Immunochemical Cross-reactions Research Articles

Immunological study of muscle parvalbumin isotypes in three African catfish during development

Eleven parvalbumin isotypes expressed during the development of clariids Heterobranchus longifilis and Clarias gariepinus and claroteid C hrysichthys auratus were purified and electrophoresed on sodium-dodecyl-sulfate polyacrylamide gels. Immunochemical cross-reactions among these proteins were investigated by immunoblotting, using purified antibodies raised against three isotypes chosen at different stages of fish development. Antibodies raised against H. longifilis PA I (larval–juvenile isotype) and against C. gariepinus PA IIIa (juvenile–adult isotype) cross-reacted to a rather similar extent despite a weaker cross-reaction of anti-PA IIIa with larval–juvenile isotypes. On the other hand, antibodies raised against H. longifilis PA IV (an exclusively adult isotype) recognized markedly only H. longifilis PA IV and C. gariepinus PA IIIb. These two adult isotypes most likely belong to the α lineage, and all the others to the β lineage. These results show that parvalbumin isotypes synthesized at different stages of fish growth differ structurally, and that the most marked difference is between larval–juvenile and adult clariid isotypes.

Purification and Characterization of Porcine Pepsinogen B and Pepsin B

Porcine pepsinogen B was prepared from extracts of adult porcine fundic mucosa. Immunoelectrophoresis showed no immunochemical cross-reactions between pepsinogen B and other porcine gastric zymogens. Pepsin B was purified after activation of the zymogen. The enzyme showed an optimum of general proteolytic activity at pH 3.0. Activation of pepsinogen B at pH 2 resulted in formation of the covalent intermediate (pseudo-pepsin B) by proteolytic cleavage of bond Met16p-Glu17p (pig pepsinogen A numbering, "p" indicates residues of the prosegment peptide). Pseudopepsin B was stable at pH 2. The intermediate was converted to pepsin B at pH 5.5. The overall activation of pepsinogen B was much slower than found for other investigated gastric zymogens. During the conversion of pepsinogen B to mature pepsin B a segment of 43 amino acid residues was cleaved from the N-terminal of pepsinogen B. The amino acid sequence of the prosegment and the first 24 residues of pepsin B was determined. Relative to porcine pepsinogen A, progastricsin, and prochymosin, the following degrees of identities were observed: 40, 55, and 51%.

Immunochemical cross-reactivity of neurotoxic phospholipase A 2 enzymes from Indian cobra ( Naja naja naja) venom using polyclonal antibodies

B. S. Basavarajappa, S. C. Guru and T. V. Gowda. Immunochemical cross-reactivity of neurotoxic phospholipase A 2 enzymes from Indian cobra ( Naja naja naja) venom using polyclonal antibodies. Toxicon 31, 1167–1177, 1993.—Rabbit antibodies were prepared against purified phospholipase A 2 (NN-XI a-PLA 2) from Indian cobra ( Naja naja naja) venom. The PLA 2 has haemolytic, neurotoxic, myotoxic, cytotoxic and oedema-inducing activities apart from the catalytic activity. The immunological cross-reactivity of structurally similar neurotoxic PLA 2s was investigated using enzyme-linked immunosorbent assay (ELISA) and immunodiffusion. Anti-NN-XI a-PLA 2 IgG cross-reacted with other purified neurotoxic PLA 2s from the same venom. Immunochemical cross-reactions of anti-NN-XI a-PLA 2 IgG with NN-XI a-PLA 2, NN-XI b-PLA 2, NN-XIII-PLA 2, NN-IV bl-PLA 2 and NN-V b-PLA 2 were shown by a very high ELISA titre and a single precipitin band on double immunodiffusion agarose plates. The catalytic activity of these PLA 2s was inhibited dose-dependently by anti-NN-XI a-PLA 2 IgG but was unable to neutralize lethality and neurotoxic symptoms in experimental animals injected with neurotoxic PLA 2. Anti-NN-XI a-PLA 2 IgG fails to neutralize myotoxicity and oedema-inducing activities of NN-XI a-PLA 2 and NN-XIII-PLA 2. Anti-NN-XI a-PLA 2 IgG inhibited cytotoxic effects of NN-XI a-PLA 2 dose-dependently, but failed to inhibit NN-XIII-PLA 2-induced cytotoxicity. Direct haemolytic activity of NN-XI a-PLA 2 and NN-XIII-PLA 2 was inhibited dose-dependently by these antibodies. The results indicate the presence of separate catalytic and pharmacologic site(s).

Cytochrome P450 in seals: monooxygenase activities, immunochemical cross-reactions and response to phenobarbital treatment

Cytochrome P450 monooxygenase activities, reflecting the expression of various subfamilies of P450, were measured in liver samples of harp and hooded seal. Differences in some of the investigated parameters were observed between the two species, between sexes, and between pups and adults. Treatment of single female pups from each species with phenobarbital (i.v. 45 mg/kg), resulted in increased levels of EROD and estradiol 2-hydroxylase activities in both species, whereas MCOD, ECOD and PROD activities were induced only in the harp seal sample. Antibodies against a dog P450 2B form (anti-dog PBD-2 IgG) gave a single band around 52kD in both species, strongest in male pups. This band seemed elevated in the PB-treated harp seal pup. Based on the single treated pup of each species, the results suggest that seals respond to PB-type treatment with a weaker response, and with different enzyme patterns, that most terrestrial mammals. Antibodies against cod P450 IAI cross-reacted with two bands in liver samples from adult seals of both species (about 54 and 52kD), but this was strongest in the hooded seal. The intensity of the bands reflected the EROD activities in the samples, suggesting the application of immunodetection in screening marine mammals for effects of environmental contamination.

ELISA measurement of immunochemical cross-reactions among Lathyrus lectins as an assessment of their phylogenetical relationship

Abstract The immunochemical cross-reactions among Lathyrus and other Vicieae lectins have been quantified by using an ELISA (enzyme-linked immunosorbent assay) technique, in order to assess their phylogenetical relationship. The data are consistent with those arising from the comparison of both the amino acid compositions of the lectins and the amino acid sequences of their light and heavy subunits. They confirm that two-chain lectins from the tribe of Vicieae are phylogenetically closely related.

Pre- α2-elastase inhibitor of the horse; a hybrid molecule between α1-proteinase inhibitor and α2- β1-glycoprotein

Pre- α 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of α 1-proteinase inhibitor and α 1- β 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free α 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre- α 1-elastase inhibitor and antibodies to α 2- β 1-glycoprotein as well as antibodies to α 1-proteinase inhibitor. Pre- α 2-elastase inhibitor could be dissociated into its parent components by dithiothreitol. After dissociation, the α 1-proteinase inhibitor portion retained its biological activity.

Studies of ribosomal proteins of yeast species and their hybrids: gel electrophoresis and immunochemical cross-reactions.

The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit. Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970). Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species. Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents. These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in eucaryotes.

Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae.

Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis.

Immunochemistry of serum albumin: VI. A dynamic approach to the immunochemical cross-reactions of proteins using serum albumins from various species as models

Antisera against bovine serum albumin were raised in two rabbits. Serial bleedings were obtained at different times after the first immunization, and antisera from these serial bleedings were not mixed but were kept and studied separately. The immunochemical cross-reactions of these antisera with serum albumins from bovine, goat, sheep, porcine, horse, human and chicken were determined by immunoadsorbent studies. These were done by titration so that the values of maximum (plateau) binding by each albumin of radioiodinated antibodies were determined. In each rabbit, the immunochemical cross-reactivity was not static but increased progressievly with time after the first immunization. In the interval 7 days to 398 days the increases in cross-reaction were extremely large. pH dissociation studies revealed that, together with the increase in cross-reactivity of a given albumin with time after immunization, there was a restriction in the antibody heterogeneity towards populations possessing higher affinity. These results provide a rational explanation for the different values of cross-reactivities for a given albumin from different laboratories. The findings are analyzed in relation to the antigenic structure of albumin and their significance in evolutionary studies discussed.

Specific antibodies against alpha-melanotropin for radioimmunoassay. Parallelism between the immunochemical cross-reactions of melanotropin structural analogues and their biological activity.

The purpose of this experimental investigation was to provide antibodies against α-melanotropin with a high serum titer and with great specificity. To this end, rabbits were immunised with a complex-antigen consisting of human serum albumin containing a rather low number of α-melanotropin molecules attached uniquely through the γ-carboxyl-group of (glutamic acid)-5 (probably mostly to ɛ-amino groups of albumin lysine residues). In such a complex-antigen the groups chemically characteristic of α-melanotropin (C-terminal valine amide and N-terminal N-acetylserine) are expected to be maximally exposed to the surroundings. The antisera had high antibody titers (1:125000 up to 1:900000). Their specificity was directed towards the C-terminal part of α-melanotropin. The cross-reaction with adrenocorticotropin was small (about 0.1%) and negligible with other peptide hormones, including β-melanotropin. The sensitivity of the assay was 40 pg/ml and the detection limit for α-melanotropin about 3 pg. Radioiodination of α-melanotropin with the lactoperoxidase method yielded a monoiodinated tracer with a specific activity of about 1500 Ci/mmol. The biological activity of structural analogues and fragments of α-melanotropin (assay in vitro with skins of Rana pipiens) paralleled their activity in the radioimmunoassay. This may indicate similar structural requirements of the melanophore receptor and antibody populations.

Immunochemical approaches to the structural chemistry of polysaccharide

Abstract

Immunochemical Cross-Reactions of Human Amyloid Proteins with Human Immunoglobulin Light Polypeptide Chains

Abstract Antisera to three human amyloid preparations were used to study antigenic relations between seven purified amyloid fibril proteins and 49 human Bence Jones proteins. Two of the amyloid proteins were antigenically similar to each other and shared antigenic determinants with 23 of 26 kappa light polypeptide chains. The other five amyloid proteins shared antigenic determinants among themselves and with 7 of 23 lambda light polypeptide chains. There was no evidence for either the presence or absence of an amyloid-specific antigenic determinant. These immunochemical data support the concept that the amyloid fibril proteins examined derive primarily from immunoglobulin light chains.

Chemical, electrophoretic and immunochemical studies of the seromucoid fraction of serum: A species comparison

1. 1. The seromucoid fraction was isolated from pooled dog, monkey, beef, horse, guinea-pig, rabbit, rat and human serums by a double-precipitation procedure employing perchloric and phosphotungstic acids. 2. 2. All fractions were characterized by a relatively high percentage of polysaccharide consisting of hexose, sialic acid, hexosamine and fucose and a high content of ash due to the formation of phosphotungstic acid conjugates. 3. 3. The major components of all fractions were α-globulins. 4. 4. Both qualitative and quantitative species differences were observed. 5. 5. Immunochemical cross-reactions occurred with all fractions.

Relationships between tobacco mosaic virus and the non-virus proteins.

1. The non-virus proteins, A4, B3, and B6, characteristically found in tobacco leaf infected with TMV exhibit specific immunochemical cross-reactions with serum prepared against the virus. The close immunochemical relations which occur among these proteins do not extend to any normal tobacco leaf proteins. 2. The rate of appearance of the non-virus proteins in newly infected cultured leaf tissue at various times after inoculation has been determined by immunochemical techniques and by direct isolation of the proteins. Both methods give comparable results. The non-virus proteins appear abruptly at about 220 hours after inoculation, when the TMV content is about one-third its final value. The amount of A4 rises rapidly and then levels off. The B6 content rises rapidly and continuously over the course of the experiment. B3 appears last, and increases in amount considerably more slowly than A4 and B6. 3. The isotope contents of TMV, B3, and B6 which appear in given intervals after inoculation in newly infected leaf cultured in nutrient containing N(15)H(4) have been compared. The isotope levels of concurrent TMV, B3, and B6 are identical within the experimental error. The isotope conditions employed in this experiment lead to the conclusion that this coincidence of N(15) levels means that the virus and non-virus proteins are probably synthesized at about the same time from the same non-protein source of nitrogen. 4. Possible interpretations of the available data on the non-virus proteins are discussed. It is likely that one or more of these proteins represents small protein units which occur in the TMV nucleoprotein.As they exist in the infected leaf, the non-virus proteins are probably no longer available to the biochemical processes which lead to TMV synthesis. They are probably not precursors of TMV protein in a temporal sense.