Tandem-crossed Immunoelectrophoresis Research Articles

Involvement of glycans in the immunological cross-reaction between α-macroglobulin and hemocyanin of the gastropod Helix pomatia

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between α-macroglobulin (αM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: αM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: α D- HpH, α N- HpH and β- HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With β- HpH, which is richer in carbohydrates than α D- HpH and α N- HpH, mainly due to a higher 3- O-methyl- d-galactose content, the cross-reaction with HpαM was highest. (ii) From the 8 functional units, designated a– h, isolated from β- HpH, two that lack carbohydrates ( c and f) were not recognized by antibodies against HpαM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both β- HpH and HpαM are further evidence that glycans are involved in the immunological relationship between HpH and HpαM. Carbohydrate analyses indicated that the glycan structures present on HpαM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3- O-methyl- d-galactose seem to be responsible for the cross-reactivity since the α-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.

Immunological analysis of apolipophorin-III in the haemolymph, ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Lipophorin (LP) was purified from haemolymph in last instar larvae of Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation and gel filtration. LP is composed of Apo-LP I and Apo-LP II with molecular weights of 230 kDa and 80 kDa, respectively. The level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the level of haemolymph LP decreases in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Actually, LP was found in the ovary by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Location of LP in the ovary was also traced by immunogold labelling. Also, LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte is larger than nurse cells—that is, when vitellogenesis is actively taking place. This fact clearly reveals that LP is synthesized by fat body and released into the haemolymph, and then taken up by the growing ovary during vitellogenesis. Also, LP was detected in testes by immunological analysis. Western blotting showed that LP was present in testicular fluid but not in the peritoneal sheath and cysts. To test whether LP is also synthesized in testes, testes and fat body tissues were cultured in vitro, indicating that fat body synthsizes LP but testes do not. The result showed that the haemolymph LP itself is taken up into the testes. © 1994 Wiley-Liss, Inc.

Comparison of lipases from different strains of the fungus Geotrichum candidum

The type A lipase and the cis-9 18:1 specific type B lipase of different strains of Geotrichum candidum were compared. Comparing the enzyme activity of crude lipase preparation and purified type A and type B lipases and the protein pattern of these preparations in denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the specific activity for cis-9 18:1 fatty acids was related to the content of the type B lipase. Tandem-crossed immunoelectrophoresis was used to demonstrate immunological identity between type A and type B lipase of G. candidum ATCC 66592. Partial immunological identity was observed between type B lipase of this strain and type A lipase of G. candidum ATCC 34614 and two commercial crude G. candidum lipase preparations (Amano and Biocatalyst), i.e., the type B lipase of G. candidum ATCC 66592 had immunogenic epitopes which are not present on the other lipases. Enzymatic deglycosylation of the lipases did not alter this pattern. After partial proteolysis of purified type A and type B lipases of G. candidum ATCC 66592, Amano and Biocatalyst, no difference between the type A lipase of the three strains was observed in SDS-PAGE. For all strains the type B lipase exhibited a distinctly different peptide pattern to that of the type A lipase. In addition, the type B lipase of G. candidum ATCC 66592 differed from the type B lipase of Amano and Biocatalyst by having an additional peptide band. The results indicate that the G. candidum ATCC 66592 should be considered a distinct strain regarding the protein chemical characteristics of its type B lipase, whereas the two commercial lipase preparations appear to be very similar.

Synthesis of nitrite reductase is regulated differently in leaf and root of barley ( Hordeum vulgare L.)

Activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE) of extracts from nitrate-treated plants indicates the presence of a single isoform of nitrite reductase in the leaf and root of barley. Crossed and tandem-crossed immunoelectrophoresis, and immunoblotting, show that polyclonal antibodies raised against the purified leaf nitrite reductase protein recognise specifically the nitrite reductase protein in both leaf and root, suggesting that the leaf and root enzymes are immunologically similar, if not identical. Green, light-grown plants maintained in the absence of added nitrate possess low levels of nitrite reductase activity (measured with dithionite-reduced methyl viologen as electron donor) and nitrite reductase cross-reacting material in both leaf and root. Addition of nitrate in the light to green, light-grown plants leads to a concomitant increase in the level of nitrite reductase activity and cross-reacting material in both leaf and root. When green, light-grown plants are treated with nitrate and transferred immediately to the dark, there is a concomitant increase in nitrite reductase activity and cross-reacting-material in the root, but neither increase in the leaf. We conclude that the observed increases in nitrate reductase activity are due to the net synthesis of new nitrite reductase molecules and that their synthesis is regulated differently in leaf and root: both light and nitrate together are required in the leaf but nitrate alone is able to bring about synthesis of nitrite reductase molecules in the root. Inability to synthesise nitrite reductase molecules in leaves in the dark after treatment of plants with nitrate is not due to exclusion of nitrate from the leaves. Possible mechanisms which underlie these observations are discussed.

A unified serotyping scheme for Moraxella bovis

Fifty-three Australian, seven British, two American and two New Zealand isolates of Moraxella bovis were classified into seven serogroups on the basis of their variable fimbrial (pilus) antigens using whole cell slide agglutination (SA), enzyme-linked immunosorbent assays (ELISA) and tandem-crossed immunoelectrophoresis (TCIE). Although results of serogroup classification by SA and ELISA were identical in 68.7% of isolates, it was found necessary to resolve the discrepancies between the two systems using TCIE. Results suggest that world-wide variation in the potentially host-protective fimbrial antigens of M. bovis may be relatively limited. It is proposed that the previous numerical classifications of British and Australian serogroups are appropriately amalgamated as a result of this latest study and are designated as serogroups A to G inclusive. A protocol for the further serotyping of fresh, fimbriate isolates of M. bovis is suggested.

Proteolytic inactivation of alpha 1-proteinase inhibitor in infected bronchial secretions from patients with cystic fibrosis

The chronic, progressively destructive bronchitis of patients with cystic fibrosis (CF) is characterized by an important imbalance between tissue destroying granulocyte proteases such as granulocyte elastase (GE) and its physiological inhibitors in bronchial secretions. Recent in vitro studies suggest, that proteases derived from bacteria or endogenous proteases may contribute to inactivation of physiological inhibitors of GE. Since only trypsin-unreactive alpha 1-proteinase inhibitor (alpha 1-PI) was detected in CF bronchial secretions, we attempted to identify the mechanism of inactivation of alpha 1-PI. We found a heat stable, serine protease-like enzymatic activity capable of degrading 125I-labelled alpha 1-PI extensively in 22 infected but not in one non-infected CF bronchial secretion. In infected secretions, only degraded alpha 1-PI, which did not migrate like oxidized alpha 1-PI in tandem-crossed immunoelectrophoresis, was detectable. We conclude, that free GE in excess as well as GE bound to bronchial mucosal inhibitor may partly account for the alpha 1-PI-cleaving activity, but that other yet unknown bacterial or host serine proteases also contribute to alpha 1-PI inactivation.

Comparative Immunological Studies on the CF1-Complex in Mutants of N. tabacum, Exhibiting Different Capacities for Photosynthesis and Photorespiration and Different Chloroplast Structures

We have compared by means of rocket immunoelectrophoresis the amount of CF1-complex in N. tabacum mutants with that in the wild type N. tabacum var. John William’s Broadleaf. Some of these mutants differ from the wild type not only by higher photosynthetic and photorespiratory activities but also by their chloroplast structure. The amount of CF1, present was related to the chlorophyll content. However, no correlation between the amount of CF1, present and a higher photosynthetic and photorespiratory activity was found. On the other hand the molar ratio of CF1, to chlorophyll seems to depend in these tobacco mutants on the morphological structure of the chloroplasts. In chloroplasts of green phenotypes, in particular in those of the mutants N. tabacum var. Consolation, var. NC 95 and var. Xanthi with high grana stacking as well as in those of the wild type N. tabacum var. JWB one CF1-complex occurs per 470-770 chlorophyll molecules. Whereas in chloroplasts of the yellow-green phenotypes with relatively more stroma thylakoids the amount of CF1 seems to be increased only by a factor of 3-4. The yellow phenotypes contain on the average 1 CF1-complex per 60 chlorophyll molecules. The CF1-complex in the investigated chlorophyll-deficient tobacco mutants is immunochemically identical to that of the wild type N. tabacum var. John William’s Broadleaf as demonstrated by means of the tandem-crossed immunoelectrophoresis. However, between the CF1-complex of tobacco and that of other higher plants such as spinach Spinacia oleracea, only partial identity is observed. Comparative quantitative determinations have shown that one CF1-complex binds on the average 8-9 CF1-antibodies which shows that the CF1-complex of the ATPase is fully exposed in the thylakoid membrane, offering a large adsorption surface to the antibodies. Furthermore, it appears that in the immediate vicinity of the CF1-complex large amounts of monogalactolipids are located. Thus, we have calculated that in the variegated NC 95 mutant, chloroplasts of the yellow leaf areas, in which only stroma thylakoids occur, exhibiting only photosystem I activity, as well as in chloroplasts of green leaf areas with a normal ratio of grana and intergrana thylakoids, the molar ratio of monogalactolipids to CF1-complex is the same. It looks as if the functionality and activity of the coupling factor of photophosphorylation depended on a certain quantity of monogalactolipid molecules.

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits ( M r 62 000, and M r 31 000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodif usion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits ( M r 63 000, 56 000, 30 000 and 26 000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H 2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All These proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration of three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH 2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH 2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.

Immunological Study on the Structural Difference between Ribulose- 1,5-bisphosphate Carboxylase/Oxygenase from Nicotiana tabacum var. John William's Broadleaf and the Tobacco Mutant Su/su

Abstract In the present paper we have attempted to characterize by means of immunological methods the molecular difference between ribulose 1,5-bisphosphate carboxylase/oxygenase from the wild type TV. tabacum var. John William's Broadleaf and the tobacco mutant Su/su. The tobacco mutant Su/su exhibits in comparison to the wild type a higher photorespiratory activity. The reagents used in the present study are monospecific antisera to the two bifunctional enzymes to be compared. We have analyzed the oxygenase activity of the two enzymes in dependance on the binding of the amount of antibodies out of the homologous and the not homologous antiserum. These analyses have shown, that the enzymes of both phenotypes were 4 0 % stronger inhibited in the equivalence regions with respect to their oxygenase function by antibodies of the antiserum to ribulose 1,5-bisphosphate carboxylase/oxygenase of the mutant than by antibodies of the wild type antiserum. It should be noted that the antiserum to the mutant enzyme exhibits a 25 % lower antibody titer, than the antiserum to the wild type enzyme. In the region of extreme antibody excess, i.e. when antibodies are mostly monovalently bound and most antigenic determinants are saturated with antibodies, the oxygenase activity of both enzymes decreases towards zero in the presence of the homologous as well as in the presence of non-homologous antibodies. In the region of excess of antigenic determinants, that is when only a few antibody molecules can bind onto the enzymes, only the oxygenase activity of the mutant enzyme is inhibited by its homologous antibodies by 40 % . This apparent difference in the molecular structure of the two bifunctional enzymes to be compared is neither caught in the double immuno diffussion test nor in the tandem crossed immuno electrophoresis, using the two antisera as test reagents. In all cases only fused precipitation bands are observed. Chemical modification of ribulose 1,5-bisphosphate carboxylase/oxygenase by hydroxylamine treatment or treatment with o-(p-nitrophenyl)hydroxylamine or simple heating of the enzyme to 50 °C are immunologically characterized. As a consequence of such treatment considerably less antibodies are adsorbed. The strongest influence exerts a treatment with o-(p-nitrophenyl)-hydroxylamine with simultaneous heat treatment of the enzyme for 20 min at 50 °C.

Immunochemical Comparative Study on RuBP Carboxylase/Oxygenase of Different Tobacco Mutants Exhibiting Different Levels of Photorespiration

By means of the immunochemical methods of double immuno diffusion and tandem crossed immuno electrophoresis we have compared the bifunctional enzyme RuBP carboxylase/oxygenase from tobacco mutants which differ with respect to their rates of photosynthesis and photorespiration. The comparative studies were carried out with a monospecific antiserum to the enzyme of the wild type Nicotiana tabacum var. John William’s Broadleaf. RuBP carboxylase/oxygenase from the green, yellow-green and yellow phenotype of the mutants namely N. tabacum Su/su, N. tabacum Su/su var. Aurea, N. tabacum var. Consolation, N. tabacum var. NC 95 and N. tabacum Xanthi (D 523) are immunochemical^ identical to the enzyme of the wild type N. tabacum var. John William’s Broadleaf. Furthermore, immunochemical identity of the RuBP carboxylase/oxygenase exists between Nicotiana tabacum and other representatives of the Solanaceae such as Solanum tuberosum, S. lycopersicum and Datura suaveolens. In contrast to this only partial identity to the enzymes of the C3-plants Antirrhinum majus, Spinacia oleracea, Sinapsis alba, Petroselinum crispum, Allium porrum, Hordeum vulgare, Avena sativa to the enzymes of the green alga Chlorella vulgaris and to the blue-green alga Oscillatoria chalybea as well as to the enzyme of the C4-plant Zea mays is observed.

Taxonomic Application of Crossed Immunoelectrophoresis

We used crossed immunoelectrophoresis, crossed-line immunoelectrophoresis, tandem-crossed immunoelectrophoresis, and crossed immunoelectrophoresis with an intermediate gel (containing antibodies) in studies of bacterial taxonomy. By these quantitative immunoelectrophoretic methods, as many as 90 antigens from a bacterial species can easily and reproducibly be analyzed for immunological cross-reactivity with antigens from other bacterial species without prior purification of antigens. The immunological cross-reactivity between various bacterial species is expressed as the matching coefficient (MC), i.e., the ratio between the number of cross-reactive antigens with another bacterial species and the total number of antigens of the reference bacterial species. The MC has been found to be significantly correlated with phenotypic similarities (biochemical characteristics) and nucleic acid hybridization results obtained with the taxa investigated so far. In general, closely related strains (serotypes within a species) have a high MC (≥0.90), while different species of the same genus have a lower MC (0.90 to 0.70 or lower in some cases); genera within a family have an intermediate MC (0.70 to 0.15), and distantly related taxa have a very low MC (≤0.15).

Alfalfa Root Nodule Carbon Dioxide Fixation

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C(4) PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C(4) plant species. Neither antiserum reacted strongly with any C(3) leaf proteins. The molecular weight of the PEPC polypeptide from C(4) leaves and legume nodules appears to be similar.

Immunological Evidence for the Presence of Proteins of the Photosynthetic Membrane in Etiolated Leaves of Phaseolus vulgaris

Summary Immunological techniques were utilized to detect the occurrence of different proteins of the electron transport chain in different fractions of etiolated bean leaves of Phaseolus vulgaris var. Commodore L: plastocyanin, cytochrome f, ferredoxin, ferredoxin-NADP + -reductase. Furthermore, the coupling factor of photophosphorylation and RuBP carboxylase/oxygenase were also shown to be present in the etiolated material. Different fractions (leaf-homogenates, etioplasts, etioplast membranes, the protochlorophyllide protein complex) were photoactive and were characterized by their low-temperature absorption and fluorescence spectra. The double diffusion test and the tandem crossed immuno electrophoresis demonstrated that all the investigated proteins were immunochemically identical in etiolated and in green leaves. In the rocket immuno electrophoresis test the relative amounts of the enzymes on a total protein basis were investigated in homogenates from etiolated, greening, and green leaves. All the electron carrier proteins as well as the RuBP carboxylase/oxygenase and the coupling factor were more abundant in green leaves than in etiolated leaves. During a 60 min illumination of etiolated leaves, the relative amount of these proteins did not markedly increase.

Glycoprotein allergens in pollen of timothy. III. Immunochemical and biological properties of a basic glycoprotein.

Crossed immunoelectrophoresis (CIE) and tandem CIE indicated that a basic glycoprotein allergen isolated from pollen of timothy is immunochemically identical to the allergen formerly denoted as antigen 30. The glycoprotein gave one band in SDS-PAGE and agarose isoelectric focusing. After transfer to nitrocellulose by electroblotting or by capillary blotting, the allergen specifically bound IgE from persons allergic to timothy pollen. The single allergen inhibited about 15% of the IgE binding to radioallergosorbent test discs of whole timothy pollen extract. Sera from patients investigated also contained detectable amounts of IgG towards the allergen. In quantitative skin prick tests, the mean activity of 2 micrograms/ml purified allergen in 7 patients was equivalent to that of a timothy pollen extract containing 10,000 BU/ml (assayed relatively to histamine in skin prick test). The ability of the allergen to form a precipitate in rocket immunoelectrophoresis was lost when it was heated to 80 degrees C or incubated with 4 M guanidine hydrochloride.

Isolation and characterization of cross-reactive allergenic components in Callistemon citrinis and Melaleuca quinquenervia pollen by trans-blot enzyme-linked crossed immunoelectrophoresis.

Antigenic extracts obtained from Callistemon citrinis (bottle brush) and Melaleuca quinquenervia (melaleuca) pollen were analyzed by crossed immunoelectrophoresis (CIE), tandem-crossed immunoelectrophoresis (TCIE), and trans-blot enzyme-linked crossed immunoelectrophoresis (TECIE). CIE analysis detected 14 and 12 antigenic components in bottle brush and melaleuca, respectively. TCIE analysis of bottle brush pollen, employing rabbit anti-melaleuca serum in the intermediate gel, demonstrated that all of the antigenic components detected were cross-reactive. Similar analysis of melaleuca using rabbit anti-bottle brush serum in the intermediate gel also showed that all of the antigenic components were cross-reactive. At least three bottle brush and two melaleuca cross-reactive pollen components were demonstrated to be allergenic by TECIE analysis of CIE and TCIE gels.

Salivary interactions with homologous and heterologous strains of oral streptococci and epithelial cells.

Twenty strains of oral streptococci resembling the species of Streptococcus mitior and Streptococcus salivarius were isolated from four subjects and were incubated with homologous and heterologous whole clarified saliva. Supernatants of bacterially absorbed and control saliva were analyzed in tandem crossed immunoelectrophoresis (TCIE) against a rabbit anti-human saliva antiserum. Six to eight antigens were detected. After incubation with the bacteria, the saliva supernatants showed a decrease in the number of antigens detectable by TCIE. For all four salivas tested, the homologous group of strains absorbed fewer antigens. Some of the antigens could be eluted from bacterial pellets with 1 M NaCl after repeated washings in PBS. Epithelial cells also absorbed fewer antigens from homologous than heterologous saliva.

Trypanosoma cruzi: surface antigenic determinants.

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37 degrees C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandem-crossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100 degrees C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.

Comparative Studies on Tree-Pollen Allergens

Gel permeation chromatography of <i>Betula verrucosa</i> (BV) crude pollenextract and pooling of the fractions after their fused rocket immunoelectrophoresis antigenic profile allowed the isolation of a purified major allergen designated BV<sup>45</sup>. This fraction was shown to be a glycoprotein with a tentative molecular weight of 10,000 daltons. The estimated molecular weight differed by approximately factor 2 from the previously reported values of birch pollen major allergens. This fraction was further purified by polyacrylamide gel chromatography (P-10), high performance liquid chomatography (HPLC) silica-base column and high-volt electrophoresis at different dimensions. The purified proteins revealed an immunochemical identity with the initial material. BV<sup>45</sup> and the previously isolated pI 5.18, though different otherwise, demonstrated a reaction of complete antigenic identity illustrated by tandem-crossed immunoelectrophoresis. Both the amino acid and carbohydrate compositions of BV<sup>45</sup> were calculated on the basis of the tentative molecular weight obtained. BV<sup>45</sup> showed a total carbohydrate concentration of 20% of the dry weight, corresponding to 13–14 residues per mol protein. The amino acid composition obtained reflected structural diversity between the BV<sup>45</sup> and pI 5.18. N-terminal analysis followed by HPLC quantitation of the DNS-amino acids for BV<sup>45</sup> and the further purification products gave two peaks having retention times corresponding to DNS-Cya and DNS-Asp. Fraction BV<sup>45</sup> was demonstrated to be a major allergen of birch pollen as assessed by RAST inhibition of 7 out of 10 reaginic sera and the specific radiostaining in crossed radioimmunoelectrophoresis of all omitt nine crossed immunoelectrophoresis plates used. RAST inhibition titration of BV<sup>45</sup> and BV crude showed qualitative identity of the allergenic determinants of both materials. The question about one or several major allergen(s) in birch pollen extract has so far been unsolved as no structural mapping of BV<sup>45</sup> and pI 5.18 has been readily available.

Serotaxonomy of Fusarium species of the sections Gibbosum and Discolor

Thirteen Fusarium species belonging to the sections Gibbosum and Discolor were compared by tandem-crossed immunoelectrophoresis to determine their serological relationships. On the basis of serological similarities, species were grouped by cluster analysis and the results expressed as a phenogram. The taxonomic relationships thus established are discussed in comparison with modern systems of morphological classification.

Crossed immunoelectrophoretic analyses of antigens from Trypanosoma lewisi and Trypanosoma musculi

Trypanosoma lewisi and Trypanosoma musculi were collected from immunosuppressed infected hosts and extracted with phosphate buffered saline. Antisera were obtained from rats repeatedly infected with T. lewisi or mice repeatedly infected with T. musculi. Cellular antigens (CAg) present in the extracts of the parasites were analyzed by microimmunodiffusion (MID), crossed immunoelectrophoresis (CIE) and tandem crossed immunoelectrophoresis (TCIE). Trypanosome extracts were absorbed with the heterologous hyperimmune antisera to examine shared and unique antigens of the parasites. Extracts of T. lewisi formed four precipitin lines when reacted with hyperimmune rat antiserum and three precipitin lines were detected by mouse anti- T. musculi serum in MID analyses. T. musculi extract formed two precipitin lines with mouse hyperimmune serum and two precipitin lines with rat anti- T. lewisi serum in the MID tests. When T. lewisi was reacted with the homologous hyperimmune rat antiserum in CIE, 14 precipitin peaks developed, while T. musculi extract formed eight peaks with homologous mouse hyperimmune serum. Seven precipitin peaks developed when T. lewisi extract was reacted with the mouse antiserum and T. musculi extract formed eight peaks during its electrophoretic migration into rat anti- T lewisi serum. TCIE clearly showed that five T. lewisi CAg could not be detected in the T. musculi extract by the rat antiserum, while mouse anti- T. musculi serum formed six precipitin peaks with the T. lewisi extract and seven peaks with the homologous extract. One of the CAg present in the T. musculi extract was not found in the T. lewisi extract. Absorptions of the extracts with heterologous antisera and subsequent CIE against the homologous antisera indicated three of the CAg of T. lewisi were not shared by T. musculi, while a single antigen of T. musculi was not detected in T. lewisi. Although concentrations of antibodies in each of the antisera and CAg in the parasite extracts were not equivalent, the data indicated that a minimum of eight CAg are shared by these rodent trypanosomes and at least three antigens appeared to be unique to T. lewisi and a single antigen to T. musculi.