A Multiple Homogenizer for the rapid
preparation of samples for immunoassays and electrophoresis
Author: Alan Devenshire.
Studies of the population genetics of small organisms, such
as insects, which rely on electrophoretic or immunoassay techniques,
require analysis of many individuals to be representative. Samples
of insects for electrophoretic analysis have been homogenized
by various homogenizers such as polypropylene microcentrifuge
tubes with close-fitting glass rods. However, homogenization
of single insects followed by loading of gels with microsyringes
is the most labour-intensive and time-consuming part of the assay
procedure. When an immunoassay was developed to quantify the
activity of the enzyme responsible for resistance to insecticides
in aphids the limitations of sample preparation became even more
evident.
A multiple homogenizer was therefore designed to fit into a
standard immunoplate, allowing 96 individual samples to be homogenized
simultaneously for immunoassay or electrophoresis (ffrench-Constant & Devonshire,
Biochemical Genetics, 25, 493-499 (1987).
The homogenizer is made from a 10mm thick Perspex backing plate
of similar dimensions to an immunoplate, into which 96 Perspex
rods are inserted at 9mm spacing, to correspond precisely with
the 96 wells of the immunoplate. The tips of the rods are ground
flat, to rest uniformly on the bases of the flat-bottomed wells.
Samples are loaded into buffer in the wells of the immunoplate
and homogenized by inserting and manually rotating the homogenizer.
Rods of 4mm diameter are optimal, both for efficient homogenization
and to leave a useful volume (200 µl) in the wells when
assembled.
The volume "lost" during homogenization, quantified
by placing a range of volumes (10-200µl of PBS/Tween plus
sucrose and bromocresol purple) in separate eight-well columns
of an immunoplate and determining the volume recoverable from
all eight wells with the multipipette after insertion, rotation,
and removal of the homogenizer rods, was only 5µl regardless
homogenisation volume, provided that the homogenizer was extracted
carefully. Thus 50% and 90% of the sample was recovered reliably
when homogenized in 10 µl and 50 µl, respectively,
covering the range of volumes typically used in electrophoresis
of small organisms.
Combs with wells at 4.5 or 9mm spacing are required if samples
are to be loaded directly from an immunoplate into the wells
of an electrophoresis gel using an eight-channel multipipette.
Purpose-made combs (now available from Burkard Scientific) with
4.5mm spacing enabled 14 samples to be loaded onto each gel of
a Pharmacia GE-2/4 apparatus (3mm thick and 72mm wide) from two
rows of an immunoplate using only seven tips of the eight-channel
pipette twice to fill alternate wells, so that 56 samples could
be analyzed simultaneously in a single "run" in one
electrophoresis tank. Samples were also loaded successfully by
multipipette into the wells of a gel cast in Hoeffer equipment
(the 15-well combs. No. SE 511-15, have 9.1mm spacing) even though
the pipette tips were too large to reach into the wells of the
1.5mm tick gel.
Insects ranging from whitefly (50µg) to houseflies (20mg)
were successfully homogenized, although quantitative assessments
of efficiency were made only on aphids (ca. 400µg), using
the immunoassay for the enzyme responsible for insecticide resistance.
The mean enzyme activity extracted from individual aphids by
the multiple homogenizer was significantly greater (P < 0.001)
and less variable (P < 0.001) than that prepared in microcentrifuge
tubes.
Besides homogenizing more effectively, sample preparation time
is approximately one tenth of that needed for individual homogenization
in microcentrifuge tubes. Other time-saving aspects of the system
include ease of sample loading data recording for the shallow
and numbered wells of the immunoplate, storage of samples in
plates stacked in a freezer, and the use of multiple pipettes
for all liquid handling. The availability of centrifuges for
microplates provides the opportunity to remove tissue debris
before analysis.
With the growth of immunological assays in surveying and diagnostic
studies, this rapid sample preparation technique should have
many biological and medical applications for tissues compatible
with the usable volume (10-200µl) of the plate wells. The
technique is well suited to electrophoretic studies provided
that appropriate combs are available. Assuming no other constraints
on comb well spacing, manufacturers should consider adopting
a standard spacing of 4.5 or 9mm so that multiple pipettes can
be used more extensively in sample preparation and loading.
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