IN VITRO AND IN SITU DETECTION OF ERWINIA AMYLOVORA WITH MONOCLONAL ANTIBODIES

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

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Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens

Blood ◽

10.1182/blood.v67.5.1257.bloodjournal6751257 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1257-1264 ◽

Cited By ~ 1

Author(s):

R Andreesen ◽

KJ Bross ◽

J Osterholz ◽

F Emmrich

Keyword(s):

Monoclonal Antibodies ◽

Cell Surface ◽

Cell Lineage ◽

Culture Conditions ◽

Human Macrophage ◽

Blood Monocytes ◽

Differentiation Antigens

We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site-specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo.

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In Situ Detection of Mycobacterium tuberculosis Transcripts in Human Lung Granulomas Reveals Differential Gene Expression in Necrotic Lesions

Infection and Immunity ◽

10.1128/iai.70.11.6330-6338.2002 ◽

2002 ◽

Vol 70 (11) ◽

pp. 6330-6338 ◽

Cited By ~ 115

Author(s):

Gael Fenhalls ◽

Liesel Stevens ◽

Lorraine Moses ◽

Juanita Bezuidenhout ◽

Joanna C. Betts ◽

...

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Transition Zone ◽

Expression Of Genes ◽

Metabolic States ◽

Necrotic Region ◽

Rna In Situ Hybridization ◽

In Situ Detection

ABSTRACT We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host.

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In situ detection of amastigotes in American cutaneous leishmaniasis, using monoclonal antibodies

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1016/0035-9203(86)90183-5 ◽

1986 ◽

Vol 80 (1) ◽

pp. 6-9 ◽

Cited By ~ 13

Author(s):

Neil R Lynch ◽

Caridad Malavé ◽

R.Benito Ifante ◽

Robert L Modlin ◽

Jacinto Convit

Keyword(s):

Monoclonal Antibodies ◽

Cutaneous Leishmaniasis ◽

American Cutaneous Leishmaniasis ◽

In Situ Detection

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Foetal Human Melanocytes: In Situ Detection, In Vitro Culture and Differentiation Characteristics at 6–11 Weeks EGA

Pigment Cell Research ◽

10.1111/j.1600-0749.1996.tb00100.x ◽

1996 ◽

Vol 9 (3) ◽

pp. 126-133 ◽

Cited By ~ 4

Author(s):

I. CAROLINE LE POOLE ◽

RENE M.J.G.J. VAN DEN WIJNGAARD ◽

RONALD P. VERKRUISEN ◽

WOUT H. LAMERS ◽

DIRK TROOST ◽

...

Keyword(s):

In Vitro Culture ◽

Human Melanocytes ◽

In Situ Detection

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Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.7.1865115 ◽

1991 ◽

Vol 39 (7) ◽

pp. 987-992 ◽

Cited By ~ 6

Author(s):

K Gerritse ◽

M Fasbender ◽

W Boersma ◽

E Claassen

Keyword(s):

Alkaline Phosphatase ◽

Synthetic Peptide ◽

Enzyme Linked Immunosorbent Assay ◽

Sufficient Degree ◽

Antipeptide Antibody ◽

In Situ Detection ◽

Antibody Secreting Cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.

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Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production

Applied and Environmental Microbiology ◽

10.1128/aem.68.7.3550-3559.2002 ◽

2002 ◽

Vol 68 (7) ◽

pp. 3550-3559 ◽

Cited By ~ 41

Author(s):

Francisco J. Moralejo ◽

Rosa Elena Cardoza ◽

Santiago Gutierrez ◽

Marta Lombraña ◽

Francisco Fierro ◽

...

Keyword(s):

Antisense Rna ◽

Aspergillus Awamori ◽

Gene Encoding ◽

Ph Values ◽

Reverse Transcription Pcr ◽

Antisense Mrna ◽

In Situ Detection ◽

In Vitro Treatment

ABSTRACT Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.

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Generation of monoclonal antibodies detecting specific epitopes in locust antennae

Journal of Experimental Biology ◽

10.1242/jeb.175.1.45 ◽

1993 ◽

Vol 175 (1) ◽

pp. 45-57

Author(s):

J. Strotmann ◽

I. Boekhoff ◽

S. Goggerle ◽

H. Breer

Keyword(s):

Molecular Weight ◽

Monoclonal Antibody ◽

Protein Synthesis ◽

Monoclonal Antibodies ◽

Embryonic Development ◽

High Molecular Weight ◽

Time Course ◽

Antigen Expression

1. Following a tissue-specific screening paradigm, monoclonal antibodies have been generated that interact with distinct subpopulations of cells in locust antennae. 2. Antigens were identified as high molecular weight components. 3. Immunoreactivity was not detectable during embryonic development, but rapidly appeared within a few hours of hatching. 4. The time course of antigen expression in antennal cells could be followed in situ as well as in vitro. 5. Expression of monoclonal antibody B14/6D2-like immunoreactivity was prevented by blocking protein synthesis with cycloheximide.

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