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1.
Plant expression of chicken secretory antibodies derived from combinatorial libraries 总被引:4,自引:0,他引:4
Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes. 相似文献
2.
A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs. 相似文献
3.
A Grubb E Méndez J L Fernandez-Luna C López E Mihaesco J P Vaerman 《The Journal of biological chemistry》1986,261(30):14313-14320
Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit. 相似文献
4.
Vu DH Schneider P Tissot JD 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,771(1-2):355-368
Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible. 相似文献
5.
A Garcia-Pardo M E Lamm A G Plaut B Frangione 《The Journal of biological chemistry》1981,256(22):11734-11738
Previous work has established that the secretory component (SC) in human secretory IgA is covalently linked to only one of the two IgA monomer subunits, but it has not been clear whether the J chain is covalently linked to one or to both of these subunits. In view of the asymmetry in the disulfide bonding between SC and the IgA subunits, an arrangement which follows disulfide interchange, several models for the disulfide linkage of J chain and the bonds between IgA subunits were envisaged and investigated. When sIgA was gel filtered through Sephadex G-200 in acetic acid, a single major symmetrical peak eluted at the front. This material contained SC, alpha and L chains, and all of the J chain. The greater resolution afforded by polyacrylamide gel electrophoresis in detergent confirmed that human sIgA contains no major noncovalently linked components in the 150,000-200,000 molecular weight range. In another series of experiments the Fc monomer, which is not covalently attached to SC, isolated after treatment of sIgA with IgA protease and cyanogen bromide, was investigated to learn whether alpha chain COOH-terminal octapeptides could be released by reduction. The results were negative. The available data thus favor a model in which J chain is disulfide-bonded to both IgA monomer subunits in sIgA. 相似文献
6.
Local immunity was evaluated in 47 patients affected by recidivant vaginal candidiasis and 33 control women. IgG, IgA, IgM and secretory component (SC) were determined by single radial immunodiffusion in samples of cervicovaginal secretion. IgG in dosable levels was detected in 17/47 samples (36.2%) and IgA in 15/47 patients (31.9%) whereas in the controls, the incidence was 31/33 (93.9%) for IgG and 24/33 (72.7%) for IgA. The difference was significative (P< 0.001) for both immunoglobulins. Significant differences were not obtained for IgM. The SC was detected in 4/47 cervicovaginal secretions of patients affected by candidiasis (8.5%) whereas in the control samples the incidence was 21/33 (63.6%) (P<0.001). In only 2/15 patients with dosable levels of IgA (13%) the secretory nature of this immunoglobulin could be shown by its reaction with anti-SC serum. In the control group, secretory IgA was detected in 19/24 cases (79%) (P< 0.001). Serum immunoglobulins levels were normal. The lack of secretory IgA and SC in the secretion could be related to the adherence capacity of the Candida albicans to epithelial cells. 相似文献
7.
B G Sanders W L Case 《Comparative biochemistry and physiology. B, Comparative biochemistry》1977,56(3):273-278
1. Chicken IgA purified from biliary fluids was chemically and immunologically characterized. 2. Chicken IgA was determined to be the only immunoglobulin class present in bile. Gel filtration studies reveal polymeric IgA e.g. 17-19S. 3. Antigenically, chicken IgA is distinct from chicken IgG, and IgM. 4. Chicken IgA does not show antigenic homology to human IgA. 5. SDS poly-acrylamide gel electrophoresis revealed IgA to possess heavy chains of 60,000 and light chains of 24,000 mol. wt, respectively. 6. Peptide mapping of tryptic digests of chicken alpha chains reveals approximately 35 peptides. The peptide map pattern is distinct from chicken gamma chains. 相似文献
8.
We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE–SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE–SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP–HPLC–TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains. 相似文献
9.
A sensitive solid phase radioimmunoassay method was established for the specific quantitative determination of secretory IgA (sIgA) by taking advantage of the dual antigenicities of sIgA, one specific for alpha-chain and the other for secretory component (SC). The sIgA and IgA in the sample were first bound by anti-IgA antibodies coated on the polystyrene tube, then the amount of bound sIgA was quantified by the use of 125I-labeled anti-SC antibodies. This method is quite sensitive and allows us to distinguish sIgA from IgA and free SC which usually coexist in exocrine secretions. Linear relationship was observed between the bound radioactivity of radioiodinated anti-SC and the amount of sIgA in the range of 5 to 60 ng of sIgA. With this method, the urinary sIgA levels in normal children and adults were measured. Urinary sIgA was detected in half of infants within 7 days after birth, while it could be demonstrated in almost all of the infants by 14th day. Then, it gradually increased and reached about a half of the adult level by the age of puberty. This method will be useful for the sensitive and specific measurement of sIgA in various exocrine secretions. 相似文献
10.
P E McKeever M E Neiders G B Nero R Asofsky 《Journal of immunology (Baltimore, Md. : 1950)》1979,122(5):1972-1977
The antigenic and molecular characteristics of the two immunoglobulins secreted by a single cell line of plasmacytoma SAMM 368 were analyzed by polyacrylamide gel electrophoresis of biosynthesized proteins. Adapted to continuous in vitro cultivation, this BALB/c plasmacytoma secretes at least 98% of its heavy chains as components of fully assembled and isotypically uniform IgG2b and IgA molecules. The IgA is secreted as monomers, dimers, and multimers with chemical properties typical of BALB/c myeloma IgA including disulfide bonded J chain and noncovalently bonded light chains. The noncovalently bonded light chains are monomers rather than dimers. Free light chains are also secreted. The ability to segregate heavy chains is attributed either to chemical, enzymatic, or compartmental regulatory factors operating within these plasma cells. 相似文献
11.
W F Riesen 《Journal of immunology (Baltimore, Md. : 1950)》1976,117(2):518-522
Recombination experiments were performed with heavy and light chains derived from a Waldenstr?m's IgM with specificity against phosphorylcholine. The recombinant molecules had an association constant for phosphorylcholine in the same order of magnitude as the native IgM; the number of binding sites at saturation was only slightly decreased in the reconstituted molecules, indicating regain of binding activity after recombination of IgM heavy and light chains. Heterologous recombinants obtained with polypeptide chains of another monoclonal IgM without demonstrable binding activity recovered only 5 to 10% of the binding activity of homologous recombinants. Hybrid molecules prepared with heavy and light chains from the phosphorylcholine-binding mouse IgA myeloma protein TEPC-15, however, regained as much as 41% of the binding activity of the homologous recombinants; these data suggest a considerable degree of structural homology shared by the human IgM and the murine IgA proteins with phosphorylcholine-binding specifity. 相似文献
12.
Butler JE 《Biochimica et biophysica acta》1971,251(3):435-449
1. Bovine secretory IgA (SIgA) from colostrum (mol. wt. about 410,000) is composed of four alpha-chains (mol. wt. 61,000), four light chains (mol. wt. 23,000) and one molecule of glycoprotein-a (mol. wt. 70,000-86,000). The alpha-chains are antigenically and physicochemically distinct from the heavy chains of IgM, IgG1 and IgG2 while the light chains are identical to those occurring on other bovine immunoglobulins. Glycoprotein-a and bovine free secretory component are identical and the former name should be abolished. Much of this protein is covalently bonded to IgA. 2. The gel filtration behavior of serum IgA suggests it is a dimer. 3. The elution behavior of IgA and SIgA from ion-exchange columns and the solubility characteristic of SIgA in the presence of Zn2+ are similar to those of human and rabbit IgA. 4. The disc electrophoretic behavior of IgA and SIgA are distinct from IgM, dimeric IgG1, 7-S IgG and glycoprotein-a. Dimeric IgG1 (s20,w = 9.5) is abundant in colostrum and is similar in size to SIgA. 5. Bovine IgA shows physicochemical and immunochemical heterogeneity when studied by gel filtration, disc electrophoresis, immunoelectrophoresis and ultracentrifugational analyses. Lacrimal and nasal SIgA possess antigenic determinants absent on colostral SIgA. 相似文献
13.
Kang SH Shin GW Palaksha KJ Shin YS Kim YR Lee EY Suh EH Huh NE Oh MJ Jung TS 《Fish & shellfish immunology》2006,20(3):295-304
The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish. 相似文献
14.
Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces. 相似文献
15.
16.
A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000–350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12–14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000–122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000–24,000) cosediment with the 21 S peak. The heavy chain composition of the 12–14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes. 相似文献
17.
Frigerio L Vine ND Pedrazzini E Hein MB Wang F Ma JK Vitale A 《Plant physiology》2000,123(4):1483-1494
Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted. 相似文献
18.
The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue. 相似文献
19.
Chumsae C Gaza-Bulseco G Sun J Liu H 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,850(1-2):285-294
Methionine (Met) oxidation is a major degradation pathway of protein therapeutics. Met oxidation of a fully human recombinant monoclonal antibody was investigated under both chemically stressed conditions using tert-butylhydroperoxide (tBHP) and thermal stability conditions where the sample was incubated in formulation buffer at 25 degrees C for 12 months. This antibody has one Met residue on each of the light chains and four Met residues on each of the heavy chains. In the thermal stability sample, only Met residues 256 and 432 in the Fc region were oxidized to form methionine sulfoxide, while Met residues in the Fab region were relatively stable. The susceptibility of Met residues 256 and 432 was further confirmed by incubating samples with tBHP, which has been shown to induce Met oxidation. Further analysis revealed that the susceptible Met residues of each heavy chain were randomly oxidized in samples incubated with tBHP, while in the thermal stability sample, the susceptible Met residues of one heavy chain were preferentially oxidized. 相似文献
20.
The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire. 相似文献