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An ELISAlike timeresolved fluorescence immunoassay for microcystin detection LaMei, YingSong Wu, Nan

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La-Mei, Ying-Song Wu, Nan-Qin Gan, Li-Rong Song. Presented By: David A. Davis ... Microcystins are cyclic heptapeptide hepatoxins produced by several species of ... – PowerPoint PPT presentation

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Title: An ELISAlike timeresolved fluorescence immunoassay for microcystin detection LaMei, YingSong Wu, Nan


1
An ELISA-like time-resolved fluorescence
immunoassay for microcystin detection La-Mei,
Ying-Song Wu, Nan-Qin Gan, Li-Rong
Song.Presented By David A. Davis Micro
405Environmental MicrobiologyTopic
Competitive ELISA for detection of environmental
microbes.
  • Department of Natural Sciences

2
What are Microcystins?
  • Microcystins are cyclic heptapeptide hepatoxins
    produced by several species of water bloom
    forming cyanobacteria. Below photos are shown of
    Microcystic aeruginosa at gross, phase
    microscopic, and electron microscopic levels.
    M.aeruginosa forms water blooms like the one in
    Balgavies Loch, Dundee, Scotland, 1981.

3
Microcystin Toxin Pathology
  • PROBLEM TO BE ADDRESSED
  • Studies have shown that (MCs) are potent
    inhibitors of protein phosphatases 1 and 2a, and
    act as tumor promoters in the liver 1,2.
  • Can you tell which one is Healthy and which has
    been devastated by microcystin toxin?

4
The Abstract
  • To Improve tradition methods of detection of
    microcystin toxins. (i.e. High Performance Liquid
    Chromatography (HPLC), protein phosphatases
    inhibition assay, bioassay, (ELISA). enzyme
    linked immnosorbent).With the use of a time
    resolved fluorescence immunoassay (TRFIA), which
    is based on anti-microcystin monoclonal
    antibodies and europium-labeled IgG conjugate.
    This method is similar to ELISA, but it is
    relatively cheaper, so what quicker, and in some
    cases more efficient.ELISA Kits can range
    anywhere from 350.00 to 550.00 and up!!! This
    kits only contain 96 reaction wells.

5
Methods Materials
  • Toxin Preparation
  • Microcystin-LR was purified from Microcystis. The
    different microcystins extracts were analyzed and
    purified with (HPLC) 6
  • Antibody Production
  • (In vivo In vitro)
  • Production of anti MCLR MAbs were prepared by a
    standard method for immunization and cell fusion,
    including immunization of BLAB/c mice with
    BSA-CLR, fusion of their splenocytes with SP2/0
    myeloma cells (hybridoma) and selection of the
    hybrids and antibody-producing clones reacting
    with PLL-MCLR. 7

6
Procedure A
  • Indirect competitive time-resolved fluorescence
    immunoassay
  • MCLR-BSA concentration established with Serial
    Dilution (4µg/ml) MAbs also determined to be
    (200ng/ml)
  • Wells were washed four times with a washing
    buffer and then coated with MCLR-BSA buffer in
    Sodium Bicarbonate (pH 9.6) and incubated
    overnight at 4oC.
  • wells were then washed again and serial dilutions
    of MCLR (0, 0.005, 0.01, 0.1, 1, 5, 10, and 50
    ng/ml) were mixed with a 100µl of Mabs were
    added.
  • After 1 hour a europium-labeled anti-mouse IgC
    conjugate and a assay buffer was added to the
    well plates. Samples were then measured by a
    multilabel reader.

7
Procedure B
  • Comparison with indirect competitive ELISA
  • The indirect ELISA is used primarily to determine
    the strength and/or amount of antibody response
    in a sample, whether it is from the serum of an
    immunized animal or the cell supernatant from
    growing hybridoma clones.
  • The same plate with same coating, washing and
    incubation steps, except for the last enzyme
    reaction was carried out. Six cyanobacterial
    samples were obtained from laboratory ordinary
    cultures the cells were harvested and broken by
    sonication, and cell debris was then removed by
    centrifugation at 10,000 x g for 10 min. The
    supernatants were either directly assayed by
    ciELISA and TRFIA, or concentrated for HPLC
    analysis.

8
Results
9
Results
10
Results
11
Discussion
  • Improved sensitivity from 0.1 to 0.01ng/ml (10x)
    Base on a second universal antibodies.
    Europium-labeled anti-mouse conjugate.
  • Safer than original (TRFIA) 8,9 method that
    involved radio isotopic labels.
  • Convenience and ease has been applied in research
    areas such as clinical diagnosis and cell
    analysis.
  • A modest answer to the problem a microcystin
    toxin detection in tap water.
  • This article will more than likely lead to the
    improvement of other detection test that are
    concurrently used.

12
Critique
  • Noble
  • Concise
  • Fundamental
  • Economical
  • Relative
  • Confluent
  • Semi-innovative

13
References
  • Indirect competitive time-resolved fluorescence
    immunoassay.
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