Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1

To gain a better understanding of the diversity of epitopes on a protein, the specificities of 103 monoclonal antibodies to a model antigen, horse cytochrome c(cyt c), were analyzed. The antibodies were generated in in vitro monoclonal, secondary antibody responses against horse cyt c coupled to hemocyanin in splenic fragment cultures. For this assay, horse cyt c-primed murine B lymphocytes were transferred to irradiated, hemocyanin-primed recipients. A panel of seven mammalian cyts c differing at one to six residues out of 104 and cyanogen bromide-cleaved fragments of horse cyt c containing residues 1-65, 1-80, and 66-104 was used to examine the specificities of the antibodies. Twenty-two distinct reactivity patterns were observed, even though the majority of the monoclonal antibodies were found to bind in the three previously identified antigenic regions of the molecule about residues 44-47, 60-62, and 89-92. The results indicate that each of the three antigenic regions consists of multiple overlapping epitopes. Few of the antibodies directed to any given antigenic region bound polypeptide fragments inclusive of the epitope sequences, demonstrating that some antibodies were more conformationally dependent than others. Only 13% of the antibodies bound to cyanogen bromide-cleaved polypeptide fragments that together encompassed the entire length of the protein. Considering the large number of antibodies analyzed and the reoccurrence of 13 of the 22 clonotypes in different lymphocyte donors, it is likely that the antibody specificities tabulated herein approach yet do not completely enumerate the total inventory of the horse cyt c-specific B cell repertoire. The remarkable diversity for epitope recognition within antigenic regions observed here is likely to pertain to protein antigens in general, and strongly supports the widely held notion that the entire surface of a protein is potentially antigenic. The restriction of the epitopes of horse cyt c to three antigenic regions where the amino acid sequences of the mammalian cyts c differ probably results from tolerance of the mice to their own cyt c.     

Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among two sites on the native protein

Antibody-secreting hybridoma cell lines produced from BALB/c mice that had been immunized with horse cytochrome c (cyt c) conjugated to hemocyanin yielded six hybridoma subclones that produced four monoclonal antibodies (mAb) with different patterns of cross-reactivity with a panel of evolutionarily variant cyt c. The recognition sites for three of these mAb lay in the same region of the intact molecule, because two of the mAb were sensitive to the amino acid residue present at sequence position 44, with one requiring threonine at position 47. The fourth mAb bound in another region of the molecule at a site that involves either residue 60 or residue 89. Synthetic peptides that included these residues did not react with these mAb, indicating that these sites may require interactions from noncontiguous regions of the molecule to bind antibody. The association constants for the interaction of the mAb with horse cyt c were very similar and of the order of 10(10) M-1. Specificity studies with anti-idiotypic sera and competition assays between mAb for binding to horse cyt c confirmed that the six positive hybridoma subclones produced from this fusion produced mAb that had one of these four distinct specificities. The idiotypes of these four mAb were serologically distinct, and were derived from Vh genes of the J558 family.     

Molecular characterization of serologic recognition sites in the human HLA-A2 molecule

The localization of the amino acid residues involved in the serologic specificity of the HLA-A2 molecule has been investigated using a combination of site-directed mutagenesis, DNA-mediated gene transfer, indirect immunofluorescence and flow cytometry techniques. Synthetic oligonucleotides were designed to introduce individual and combined amino acid substitutions in both the alpha 1 (positions 9, 43, and the highly polymorphic cluster of residues from aa 62 to 83) and alpha 2 (positions 107, 152, and 156) domains to investigate the effect of the specific mutation on the recognition of the molecule at the surface of transfected human and mouse cell lines by a panel of mAb that recognize monomorphic or polymorphic determinants in MHC class I molecules. At least three non-overlapping serologic epitopes were identified. Mutations in the highly polymorphic region at aa 62 to 66 completely eliminated binding of mAb MA2.1 (A2/B17 cross-reactive). Mutation at position 107 resulted in complete loss of binding of the A2/Aw69-specific mAb PA2.1 and MA2.2 and partial loss of mAb BB7.2 binding. The recognition by other allotypic mAbs was not affected by these mutations and they therefore represent at least a third serologic epitope. Mutations at positions 152 and 156, known to be important for T cell recognition, did not affect serologic recognition. Introduction of residues of HLA-B7 origin in the polymorphic segment spanning aa 70 to 80 created a molecule carrying the -Bw6 supertypic determinant as demonstrated by mAb SFR8-B6 binding.     

The specificity of human anti-cytochrome c autoantibodies that arise in autoimmune disease.

Cytochromes c (cyt c) are among the best characterized model Ag because their amino acid sequences and tertiary structures are well defined. One unique aspect of cyt c as an immunogen is its ability to induce autoantibody responses in animal models, although no pathology resulting from these responses has been reported. In this study, the presence and specificity of autoantibodies to cyt c were investigated in patients with SLE and related connective tissue diseases. Anti-cyt c antibodies were found in approximately 7% of patient sera and were statistically associated with the expression of antimitochondrial antibodies but were not statistically associated with any disease subset among those represented. Anti-cyt c was not associated with the presence of autoantibodies to DNA, histones, Ro, La, or Sm autoantigens. Most of the autoantibodies were specific for native or native-like forms of cyt c but antibodies to denatured forms were also apparent. Autoantibody binding was shown to be directed predominantly at selected sites of evolutionary variability within cyt c. The specificity of the human anti-cyt c autoantibodies appear to be similar to that of mouse anti-human cyt c antibodies and to autoantibodies elicited in mice against rat (mouse) cyt c.     

pH-Dependent interaction of cytochrome c with mitochondrial mimetic membranes: the role of an array of positively charged amino acids

The interaction of cytochrome c (cyt c) with mitochondrial mimetic vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and heart cardiolipin (PCPECL) was investigated over the 7.4-6.2 pH range by means of turbidimetry and photon correlation spectroscopy. In the presence of cyt c, the decrease of pH induced an increase in vesicle turbidity and mean diameter resulting from vesicle fusion as determined by a rapid decrease in the excimer/monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidylcholine (PyPC). N-acetylated cyt c and protamine, a positively charged protein, increased vesicle turbidity in a pH-independent manner, whereas albumin did not affect PCPECL vesicle turbidity. pH-dependent turbidity kinetics revealed a role for cyt c-ionizable groups with a pK(a)((app)) of approximately 7.0. The carbethoxylation of these groups by diethylpyrocarbonate prevented cyt c-induced vesicle fusion, although cyt c association to vesicles remained unaffected. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that Lys-22, Lys-27, His-33, and Lys-87 cyt c residues were the main targets for carbethoxylation performed at low pH values (<7.5). In fact, these amino acid residues belong to clusters of positively charged amino acids that lower the pK(a). Thus, at low pH, protonation of these invariant and highly conserved amino acid residues produced a second positively charged region opposite to the Lys-72 and Lys-73 region in the cyt c structure. These two opposing sites allowed two vesicles to be brought together by the same cyt c molecule for fusion. Therefore, a novel pH-dependent site associating cyt c to mitochondrial mimetic membranes was established in this study.     

Antigenic analysis of testicular cytochromes c using monoclonal antibodies

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.     

The T lymphocyte response to cytochrome c. IV. Distinguishable sites on a peptide antigen which affect antigenic strength and memory

The murine T cell proliferative response to the carboxyl terminal cyanogen bromide cleavage fragment 81-104 of pigeon cytochrome c (cyt) has been studied. Two interesting properties of this response have been previously described. First, T cells from B10.A mice primed with pigeon cyt 81-104 show more vigorous proliferation when restimulated with moth cyt 81-103 than when stimulated with pigeon cyt 81-104; that is, the B10.A T cell response to pigeon shows heteroclitic restimulation by moth. Second, T cells primed with the acetimidyl derivative (Am) of pigeon cyt 81-104 did not cross-react with the unmodified cyt fragments, but Am-moth cyt 81-103 still stimulated Am-pigeon cyt 81-104 primed T cells better than the Am-pigeon cyt 81-104 fragment. These results raised the issue of whether the antigenic sites on the fragments responsible for the specificity of T cell priming in vivo differed from the residues that contributed to the heteroclitic response of pigeon (or Am pigeon)-primed T cells to moth cyt c fragments. In this paper, synthetic peptide antigens were tested in order to identify which residues caused the heterocliticity of the moth fragment and which residues were involved in the antigenic differentiation of native and derivatized fragments. The heterocliticity of the T cell response to moth fragment 81-103 was found to be due to the deletion of the penultimate residue (Ala103) from the pigeon fragment. However, the ability to cause heterocliticity was not uniquely a property of this deletion. T cells from animals primed with peptides containing substitutions at positions 100 or 102 were also heteroclitically stimulated by the moth-like antigen. The observation that T cells could not be primed for recognition of the changes in peptide sequence that caused heteroclitic stimulation suggests that T cells do not directly recognize determinants in this region. The antigenically significant site of derivatization for T cell priming was found to be Lys99. Furthermore, substitution of a Gln at position 99 also resulted in elicitation of yet a third set of T cell clones specific for the presence of that residue. That is, the specificity of the primed T cell population was found to be altered by changes at residue-99, but no such alterations in specificity were demonstrable when T cells primed with peptides altered at residue-103, residue-102, or residue-100 were compared. Overall, the results demonstrate that the antigen can be divided into two functionally distinct sites that are in close physical proximity.     

Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme.

We tested 49 BALB/c antilysozyme mAb from seven intervals during the immune response to lysozyme for patterns of specificity and avidity. We found that the antibody epitopes in composite covered at least 80% of the lysozyme surface, and their patterns of overlap suggest a continuum of potential antibody epitopes. Previously observed regional specificities, which emerged at different times in the immune response, were more discretely defined in late response antibodies, when the majority of mAb could be assigned to one of three functionally nonoverlapping complementation groups. The area covered by each antigenic region may be greater than an individual epitope, and may include multiple epitopes that overlap structurally and functionally to varying degrees. Connectivity between antigenic regions was seen in interactions among early and late stage antibodies, and among secondary stage mAb, but not among tertiary stage mAb from hyperimmunized mice. Patterns of overlap of early and late response antibodies suggest that the organization of antibody specificities change during the progression from primary to secondary to tertiary response. Over the same period in the response, the average relative avidity of IgG1 kappa mAb did not increase, suggesting that "affinity maturation" of serum antibodies reflects an increase in the number and diversity of antibodies, rather than an overall increase in the avidity of individual antibodies.     

Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad. Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position. To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2). The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p. The remaining mutant signal peptides are cleaved inefficiently or not at all. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Diverse VH and VL genes are used to produce antibodies against a defined protein epitope

mAb secreting hybridomas were produced from mice hyperimmune to the model Ag tobacco mosaic virus protein. Six mAb were selected for their ability to bind synthetic peptides corresponding to amino acid residues 103-112 and 97-107 of tobacco mosaic virus protein. These mAb were analyzed for their fine specificity by measuring binding to synthetic analogs of the decapeptide, and cDNA sequences encoding the mAb V regions were determined. These analyses revealed that a wide range of different V regions are capable of binding with the same decapeptide epitope, and that these antibody sequence differences generally coincided with different binding fine specificities. This diverse antibody response with specificity for the same epitope demonstrates both the breadth of potential of the immune system and the lack of exclusivity in specific protein:protein interactions.     

Comparison of base specificity and other enzymatic properties of two protozoan ribonucleases from Physarum polycephalum and Dictyostelium discoideum

Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.     

Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.     

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.     

Topographic determinants on cytochrome c. I. The complete antigenic structures of rabbit, mouse, and guanaco cytochromes c in rabbits and mice1.

Rabbit, mouse, and guanaco cytochromes c differ from each other by only two amino acid residues. The identification is described of all of the antigenic determinants of mouse and guanaco cytochrome c that elicit an antibody response in rabbits, and those of the rabbit and guanaco proteins that elicity antibodies in the mouse. All except one of these sites center around single amino acid residue differences between the antigen and the host cytochrome c. The corresponding antibody popylations bind only to the areas of the protein in which the substitutions occur. Such antigenic determinants manifested in rabbits by quanaco and mouse cytochromes c are centered around residues 62 and 89, and residues 44 and 89, respectively. Similarly, the mouse recognizes sites containing residues 44 and 62 in guanaco cytochrome c, and residues 44 and 89 in rabbit cytochrome c. In none of these instances has a change in sequence failed to produce an antibody response. Each of these determinants appears to elicit and bind to its antibody, independently of other determinants present on the protein. In addition, two different autoantigenic responses have been detected. The antibodies produced against the determinant formed by glutamyl residue 62 of the guanaco protein in both rabbits and mice, the cytochromes c of which carry an aspartyl residue in that position, also bind to the aspartyl-containing region but with lower affinity. However, mouse and rabbit cytochrome c also elicit antibodies to the area of residue 62 in rabbits and mice, respectively, and these antibodies still bind more strongly to the glutamyl-than to the aspartyl-containing determinant. This last response occurs only when there are residue substitutions elsewhere in the molecule, because mice and rabbits fail to respond to their own cytochrome c. Antibodies produced in mice against the change from alanyl to valyl residue 44 by rabbit and guanaco cytochromes c also bind to the alanyl-containing determinant, except less tightly than to the valyl region. Conversely, antibodies raised in rabbits against the change from valyl to alanyl residue 44 only bind to this region when it carries an alanine. It is suggested that antigenic determinants that arise as a result of amino acid residue substitutions between the immunizing and the corresponding host protein, without a change in the spatial arrangement of the polypeptide backbone, be termed topographic determinants.     

Comparing substrate specificity between cytochrome c maturation and cytochrome c heme lyase systems for cytochrome c biogenesis

Hemes c are characterized by their covalent attachment to a polypeptide via a widely conserved CXXCH motif. There are multiple biological systems that facilitate heme c biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems. System III, cytochrome c heme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophilus cytochrome c(552) variants was successfully expressed and matured by the CCM system with CX(n)CH motifs where n = 1-4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1-3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemes c with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.     

The amino acid sequence of zinc-carboxypeptidase from Streptomyces griseus

The amino acid sequence of a zinc-carboxypeptidase from S. griseus (Cpase SG) was determined by automated Edman degradation and carboxypeptidase digestion of the S-carboxymethylated protein and by sequence analyses of peptides produced by cyanogen bromide cleavage and by lysyl endopeptidase digestion of the S-carboxymethylated protein. This enzyme is characterized by a uniquely broad substrate specificity which combines the specificities of mammalian Cpase A and Cpase B (J. Biochem. 86, 683-694, 1979). Cpase SG consists of 328 amino acid residues. The amino acid sequence of Cpase SG is partially similar to those of bovine Cpase A and Cpase B (sequence identity, 28-29%). In the sequence of Cpase SG, residues that are functionally important in mammalian Cpase A and Cpase B were all found at the corresponding positions. Residue 255 (according to the numbering system for bovine Cpase A), which, in the other Cpases, contributes to the difference in specificity between Cpase A (Ile-255) and Cpase B (Asp-255), was Asp. However, residue 254 was Ile, in contrast to Ser or Thr in all of the forms of Cpase A and Cpase B examined to date. The increase in hydrophobicity caused by the change at position 254 and the presence of negative charge at position 255 is probably one of the reasons for the broad substrate specificity of Cpase SG.     

Antigenic and size differences between somatic and testicular cytochromes c

Enzyme-linked immunosorbent assays (ELISA) of somatic (cs) and testicular (ct) cytochromes of rat, mouse, rabbit, and beef with rabbit anti-rat cyt ct antibody exhibited two different antigenic profiles, indicating the presence of two different antigenic structure between cyt ct and cs. SDS-polyacrylamide gel electrophoresis of cyt c showed that the molecular size of rat, rabbit and beef cyt ct is slightly smaller than that of their cyt cs. However, the electrophoretic mobility of mouse cyt ct is almost identical to that of mouse cyt cs, but slightly slower than that of rat cyt ct. These results indicate that mouse and rat cyt ct are different despite the identical amino acid sequences for both rat and mouse cyt cs.     

活化相关分泌蛋白1单克隆抗体的制备及其在保守结构域鉴定中的应用

目的:制备重组活化相关分泌蛋白1(ASP-1)的单克隆抗体,并用其鉴定保守结构域。方法:用原核表达并纯化的重组ASP-1不加佐剂免疫BALB/c小鼠,采用杂交瘤技术及有限稀释传代法筛选稳定分泌特异性抗体的杂交瘤细胞株,制备单抗腹水后用间接ELISA进行抗体特异性鉴定和效价检测,利用肽结合ELISA和Western印迹鉴定单抗识别的保守结构域。结果:获得5株能稳定分泌抗ASP-1单克隆抗体的杂交瘤细胞株,且5株单抗的识别区域均为21~28氨基酸残基的保守性结构域。结论:制备了抗ASP-1的单克隆抗体,为深入研究ASP-1佐剂的活性功能区及作用机制提供了有效工具。