Methods for derivation and detection of anti-parasite monoclonal antibodies

Pearson, Terry W.; Pinder, Margaret; Roelants, Georges E.; Kar, Sasmita; Lundin, Lennart; Mayor-Withey, Kathleen S.; Hewett, Rosemary S.

doi:10.1016/0022-1759(80)90168-4

Cited by 91 publications

(28 citation statements)

References 14 publications

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“…Hybridomas secreting MAbs to C. jejuni VC74 flagellin were prepared by the procedure of Pearson et al (26). Briefly, a BALB/c mouse was injected intraperitoneally with an emulsion containing Freund complete adjuvant and 50 ,ug of flagellin isolated by the procedure outlined above.…”

Section: Methodsmentioning

confidence: 99%

Location of epitopes on Campylobacter jejuni flagella

Logan¹,

Trust²

1986

J Bacteriol

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Flagella were isolated from strains of Campylobacterjejuni belonging to different heat-labile serogroups and from a strain of Campylobacter fetus, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the flagellin molecular weights (Mr) were approximately 62,000. The flagellins were cleaved by hydrolysis with cyanogen bromide, and sodium dodecyl sulfate-urea peptide gel electrophoresis showed that the C. jejuni flagellins were structurally similar, and differed from C. fetus flageUin. Immunochemical analysis by Western blotting, enzyme-linked immunosorbent assay, immune electron microscopy, and immunoprecipitation with polyclonal and monoclonal antibodies revealed the presence of both internal and surface-exposed epitopes. The internal epitopes were antigenically cross-reactive and linear, and in the case of C. jejuni flagellin were located on cyanogen bromide peptides of apparent Mr 22,400 and 11,000. Antigenically cross-reactive epitopes were also present on an Mr 43,000 cyanogen bromide peptide of C. fetus flageilin. The Mr 22,400 peptide of C. jejuni VC74 flageilin also carried closely positioned internal linear epitopes for two monoclonal antibodies. One epitope was strain specific, while the other was shared by some but not all Campylobacter flageilins. The flagella of C. jejuni VC74 also displayed both surface-exposed antigenically cross-reactive and surface-exposed serospecific epitopes. Both linear and conformational epitopes contributed to the serospecificity of C. jejuni VC74 flagella, and a linear serospecific epitope was located on a cyanogen bromide peptide of apparent Mr 4,000.

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Section: Methodsmentioning

confidence: 99%

Location of epitopes on Campylobacter jejuni flagella

Logan¹,

Trust²

1986

J Bacteriol

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“…After 3 to 5 days of incubation in growth medium (GM) containing RPMI 1640 (Flow Laboratories), 10 % heat-inactivated foetal calf serum, 20 mM-HEPES buffer, 20 mM-glutamine, 5 x 10 -5 M-2-mercaptoethanol, antibiotics and 20% T cell growth factor (TCGF), the cultures were assayed for lytic activities, and active bulk CTL cultures expanded for 4 weeks and then cloned first by soft agarose plating and then by the limiting dilution technique in the presence of TCGF (Lotze et al, 1980). For soft agarose cloning, 4 ml amounts of 0.5% agarose medium (Pearson et al, 1980) containing 20% TCGF were plated into 35 mm Petri dishes, and the plates incubated at 4 °C for 10 min. The cells from bulk CTL cultures, usually 103 or 104, were suspended in 0.1 ml of GM-TCGF and mixed with 0.5 % agarose medium.…”

Section: Discussionmentioning

confidence: 99%

Protection of Mice from Fatal Herpes Simplex Virus Type 1 Infection by Adoptive Transfer of Cloned Virus-specific and H-2-restricted Cytotoxic T Lymphocytes

Sethi

Omata

Schneweis

1983

Journal of General Virology

137

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“…A BALB/c mouse was inoculated intraperitoneally with 6, 16, and 16 tick equivalents of live purified sporozoites on days 0, 14, and 42, respectively. Three days after a final intravenous inoculation of 4 tick equivalents of sporozoites on day 204, the mouse was killed, its spleen was removed, and splenocytes were fused with X63-Ag8.653 myeloma cells (21) by a method based on a standard protocol (22). Hybridoma cultures secreting antisporozoite antibody, as detected by indirect immunofluorescence on formalin-fixed sporozoites (4), were selected for expansion and cloning.…”

Section: Methodsmentioning

confidence: 99%

Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody.

Williamson

Tait

Brown

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Theileria annulata is an economically important protozoan parasite that threatens an estimated 250 million cattle with the disease tropical theileriosis. Development of a defmed subunit vaccine is one means of trying to develop control measures against the disease. To this end we have characterized a surface antigen complex of the infective stage (sporozoite), by using a monoclonal antibody that neutralizes sporozoite infectivity in vitro. We have cloned the gene coding for this complex and have demonstrated that a fusion protein expressed from a fragment of this gene elicits strong neutralizing antibodies. Furthermore we provide data on the structure and expression of this gene. In particular we show that the region of the gene, expressed in one clone, codes for a protein segment relatively rich in proline residues. Also we demonstrate that expression of this gene appears to be stage specific, transcripts being present only in the sporoblast and sporozoite stages. The relevance of these rmdings to the production of a defined subunit vaccine is discussed.Tropical theileriosis threatens an estimated 250 million cattle in the Mediterranean littoral, North Africa, the Middle East, the Indian subcontinent, and Central Asia and acts as a major constraint on livestock production and improvement in many developing countries (1). The disease is caused by a tickborne protozoan, Theileria annulata, which enters the bovine host as the infective sporozoite stage in the saliva of a feeding tick. These sporozoites rapidly invade mononuclear cells in which the macroschizont stage develops; subsequently these parasites are liberated, whereupon they invade erythrocytes producing the piroplasm stage of the life cycle (2). Present methods of vaccination depend upon establishing active infection (3) and thus incur both the risk of causing clinical disease and the problems of handling live parasite material under tropical conditions. For these reasons we are actively engaged in identifying parasite antigens capable of inducing a protective immune response (4-6) using similar approaches to those followed by investigators working on Theileria parva, the cause of East Coast fever (7-11). Here we describe an antisporozoite monoclonal antibody (mAb) that exhibits significant sporozoite neutralizing activity and provide data on the nature of the polypeptides recognized by this antibody. In addition we describe the isolation of two recombinant DNA clones that express the epitope defined by this mAb and we report our findings on the structure and expression of this gene(s). § MATERIALS AND METHODSParasite Material. Three uncloned isolates of Theileria annulata were used in this study: they are called Ankara, from Turkey (12); Hissar, from India (13); and Gharb, from Morocco (14). Sporozoites for mAb production, immunofluorescence tests, inhibition assays, and immunoblotting were obtained from Theileria annulata-infected adult ticks (Hyalomma anatolicum anatolicum). These ticks were fed on rabbits for 3 days to stimulate sporozoite m...

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Methods for derivation and detection of anti-parasite monoclonal antibodies

Cited by 91 publications

References 14 publications

Location of epitopes on Campylobacter jejuni flagella

Location of epitopes on Campylobacter jejuni flagella

Protection of Mice from Fatal Herpes Simplex Virus Type 1 Infection by Adoptive Transfer of Cloned Virus-specific and H-2-restricted Cytotoxic T Lymphocytes

Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody.

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