A Modification of the Mycoplasma Serum-Drop Growth Inhibition Test

A modification of the serologic mycoplasma growth inhibition test is described. It is based on application of a drop of antiserum directly onto the centre of an inoculated agar plate provided with a slightly concave agar surface. The special apparatus devised for the preparation of these plates is described. This modified serum-drop method is more sensitive than other growth inhibition tests used today and is equally specific.     

Assessment of the specific activity of commercial allergen preparations made of house dust in the bacteriosorption reaction: a comparison with the results of the radioallergosorbent inhibition test

The activity of the commercial batches of house-dust (HD) allergens was compared in the inhibition of the radioallergosorbent test (RAST) and in the direct bacteriosorbent test (BST), detecting IgG to the antigens by adsorption on the complexes of whole staphylococcal cells containing protein A. BST was made with rabbit antiserum to HD allergen. This antiserum inhibited RAST by 76% and, therefore, contained antibodies to most of the allergenic determinants of HD. At the same time, no significant correlation between the activity of 15 batches of HD allergen was revealed in RAST inhibition and in BST with the above antiserum. Nevertheless, the exhaustion of the antiserum with a batch of HD allergen showing low activity in RAST inhibition, but high activity in BST made it possible to obtain BST results significantly correlating with the data resulting from RAST inhibition in two series of experiments.     

Growth Inhibition Test for Identification of Mycoplasma Species Utilizing Dried Antiserum-Impregnated Paper Discs

The growth inhibition test for identifying Mycoplasma species has been modified by drying antibody-impregnated paper discs at 5 C. When stored at -20 C, these discs have been found to retain their inhibitory activity for longer than 7 months. Since these discs can be stored for long period of time, significant advantages over present methods result. When, for example, discs are arranged on a ring, a single test can be used for the identification of an unknown human species. Valuable antisera can be distributed to other laboratories on paper discs in much less volume than can fluid antiserum. Considerable savings of time result from prior preparation of many discs that can then be stored and used over a long period of time. The growth-inhibiting antibody is stable, and the activity is not enhanced by a heat-labile accessory factor from fresh guinea pig serum which increases the antibody titer in the metabolic inhibition test.     

Isolation and Characterization of a New Porcine Mycoplasma

In a study on the distribution of Mycoplasma sui- (hyo-) pneumoniae (M. suip.) among Danish swine it was found that most isolates possessing the characteristic colonial morphology of M. suip. would be inhibited significantly in the growth inhibition (g.i.) and metabolic inhibition (m.i.) tests by antiserum for a type strain* of this species. However, a few isolates were found to be completely unaffected by this antiserum. Five such strains have been recovered, viz. 4 from cases of catarrhal pneumonia in bacon pigs, 1 from the nasal cavity of a 40-kg pig. The pigs in question originated from 5 different herds. The recovery of 1 of the strains has been reported (Friis 1971b).     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.     

Antigenic studies of a VH fragment: demonstration of three sets of antigens, idiotypic, VH subgroup, and VH framework-specific antigens.

The antigenic properties of the VH region of immunoglobulin heavy chains were studied by means of a fragment corresponding to the variable part of the heavy chain of an IgG3 myeloma protein (KUP) and an antiserum made against this fragment. By hemagglutination, hemagglutination inhibition, and immunofluorescence techniques, it was shown that the anti-VH antiserum detected three sets of antigens in the VH region, namely idiotypic antigens, VH subgroup-specific antigens, and VH domain-(framework) specific antigens. The VH fragment inhibited in a VHII subgroup-specific hemagglutination inhibition test system. The VH fragment was thus antigenically similar to the tvh region found in the intact molecules and the light chains were not needed to express the VH subgroup antigens or the VH framework antigens.     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.     

Immunochemical studies on glutamic acid decarboxylase (EC 4.1.1.15) from mouse brain

Purified homogenous glutamic acid decarboxylase (GAD) from mouse brain and rabbit antiserum prepared to partially purified GAD gave only one sharp precipitin band in the Ouchterlony double diffusion test. GAD activity was inhibited partially by incubating with the antiserum. The maximal extent of inhibition was approximately 50 per cent. In the presence of antiserum all enzyme activity could be precipitated. The precipitates formed by GAD and antiserum had about 50 per cent of the enzyme activity and the K_m values for both glutamic acid and pyridoxal phosphate were significantly higher than those of the control system. Pyridoxal phosphate protected GAD from inhibition only slightly, even at very high concentrations. The results suggest that the antibodies may not react with the catalytic site, but rather that the inhibition of enzyme activity is attributable to indirect effects.     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

Purification of tyrosine hydroxylase from pheochromocytoma tumors.

Tyrosine hydroxylase was purified from human pheochromocytoma tumors. Polyacrylamide disc gel electrophoresis of the enzyme preparation obtained after sucrose density gradient centrifugation revealed a single enzymatically active protein band. A specific antiserum to purified human pheochromocytoma tyrosine hydroxylase was produced in rabbits. The specificity of the antiserum was demonstrated by immunoelectrophoretic analysis as well as by the specific inhibition of tyrosine hydroxylase. Enzyme inhibition studies revealed extensive cross-reactivity between the antiserum and tyrosine hydroxylases from bovine and rat adrenals and from rat striatum. The kinetic properties of the purified pheochromocytoma enzyme are similar to those of the bovine adrenal enzyme.     

新生幼犬的被动免疫研究

新生幼犬通过初乳获得的被动免疫,对它的生存非常重要,本研究用犬的多价免疫血清代替初乳作为幼犬保护性免疫球蛋白,对生后2 d内不能获得初乳的幼犬,以及虽然获得初乳但初乳中特异性免疫球蛋白含量较低的幼犬进行了被动免疫试验。对5窝35只幼犬在生后,随机分为吃初乳的对照组、口服血清组、皮下注射血清组、以及口服和皮下注射血清又吃初乳5个组,在幼犬出生时、及出生后48 h和第5天采血,测定犬细小病毒、犬传染性肝炎、和犬副流感的血凝抑制（HI）抗体效价。结果表明：对没有吃到初乳的仔犬,通过早期口服或皮下注射途径给予成年犬的血清,均能代替初乳供给幼犬免疫球蛋白。对吃到初乳的仔犬,能进一步提高幼犬体内特异性抗体水平,但以皮下注射途径最佳。     

The effect of dexamethasone on formation of a fibronectin extracellular matrix by rat hepatocytes in vitro

Monolayer cultures of newborn rat hepatocytes were initiated in the presence or absence of dexamethasone, and fibronectin was analysed by indirect immunofluorescence microscopy using a specific antiserum raised in rabbit. Dexamethasone-treated hepatocytes produce a well defined extracellular matrix of fibronectin that begins to form as early as 24 h after treatment. Non-treated hepatocytes exhibit very little immunofluorescence staining. Moreover, examinations by phase contrast microscopy reveal a very good preservation of the hepatocyte typical epithelial morphology and a drastic inhibition of fibroblast growth in the treated cultures.     

Antibodies raised against the extracellular tail of the CCKB/gastrin receptor inhibit gastrin-stimulated signalling

INTRODUCTION: Gastrin acts to stimulate gastric acid secretion and is an acknowledged growth factor for human gastrointestinal (GI) cancer. The identity of the exact receptor type mediating the growth promoting effects of gastrin in tumours is uncertain. However, the best-characterised gastrin receptor is the CCK receptor type B (CCKB)/gastrin receptor. The anti-GRE1 antibody is a polyclonal, affinity-purified antibody raised against GRE1, a synthetic 21 amino acid peptide homologous to part of the extracellular, N-terminal tail of the CCKB receptor. We have recently proven that GRE1 antiserum specifically localises CCKB receptors on CCKB receptor transfected NIH3T3 cells and human gastrointestinal tumour cells by Western blotting and immunocytochemistry. GRE1 antiserum also inhibits liver invasion in the C170HM2 colorectal liver-metastasis model. AIM: To relate the ability of GRE1 antiserum to displace G17 from CCKB receptors with its impact on cellular transduction effects. METHODS: Radioligand binding studies were performed with 125IG17 and Calcium mobilisation studies by use of the fluorescent dye Fura 2-am. RESULTS: GRE1 antiserum competitively displaced 50% radiolabelled gastrin-17 from whole cell NIH3T3 CCKB transfectants at a protein concentration of 250 microg x ml(-1). GRE1 antiserum did not stimulate calcium ion influx in the transfectant NIH3T3 cells when used at a range of protein concentrations. Pre-incubation with GRE1 antiserum was required to inhibit gastrin-stimulated calcium ion influx. This was found to be concentration-dependent, with inhibition shown at 30 and 5 microg x ml(-1) but not at 500 ng x ml(-1) or below. CONCLUSION: The GRE1 antiserum is specific for the CCKB receptor and may act to inhibit gastrin-stimulated signalling in tumour cells.     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

Influence of Food Microorganisms on Staphylococcal Growth and Enterotoxin Production in Meat

Forty-four microorganisms were studied for their influence on staphylococcal growth and enterotoxin production. Inhibition was found to be more common than stimulation. Two types of inhibition were observed: inhibition of staphylococcal growth, and inhibition of enterotoxin formation with no apparent effect on growth. By use of a plate test, 12 of the 44 food microorganisms were found to inhibit staphylococcal growth at 35 C. Of the 12, 3 also inhibited growth at 25 C. No significant differences in inhibition were observed with the 15 strains of enterotoxigenic staphylococci. In meat slurries, inhibition of staphylococcal growth was found to be greater at 25 C than at 35 C. Results on inhibition obtained from the plate test could not be correlated with the effect of the organisms in slurries. Environmental conditions were found to affect markedly the influence of food microorganisms on staphylococci. Of the 44 food microorganisms studied, only Bacillus cereus was observed to stimulate significantly staphylococcal growth and enterotoxin formation. Stimulation was more pronounced with Staphylococcus aureus 196E than with other strains of enterotoxigenic staphylococci. Bacillus megaterium and Brevibacterium linens were inhibited by staphylococci. These organisms were completely inhibited when inoculated in mixed cultures with staphylococci. In pure cultures, good staphylococcal growth was found to be accompanied by enterotoxin production; however, in the presence of food microorganisms, good staphylococcal growth occurred without the formation of detectable levels of enterotoxin A.     

Relation between bacterial strain resistance to solvents and biodesulfurization activity in organic medium

Microorganisms used in biodesulfurization of petroleum products have to withstand high concentrations of hydrocarbons. The capacities of seven desulfurizing strains of Rhodococcus to be active in the presence of solvents were evaluated. Octanol and toluene (log P=2.9) were selected as toxic solvents. The effect of the solvents was determined by measuring either inhibition of growth or the decrease in respiratory activity of the cells. Differences among strains in their resistance to solvent responses were observed, but these variations were dependent on the test used. Resistance to solvents was then compared to the capacity of the different strains to retain biodesulfurization activity in the presence of hexadecane. Inhibition of desulfurization by high concentrations of hexadecane was found to be well correlated to the sensitivity of the strains to respiration inhibition by toluene, but not to growth inhibition. This result also showed that the respirometric test was a rapid and reliable test to select solvent-resistant strains for use as resting cells in biocatalysis processes, such as biodesulfurization, in organic media.     

Preparation and specificity of avian anti-GM2(NeuGc) ganglioside antiserum

Antibodies to N-glycolyl neuraminic acid-containing GM2 ganglioside, GM2(NeuGc), were prepared by immunizing chickens. The specificity of the antibodies was examined by the double immunodiffusion test and solid-phase radioimmunoassay (RIA). One(C-4) of two antisera produced did not cross-react with GM3(NeuGc) but the other(C-3) did as assessed by the double immunodiffusion test. In RIA, the antibody activity of C-4 antiserum was detected only in the IgG fraction. Specificity of the serum was examined using authentic glycolipids which were structurally related to GM2(NeuGc). The antiserum showed a high specificity for the homologous ganglioside by either an RIA or an inhibition assay. This antiserum is a useful tool for the detection of GM2(NeuGc) in human and animal tissues under normal and/or disease condition.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Localization,Accumulation, and Antifungal Activity of Chitinases in Rye (Secale cereale) Seed

In order to understand a physiological role of chitinases in rye, the localization and accumulation of rye seed chitinase-a and -c (RSC-a and -c) in the seeds were studied by immunochemical methods. An antiserum specific to the chitin-binding domain (CB-domain), which is an N-terminal part of RSC-a, and an antiserum specific to the catalytic region of RSC-a and RSC-c were used. An immunoblot analysis detected both RSC-a and RSC-c in the endosperm of the rye seed. Immunohistochemical staining indicated that RSC-a was localized in only the aleurone cells, whereas RSC-c existed at least in the starchy endosperm and was also likely to exist in the aleurone cells. It was found by ELISA and an immunoblot analysis that RSC-a and -c accumulated in the seed during the later stage of development. Both chitinases and the Cat-domain exhibited antifungal activity toward Trichoderma species, while the CB-domain did not. Observation of the inhibition of hyphal growth of the T. species suggests that the two chitinases acted in different ways.