Different adhesion inhibiting activities of antisera against plasma membranes of liver and Morris hepatoma 7777

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against plasma membranes of liver (anti-liver antiserum) and Morris hepatoma 7777 (anti-hepatoma antiserum). Similar concentrations of both antisera inhibited adhesion on collagen. Anti-liver antiserum also inhibited the adhesion of hepatocytes on plastic, whereas anti-hepatoma antiserum was only able to inhibit the adhesion on collagen completely. These results suggest the existence of at least two different adhesion-involved molecules. Cells adhere to plastic by means of both molecules, whereas adhesion on collagen is mediated by only one of them. The results further suggest that hepatoma cells lost the molecule involved in adhesion on plastic.     

Towards the protein phosphatase-based biosensor for microcystin detection

A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol-gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and alpha-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach.     

Immunocytochemical localization of water-soluble glycoproteins, including Group 1 allergen, in pollen of ryegrass,Lolium perenne, using ferritin-labelled antibody

Summary The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization.Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.     

Immobilisation of DNA probes for the development of SPR-based sensing

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).     

Colloidal gold immunostaining on deplasticized ultra-thin sections

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.     

Efficient immobilization of enzymes on microchannel surface through His-tag and application for microreactor

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.     

Use of epoxysepharose for protein immobilisation

Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However, a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8.5. Increasing ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation. This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90% even after 60 days of storage in aqueous suspension at 4°C. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose.     

Hydroxyapatite - a reagent for the separation of free and antibody-bound steroid during the radioimmunoassay.

The use of hydroxyapatite to absorb antibody-bound steroid and thus separate free and antibody-bound steroid during radioimmunoassay has been examined using three steroid antisera (to testosterone, to 17-hydroxyprogesterone and to estradiol-17beta). For all three antisera studied the separation was shown to be independent of length of time in contact with hydroxyapatite (up to 1h); temperature variations from 4 degrees -37 degrees and pH over the range 4.9-8.0. The presence of protein affected the absorption of antibody-bound steroid but this effect could be overcome by the addition of increasing amounts of hydroxyapatite. Further increase in the amount of hydroxyapatite added had no effect on the separation of free and bound steroid. Sodium phosphate buffers of molarity greater than 0.01M eluted antibody-bount steroid from hydroxyapatite, but Tris-HC1 buffers up to molarities of 0.1 M had no effect. Hydroxyapatite when used as a dry powder had the same effects as suspensions. No effect on the cross-reactivities of the antisera used could be demonstrated when hydroxyapatite was used and plasma testosterone assays on 22 plasma samples using hydroxyapatite gave essentially the same results as assays on the plasma using a coated-tube assay. Hydroxyapatite can also be successfully pumped along small bore plastic tubing without settling and can thus be used in automated immunoassay systems.     

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.     

玉米素核苷的酶标免疫测定法

牛血清白蛋白-玉米素核苷(BSA-ZR)的兔抗血清对玉米素核苷具有很高的亲和性,而且专一性强,除了玉米素外,对其它一些细胞分裂素如激动素(KT)的交叉反应甚微。用辣根过氧化物酶(HRP)作为标记物的酶标免疫法,由于它的灵敏度高,相当于几十毫克量的样品就可以测出细胞分裂素的含量。测定范围在0.25—50pmol之间,测定范围较广。由于该方法专一性高,植物组织的粗提取物可以直接用于测定。避免了提取分离的繁琐程序,使得测定方法较简便、快速,可成批进行,适用于一般实验室。用该方法测得风信子(Hyacinthus orientalis L.)各部分的细胞分裂含量(以玉米素核苷计)在10—60×10~(-9)克/克鲜重,即10—60ng/g F.W.。     

Antibody immobilisation on fibre optic TIRF sensors

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.     

The production and assessment of a plastic rod for the Chinese Reference Preparation for Opacity

A reference preparation for opacity consisting of a plastic rod was introduced by Perkins et al. in 1973. It was adopted as the International Reference Preparation for Opacity in 1975. This plastic rod opacity reference preparation has been used to standardize the Chinese National Bacterial Opacity Standard. The material was prepared from plastic sheet by a water-bath method and by a dry-heat method; the sheet was then machined into the plastic rods. We have studied the technical processes and set up methods for the examination of the sheets and rods. The water-bath method was found to be better than the dry-heat method in our tests. Collaborative assays in research institutes of biological products have shown that the plastic rod can replace the glass-powder suspension. The duration of validity of the plastic rod opacity reference preparation and that of the glass-powder suspension used for the Chinese National Bacterial Opacity Standard were studied and found to be similar. For this reason the plastic rod opacity reference preparation has not been widely used in China.     

Characterization of an immunofluorescence technique for the demonstration of coexisting neurotransmitters within nerve fibers and terminals

Neurotransmitters have been shown to coexist in cell bodies, but demonstrating their coexistence within nerve fibers and terminals has been more difficult. However, two recent reports outlined a simple light-microscopic method by which two neurotransmitters can be shown to coexist in fibers and terminals. The method was identical to that used for immunohistochemical localization of one antigen, except that two primary--secondary antibody systems labeled with two different fluorochromes were used simultaneously. In the present study, a method for the simultaneous visualization of serotonin and substance P was characterized. This method employed an antiserum to serotonin generated in goat in combination with a rabbit-generated antiserum to substance P. These antisera were visualized with secondary antisera raised in swine and conjugated with rhodamine and fluorescein respectively. Spinal cord sections stained by this protocol showed large numbers of fibers fluorescing both red and green. Many of them were in the ventral horn, fewer were around the central canal, and virtually none were in the dorsal horn. The apparent double labeling could be shown not to be the result of cross-reactivity among the antisera, of any inappropriate affinity among the antisera, of green fluorescence by rhodamine, or of red fluorescence by fluorescein. It is concluded that the method provides a simple technique for visualizing fibers and terminals in which serotonin and substance P coexist.     

Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase

This work presents a biosensor for organophosphorus pesticides based on immobilisation of a highly sensitive genetically engineered acetylcholinesterase (B394) by affinity interactions on metal chelate-functionalised magnetic microbeads. The developed sensor has been compared with those based on the widely used Electric eel cholinesterase and a classical entrapment procedure in a polyvinylalcohol-based matrix. The use of the B394 enzyme allowed lowering both IC50 and LOD by a factor of 100 when compared with Electric eel enzyme sensor. The oriented and site-specific immobilisation combined with the high specificity of the B349 mutant allows a more sensitive detection of insecticides, concentrations as low as 1.31(-11)M (IC10) being detected for both pesticides chlorpyriphos-oxon and chlorfenvinphos.     

Observations on Schizothrix mexicana Gomont (Cyanophyta) under culture conditions

Using agar gel électrophoresis, the number and relative mobility of seric protein fractions has been determined for twelve species of fishes belonging to the Elasmobranchii, Dipnoi and Actinopterygii.The study of relative mobilities has shown both similitudes and divergences between some of the proteinograms. Immunoelectrophoretic cross tests using the twelve sera and five antisera have shown that these similitudes did not result from protein homology and thus agar gel electrophoresis could not be used to determine phylogenetic relationships between the species considered.

Laboratoire d'Hydrobiologie et de Pisci culture, Université de Kinshasa (Zaïre)     

Use of immobilised biocatalysts in the processing of cheese whey

Food processing industry operations need to comply with increasingly more stringent environmental regulations related to the disposal or utilisation of by-products and wastes. These include growing restrictions on land spraying with agro-industrial wastes, and on disposal within landfill operations, and the requirements to produce end products that are stabilised and hygienic. Much of the material generated as wastes by the dairy processing industries contains components that could be utilised as substrates and nutrients in a variety of microbial/enzymatic processes, to give rise to added-value products. A good example of a waste that has received considerable attention as a source of added-value products is cheese whey. The carbohydrate reservoir of lactose (4–5%) in whey and the presence of other essential nutrients make it a good natural medium for the growth of microorganisms and a potential substrate for bioprocessing through microbial fermentation. Immobilised cell and enzyme technology has also been applied to whey bioconversion processes to improve the economics of such processes. This review focuses upon the elaboration of a range of immobilisation techniques that have been applied to produce valuable whey-based products. A comprehensive literature survey is also provided to illustrate numerous immobilisation procedures with particular emphasis upon lactose hydrolysis, and ethanol and lactic acid production using immobilised biocatalysts.     

Dye removal by immobilised fungi

Dyes are widely used within the food, pharmaceutical, cosmetic, printing, textile and leather industries. This has resulted in the discharge of highly coloured effluents that affect water transparency and gas solubility in water bodies. Furthermore, they pose a problem because of their carcinogenicity and toxicity. Therefore, removal of such dyes before discharging them into natural water streams is essential. For this, appropriate treatment technologies are required. The treatment of recalcitrant and toxic dyes with traditional technologies is not always effective or may not be environmentally friendly. This has impelled the search for alternative technologies such as biodegradation with fungi. In particular, ligninolytic fungi and their non-specific oxidative enzymes have been reported to be responsible for the decolouration of different synthetic dyes. Thus, the use of such fungi is becoming a promising alternative to replace or complement the current technologies for dye removal. Processes using immobilised growing cells seem to be more promising than those with free cells, since the immobilisation allows using the microbial cells repeatedly and continuously. This paper reviews the application of fungal immobilisation to dye removal.     

A new covalently modified support for gas-liquid phase sequencing

A new method for activation of glass fiber supports for immobilisation of proteins and peptides in gas-liquid phase sequencing is described. The new support offers several advantages over the presently used carrier polybrene: no precycling is required, initial yields are improved and background contamination is lower. This leads to an overall increase in detection sensitivity. The derivatisation method includes acid activation and subsequent covalent coating of glass fibers with quaternary ammonium groups thereby giving the glass surface a high binding capacity for both proteins and peptides. The activated glass has been successfully used for sequencing proteins and peptides isolated by HPLC as well as by electroelution from polyacrylamide gels.     

On a newly modified method for plastic reconstruction of the face using the skull (based on Kollman data)

Since Kollmann & Büchly (1898) presented for the first time a plastic reconstruction of the face on the skull, which was based on empiric data, this method has been improved continuously. Up to now, however, doubts on its reliability could not be removed completely. While in the meantime the reconstruction of the large organs of the face could be based on a more secured empiric basis, this was not possible concerning the remaining surface of the face. This could be surmounted using the measurements of the soft parts of the living body (34 items) proposed by Helmer (1980). Basing on these measurements the author presents a newly modified method for the plastic reconstruction of the face on the skull. Among others he accepts as basic pattern the principle of parallel profile lines suggested by Helmer (to identify skulls by means of superposition of electronic pictures). By that the author is of the opinion that he has improved the possibility of verifying and reproducing acquired results. It is recommended to fill still uncertain methodological gaps by distinct working hypothesis. However, on plastic reconstructions unclear details should be treated in such a way that they are standing out against the remaining surface. The potential scope of the plastic reconstruction of the face on the skull has been limited by Helmer's method (identification of skulls), but is basically still unchanged.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.