Earl A. Timm, Jr.
timm at sc3101.med.buffalo.edu
Tue Sep 26 11:04:45 EST 2000
Hello! To all those interested in the Luminex technology,
Our laboratory has been using the Luminex system for at least 1 and 1/2
years. We presently read the samples on our FACScan. In our laboratory,
we have concentrated on screening for soluble human cytokines although we
have screened for a few mouse cytokines. The largest combination of human
cytokines in one sample has been 28. The beads are from the original lots
sold by Luminex for their assay system which was designed for the FACScan.
These beads have dyes the emit in the orange and red regions to produce
distinct populations in the FL2 vs FL3 bivariate. The system has worked
well for us. It is very easy to couple any protein to the carboxylated
beads as we have done not only for the anti-cytokine antibodies but for
other proteins such a anti-isotype antibodies used for isotype
determination. The coupling protocol takes one 8 hour day.
The sensistivity of the assay on individual beads (singleplex) approaches
and sometimes matches or surpasses that from ELISA data specified from the
manufacturer of the ELISA kits. As there is no data for multiplex ELISA
assays it is hard to make a comparison with bead multiplex sensitivity.
Costwise, the multiplex bead assay is better. Multiple screening assays
such as for cytokines can be done in a single tube using as little as 1000
microbeads per cytokine per 100 ul sample. Many times the sample volume is
limited and therefore the amount of screens using an ELISA is limited.
It is our understanding that the old bead lots will not work with the new
Luminex instrumentation and the readout for the newer beads come from
emissions which are different from the old lot beads. As I understand,
the newer beads cannot be used with a FACScan thereby necessitating the
purchase of the new instrument. I believe it would be a worthwhile
purchase. The New Luminex system uses a 96 well filter plate technology
with appears to greatly enhances throughput by semi-automating the assay
procedure. The one caveat is that there is no companion data analysis
software with the present system we use and it appears there is none with
the new system. Presently we use Microsoft Excel to produce the titer
curves and plot analysis of the raw data in mean channel fluorescence (MCF).
The best aspect of this system is that it is very flexible in terms of
assay development. Any capture antibody or other protein can be coupled to
the beads. There is no restriction as with kits in purchasing required
reagents or getting unwanted tests along with the specific tests needed.
Our cytokine beads have been very stable our the long term (1+ years)
interms of titer and sensitivity in various media such as PBS, culture and
Information about the Luminex bead system and results from laboratories
that have used the Luminex technologies can be seen at the GLIIFCA meeting
in Detroit, Michigan on October 13-15, 2000.
Earl A. Timm, Jr.
Laboratory of Flow Cytometry
Roswell Park Cancer Institute
Buffalo, New York 14263
More information about the Cytometry