polysaccharide-based) serotype. Among these mAbs, MH-4H7 and OM-1D6 showed a high level of protective activity against P. aeruginosa in an experimental infection model using normal mice. In vivo protective activity was shown to be closely related to in vitro binding activity to whole cells as determined by agglutination and flow cytometry, but not ELISA.     

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.     

Modulation of apolipoprotein B antigenic determinants in human low density lipoprotein subclasses

To investigate the effect of low density lipoprotein (LDL) heterogeneity on the conformation of LDL apolipoprotein B (apo-B), the immunoreactivities of 6 monoclonal antibodies against LDL apo-B were measured in 3 LDL subfractions isolated by equilibrium density gradient ultracentrifugation. To ensure a broad range of LDL particles, the LDL subfractions were prepared from normal subjects and patients with hyperapobetalipoproteinemia. With 3 of the antibodies, 1D1, 5E11, and 3A10, LDL fractions 1 (the most buoyant), 2 (the intermediate), and 3 (the densest) were equally immunoreactive and competed similarly with reference whole LDL. In contrast, with 3 other antibodies, 2D8, 3F5, and 4G3, fraction 1 was significantly more reactive than fraction 3; that is for each in turn, 290, 250, and 150% more of the densest LDL protein was required to achieve the same displacement as with fraction 1. Further, the immunoreactivities of the 3 LDL fractions with antibodies 2D8, 3F5, and 4G3 were negatively correlated with their LDL cholesterol to LDL protein ratio with r values of 0.727, 0.898, and 0.870, respectively, suggesting that as LDL particle size decreases, the conformation of the LDL apo-B changes progressively. It is of interest that the antigenic determinants recognized by 3F5 and 4G3 are close to the LDL receptor recognition site on LDL apo-B. Therefore, it is possible that the reduced immunoreactivity of these determinants in dense LDL may be the in vitro correlate of the reduced fractional catabolics rate of dense LDL compared to buoyant LDL previously observed in vivo.