The overall goal of this procedure is to determine the presence of multiple cyanobacterial strains in liquid and solid samples using a fluorescence microarray immunoassay. This method can help answer key questions in the environmental, monitoring and water management fields, such as the presence of toxins produced protoxin-producing bacteria before the blooms become visible to the naked eyes. The main advantage of this technique is that multiple cyanobacterial strains and multiple samples can be monitored simultaneously in a three, four-hour period, both in situ or in the laboratory.
So, these method can provide insight into biodiversity. It can also be applied to other fields, such as monitoring agricultural installations, life detection in strain environments or planetary exploration. Generally, individuals new to this method will not struggle.
Because of the simplicity in the sample processing and the easier the interpretation of the results. After producing and purifying antibodies according to the text protocol, add 100 microliters of DMSO into a commercial vial containing dye for labeling, one milligram of protein. Once the dye is dissolved, label the purified antibodies by adding two microliters of the dye to 50 microliters of each antibody preparation.
Maintain the labeling reactions at ambient temperature under continuous agitation on a vibrating platform for one hour. After purifying the labeled antibodies according to the text protocol, load a small aliquot into a spectrophotometer and measure the absorbance at 280 and 650 nanometers. Calculate the labeling efficiencies by following the supplier recommendations.
To make a CYANOCHIP, prepare 30 microliters of each purified antibody by mixing one milligram per milliliter of antibody in a 1X protein printing buffer with 01%volume per volume of Tween 20. Prepare 30 microliters of the following printing solutions as controls. 1X protein printing buffer with 01%of volume per volume of Tween 20.
Each protein A purified preimmune serum at one milligram per milliliter in 1X protein printing buffer with 01%Tween 20 and one millgram per milliliter of BSA and 1X protein printing buffer with 01%Tween 20. Add 30 microliters per well of the prepared printing solutions to the high-quality 384 well microplates. After setting up the printing robot and slide substrates according to the text protocol, spot all the antibodies and the controls onto the slides in a triplicate spot pattern approximately 180 to 200 micron spot diameter.
For sampling in the environment, use a sterile syringe to collect one to 100 milliliters of liquids and concentrate the cells by passing the sample through a three micron pore size filter. If needed, collect solid samples from rocks or soils and keep them in sterile tubes. Samples can be assayed in a small field lab.
Add one milliliter of extraction incubation buffer to recover the biomass collected in the filter and use a spatular to scrape the filter into a 15 milliliter tube. With a handheld ultrasonic processor, or by pipetting up and down several times, this aggregate and homogenize the sample. In addition, weigh up to 0.5 grams of the solid sample in a separate 10 milliliter tube and add up to two milliliters of extraction buffer.
Immerse a sonicator probe into the tube and sonicate with a handheld ultrasonic processor or other ultrasonication device. While keeping the sample on ice, perform at least five 30 second cycles at 30 kilohertz, stopping for 30 seconds in between sonications. To prepare a filtrate extract for the immunoassay, use a 10 milliliter syringe coupled to a five to 20 micron pore size nylon filter holder to filter the solution into a 1.5 milliliter tube to remove sand, clay and other coarse material.
With the microarray spots facing down, lay the slide onto a 100 to 200 microliter drop of blocking solution and incubate for three to five minutes. Using plastic-tipped forceps, and avoiding the microarray zones, carefully pick up the slide. Immerse the slide into a fresh blocking solution and incubate the slide with mild agitation for 30 minutes.
To dry the slide, place it in a centrifuge adapted for microscope slides and briefly spin it. Set up the slide in a three by eight well microarray hybridization cassette following the provider's instructions. Alternatively, after modifying the antibody printing pattern, set up a custom-made cassette, such as the one shown here, or simply use cover slides for the setup.
Following slide and cassette assembly, pipette up to 50 microliters of each sample extract or dilution of it in extraction incubation buffer into each well of the cassette. Alternatively, load samples into other incubation devices or simply use cover slides. As a blank control, pipette 50 microliters of TBSTRR buffer into at least two separate wells of the cassette.
Incubate the samples at ambient temperature for one hour and mix by pipetting every 15 minutes, or incubate at four degrees Celsius for 12 hours. To wash the microarrays, place the cassette down and carefully tap it onto clean absorbent paper. Then wash the wells by adding 150 microliters of extraction incubation buffer to each one and remove the buffer by tapping on absorbent paper as before.
Repeat the wash three more times. To each well, add 50 microliters of the fluorescent antibody mixture that contains the 17 anti-cyanobacterial strain antibodies. Incubate the samples at ambient temperature for one hour or at four degrees Celsius for 12 hours.
To wash the microarrays, carefully place the cassette down and gently tap it onto clean absorbent paper. Then add 150 microliters of extraction incubation buffer before removing it by tapping onto a paper towel. Repeat the wash three times.
Then disassemble the cassette and immerse the slide in 0.1X PBS solution for a quick rinse. Quickly centrifuge as before to dry the slide. Using a fluorescent scanner, scan the slide for fluorescence at the maximum emission peak for far red fluorescent dye.
Take several images of the microarrays at different scanning parameters by changing the gain value. Finally, load the grid, quantify, then process and analyze the data according to the text protocol. The CYANOCHIP multiplex immunoassay demonstrated in this video, was used to test for the simultaneous identification of the most relevant freshwater cyanobacterial species as listed in this table.
Water samples collected at the shore of a freshwater reservoir, were analyzed using the microarray assay. The main positive immuno reactions corresponded to antibodies to planktonic cyanobacteria microcystis species and to the benthic leptolyngbya species. Lower fluorescent signals were obtained for planktonic nostocales aphanizomenon species, anabaena species, microcystis flos-aquae and the benthic rivularia species.
The microarray was also validated by assaying an antarctic dry microbial mat to test for the presence of cyanobacterial markers. Positive reactions in the antibodies were produced to the benthic cyanobacteria, anabaena species and leptolyngbya species. Additional positive immuno reactions were detected with antibodies to planktonic cyanobacteria, such as microcystis species, aphanizomenon species and planktothrix rubescens species.
Finally, those signals were obtained from antibodies to other benthic species, including tolypothrix species and anabaena species. Once mastered, this technique can be done in three, four hours, if it is performed properly, excluding antibody production and leveling. By simultaneous incubation of sample, and fluorescent antibodies, it can be save one hour.
While attempting these procedures, it is important to remember to get the fluorescent antibody mixture ready with its antibody at its optimal working dilution. Following these procedures, other methods, like optical microscopy or DNA extraction and a specific PCR amplification, can be perform in order to answer additional questions, like the soil concentration or to confirm the type of strain detected. After its development, this technique will pave the way for researchers in the field of environmental monitoring to explore water bodies, in a biculture processes, fresh water management or microbial ecology studies, regardless of the type of the sample.
After watching this video, you should have a good understanding of how to label antibodies with fluorescence, manipulate antibody microarrays, perform multiplex immunoassay and analyze and interpret microarray immunoassay results. Don't forget that working with ultrasounds can hazardous and precautions such as wearing ear-protection devices or other specific care measures should always be taken while performing these procedures.
