A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Overview

This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

Protocol

1. Preparation of the Immunogens

1. Grow each cyanobacterial strain in the corresponding culture medium under conditions described in Table 1.  
   NOTE: Growth medium and culture conditions for each cyanobacteria strain are listed in Table 1. All the cyanobacterial strains, with the exception of K17, belong to Antonio Quesada's group from Autonoma University (Madrid, Spain). The antibody against Planktothrix rubescens was generated from a natural sample of the monospecific bloom of this cyanobacterium from the Vilasouto reservoir (northern Spain).
2. Quantify the number of cells using a cell counting chamber by optical microscopy to obtain approximately 10⁸ cells/mL from a late exponential or stationary growth phase culture.
3. Harvest cells from 5 mL of culture by centrifugation at 2,000 x g for 5 min.
4. Discard the supernatant and resuspend the cells in a 10-mL tube with 5 mL of 1x phosphate-buffered saline (1x PBS) to obtain around 10⁸ cells/mL.
5. Homogenize and lyse the cells of the suspension by sonication for 5 cycles, 30 s each, with a 30- to 60-s pause on ice, using a portable, hand-held ultrasonic processor or by dipping the tube into the water bath of a cell disruptor at maximum amplitude (30 kHz).
6. Repeat steps 1.3 - 1.5 for each strain of cyanobacterium.     
   NOTE: Sonication produces cell disruption and cellular content release. While proteins and polysaccharides are good immunogens, specific molecules, such as lipids and nucleic acids, do not induce a humoral immunogenic response by themselves. Therefore, they must bind to carriers, such as polysaccharides or proteins, to increase the molecular complexity. In addition, sonication releases intracellular material that can trigger antibody production.

2. Production of Polyclonal Antibodies

1. Prepare the first immunogen dose by mixing 0.5 mL of the ultrasonicated cell lysate obtained in step 1.5 with 0.5 mL of complete Freund's adjuvant. Deliver it to the operator of an animal facility for polyclonal rabbit antibody production.
2. Prepare three more doses as before for further use as memory boosts in the antibody production process by mixing 0.5 mL of the same homogenate/lysate obtained in step 1.5 with 0.5 mL of incomplete Freund's adjuvant. Give them to the animal facility to fulfill the antibody production process.
3. Repeat steps 2.1 and 2.2 for each new antibody production process.         
   NOTE: Normally, antibody production is entrusted to specialized animal facilities or companies because appropriate licensing and training are required to work with animals. The companies supply a relative enzyme-linked immunosorbent assay (ELISA) measurement of the quantity of antigen-specific antibodies present in the serum sample.

3. Antibody Purification

1. Purify the immunoglobulin G (IgG) fraction from both the immune and the pre-immune serum collected in step 2 by protein-A affinity chromatography. Some commercial purification kits, based on cartridge systems, work well; follow the provider instructions.
2. When the purification kit does not provide a desalting system, change the buffer after the elution of the purified antibodies to 0.1x PBS, either by dialysis or by using centrifugal filter devices with a 100-kDa (or lower) membrane pore size.
3. Determine the antibody concentration by measuring the absorbance at 280 nm or by using colorimetric methods, such as Bradford, Lowry, or Bicinchoninic acid (BCA).
   NOTE: It is important to avoid including amine groups in the elution buffer (e.g., Tris buffers) because they compete with the antibody for binding to solid surfaces activated with epoxy groups. After purification, it is important to test the antibody activity with ELISA.

4. Fluorescence Antibody Labeling

1. Label the purified antibodies obtained in section 3 with a fluorochrome (e.g., a far-red fluorescent dye) by dissolving a commercial vial containing dye for labeling 1 mg of protein into 100 µL of dimethyl sulfoxide (DMSO). Add 2 µL of the dissolved dye to each antibody preparation at a concentration of 2 mg/mL in a final volume of 50 µL in 50 mM phosphate-buffered saline (pH 8.5).
2. Maintain the labeling reactions under continuous agitation for 1 h at ambient temperature and 1,200 rpm on a vibrating platform.
3. Purify the labeled antibodies by size exclusion chromatography (e.g., using a gel with a fractionation range between 1.5 and 30 kDa trapped into a column), following supplier recommendations.
4. Measure the absorbance at 280 nm and at 650 nm in eluates and calculate the labeling efficiencies following supplier recommendations.         
   NOTE: Up to 50 x 50-µL antibody-labeling reactions can be done with a single vial of the fluorescent dye for labeling 1 mg of protein. It is recommended to cover the tubes with aluminum foil or, alternatively, to use opaque 0.5-mL tubes to avoid quenching processes after step 4.3. For IgG antibodies, optimal labeling is achieved with 3-7 mol of the dye per mol of antibody.

5. CYANOCHIP Production

1. Antibodies and controls in printing solution
   1. Prepare 30 µL of each purified antibody in printing solution by mixing each antibody at 1 mg/mL in a commercial 1x protein printing buffer with 0.01% (v/v) Tween 20 (a non-ionic detergent), all as final concentrations.
      NOTE: Alternatively, an antibody solution may contain 20% glycerol, 1% (w/v) sucrose (or trehalose, as a preservative), and 0.01% (v/v) Tween 20 in carbonate buffer (pH 8.5).
   2. Prepare 30 µL of the printing solution as controls: (a) 1x protein printing buffer with 0.01% (v/v) Tween 20, (b) protein-A purified pre-immune serum at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20, and (c) bovine serum albumin (BSA) at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20.
   3. Prepare 30 µL of a fluorescently labeled, purified pre-immune serum at different concentrations (e.g., from 50 µg/mL to 1 µg/mL) in 1x protein printing buffer with Tween 20, as in step 5.1.1. These samples will be used as fluorescent frame markers and for relative fluorescence quantification after spotting.
   4. Add 30 µL per well of the printing solutions prepared in steps 5.1.1, 5.1.2, and 5.1.3 to the highest-quality 384-well microplates for microarray manufacturing, such as polypropylene. 
      NOTE: Protein printing buffer increases the quality and stability of the antibodies, and Tween 20 homogenizes spot morphology and builds up protein coupling up. It is recommended to maintain the 384-well microplate at 4 °C before use. Store it at -20 °C for long periods of time. Polypropylene has low DNA, protein, cell extract, and small-molecule intrinsic binding.
2. Antibody printing onto a microscope slide
   NOTE: Print the antibodies onto activated microscope slides by using different array platforms, like contact, split-needle, or non-contact devices, such as piezoelectric printers or "ink jet" technologies. In this work, the CYANOCHIP has been routinely printed by contact using a robotic system (arrayer) capable of spotting nL quantities of the antibodies at the µm scale.
   1. Set the environmental conditions of the printing room to 20 °C and 40 - 50% relative humidity.
   2. Set up the slide substrates (e.g., 75- x 25-mm epoxy-activated microscope glass slides) to perform several identical antibody arrays on each slide.
   3. Spot each purified antibody, including controls and the reference frame, in a triplicate spot pattern; under these conditions, the spots are 180 - 200 µm in diameter.
   4. After printing, leave the slides for at least 30 min at ambient temperature to let them dry, and then store them at 4 °C; for working in the field, the slides can be transported and stored at ambient temperature for several months.
      NOTE: The CYANOCHIP is printed in a triplicate spot microarray format with 3 x 8 identical microarrays per slide or 9 identical arrays in a 1 x 9 microarray format. Each array size must not be higher than the reaction chamber dimensions for a 24-well gasket (usually 7.5 x 6.5 mm in a 3 x 8 hybridization chamber).
   5. Before using the microarray to analyze environmental samples, use fluorescent sandwich microarray immunoassay (FSMI) to determine the working dilution for each antibody in a titration curve. For each antibody, use a standard concentration of the corresponding immunogen (10³ - 10⁴ cells/mL) and serial dilutions of the fluorescent antibody (between 1:500 and 1:32,000). The optimal antibody concentration corresponds to 50% of the maximum signal intensity obtained in the titration curve. Also, the sensitivity and specificity for each antibody must be determined, as described in Blanco et al.

6. Preparation of Environmental Multianalyte Extracts for the Fluorescent Sandwich Microarray Immunoassay (FSMI)

1. Multianalyte extract from a liquid sample
   1. Take 1 - 100 mL of the liquid sample with a sterile syringe (e.g., water from the shore of a water reservoir); the amount of sample is greatly dependent upon the potential concentration of the targets.
   2. Concentrate the cells by passing the water sample through a 3-µm pore size, 47-mm diameter polycarbonate filter; a cellular concentration between 10³ - 10⁸ cells/mL is desirable for positive detection, but the actual concentration is unknown.
   3. Recover the biomass collected in the filter with 1 mL of a modified Tris-buffered saline, Tween 20-reinforced buffer (TBSTRR; 0.4 M Tris-HCl (pH 8), 0.3 M NaCl, and 0.1% Tween 20) by scraping it with a spatula into a 15-mL tube.
   4. Homogenize and disaggregate by using a hand-held ultrasonic processor, as described in step 1.5, or just by pipetting up and down multiple times; this prepares the sample for analysis by the microarray.
2. Multianalyte extract from a solid sample
   1. Weigh up to 0.5 g of the solid sample (e.g., rock, soil, or sediments) into a 10-mL tube and add up to 2 mL of TBSTRR.
   2. Sonicate by immersing the sonicator probe in the tube, by dipping the tube into the water bath of a powerful sonicator horn, or by using a hand-held sonicator. Perform at least 5 x 30-s cycles at 30 kHz, stopping for 30 s while on ice.
   3. Filter to remove sand, clay, and other coarse material with a 10-mL syringe coupled to a 10- to 12-mm diameter, 5- to 20-µm pore size nylon filter holder. Push the sample through the filter into a 1.5-mL tube. If the filter saturates, agitate the suspension in the syringe and take it to a new one; this prepares the filtrate material for the immunoassay (step 7).     
      NOTE: The buffering capacity of TBSTRR depends on the type of sample. It is important to carry out the immunoassay immediately after the preparation of the environmental extract to avoid the effect of enzymatic degradation on the analytes. Alternatively, add protease inhibitors into the environmental extract and freeze at -80 °C until the next step.

7. Fluorescent Sandwich Microarray Immunoassay (FSMI)

1. Blocking the CYANOCHIP        
   NOTE: Immediately before use, treat the printed slides to block all free epoxy groups on the slide and to remove the excess of non-covalently bound antibodies.
   1. Immerse the microarray into 0.5 M Tris-HCl (pH 9) with 5% (w/v) BSA solution on a clean surface (e.g., Petri dish or a 50-mL tube) with mild agitation from a rocker platform for 5 min. Alternatively, lay the slide down onto a 100- to 200-µL drop of the above solution with the microarray spots facing it. Leave for 3 - 5 min and then proceed.
   2. Carefully pick up the slide using plastic-tipped forceps; try to avoid touching microarray zones. Eliminate the excess of liquid by softly knocking the slide onto a paper towel. Immerse it in 0.5 M Tris-HCl (pH 8) with 2% (w/v) BSA solution for 30 min with mild agitation from a rocker platform.
   3. Dry the slide by performing a short centrifugation (200 - 300 x g for 1 min) using a commercial microcentrifuge adapted for microscope slides. Alternatively, dry the slide by softly knocking onto a paper towel.
2. Incubation of the multianalyte sample extract with the microarray
   1. Set up the slide in a commercial microarray hybridization cassette with 24 wells for multiple microarrays; follow the provider instructions.
   2. After slide and cassette assembly, pipette up to 50 µL of the sample extract or a dilution of it in TBSTRR into each well of the cassette.
   3. Repeat step 7.2.2 for each sample to be analyzed.
   4. As a blank control, pipette 50 µL of TBSTRR buffer into at least two separate wells of the cassette.
   5. Incubate at ambient temperature for 1 h with mixing by pipetting every 15 min or by leaving it under mild shaking. Alternatively, incubate for 12 h at 4 °C.
      NOTE: Use other incubation gaskets as a function of the microarray pattern. The time and temperature of the incubation in step 7.2.5 are empirical parameters that normally depend on the affinity and binding kinetics of each paired antigen-antibody.
3. Washing
   1. Remove the samples by putting the cassette down and carefully knocking it onto a clean, absorbent paper.
   2. Wash the wells by adding 150 µL of TBSTRR to each one, and eliminate the buffer, as above.
   3. Repeat step 7.3.2 three more times.
4. Incubation with fluorescent detector antibodies
   1. Add 50 µL of an antibody mixture containing the 17 anti-cyanobacterial-strain antibodies, each labeled with the fluorochrome in TBSTRR with 1% (w/v) BSA. Determine the concentration of each fluorescent antibody in the mixture (from 0.7 to 2 µg/mL) by performing titration experiments of each antigen/antibody pair.
   2. Incubate for 1 h at ambient temperature, as described in step 7.2.5, or for 12 h at 4 °C.
5. Washing out the fluorescent antibodies
   1. Remove the fluorescent unbound antibodies, as in step 7.3.
   2. Disassemble the cassette and immerse the slide into 0.1x PBS (e.g., in a 50-mL tube) for a quick rinse.
   3. Dry the slide as in step 7.1.3.

8. Scanning for Fluorescence

1. Scan the slide for fluorescence at the maximum emission fluorescence peak for far-red fluorescent dye in a scanner for fluorescence. Take several images of the microarrays at different scanning parameters, generally by lowering the laser gain value.
   NOTE: Avoid saturated spots (> 65,000 fluorescence counts)—they are out of scale and may introduce quantification errors.

Table 1. List of the Antibodies (Abs) and the Cyanobacterial Strains Used to Produce the CYANOCHIP.

Ab code	Immunogen (strain)	Order	Habitat	Medium	Culture conditions
K1	Anabaena sp.	Nostocales	unknown	BG11 and nitrate	30 ºC, continuous light
K2	Anabaena sp.	Nostocales	unknown	BG11o	30 ºC, continuous light
K3	Microcystis flosaquae	Chroococcales	planktonic	BG11	28 ºC, continuous light
K4	Microcystis novacekii	Chroococcales	planktonic	BG11	28 ºC, continuous light
K5	Microcystis aeruginosa	Chroococcales	planktonic	BG11	28 ºC, continuous light
K6	Aphanizomenon ovalisporum	Nostocales	planktonic	BG11o	28 ºC, continuous light
K7	Phormidium sp.	Oscillatoriales	benthic	BG11	18 ºC, 16-8 photoperiod
K8	Rivularia sp.	Nostocales	benthic	CHU-D	18 ºC, 16-8 photoperiod
K9	Chamaesiphon sp.	Chroococcales	benthic	BG11	18 ºC, 16-8 photoperiod
K10	Leptolyngbya boryana	Oscillatoriales	benthic	BG11	18 ºC, 16-8 photoperiod
K11	Tolypothrix distorta	Nostocales	benthic	BG11o	18 ºC, 16-8 photoperiod
K12	Aphanizomenon aphanizomenoides	Nostocales	planktonic	BG11o	28 ºC, continuous light
K13	Nostoc sp. (Antarctica)	Nostocales	benthic	BG11o	13 ºC, 16-8 photoperiod
K14	Anabaena sp.	Nostocales	benthic	BG11o	13 ºC, 16-8 photoperiod
K15	Leptolyngbya sp.	Oscillatoriales	benthic	BG11	13 ºC, 16-8 photoperiod
K16	Tolypothrix sp.	Nostocales	benthic	BG11	13 ºC, 16-8 photoperiod
K17	Planktothrix rubescens	Oscillatoriales	planktonic	none	none

Materials

Name	Company	Catalog Number	Comments
0.22 mm pore diameter filters	Millipore	GSWP04700	For preparation of immunogens
Eppendorf  5424R microcentrifuge	Fisher Scientific		For preparation of immunogens
Phosphate buffer saline (PBS) pH 7.4 (10X)	Thermofisher Scientific	70011036	50 mM potassium phosphate, 150 mM NaCl, pH 7.4
Ultrasonic processor UP50H	Hielscher		For preparation of immunogens
Complete Freund's adjuvant	Sigma-Aldrich	F5881	Immunopotentiator
Incomplete Freud's adjuvant	Sigma-Aldrich	F5506	For boost injections
Protein A antibody purification kit	Sigma-Aldrich	PURE1A	For isolation of IgG
Centrifugal filter devices MWCO<100 KDa	Millipore	UFC510096-96K	For isolation of IgG
Dialysis tubings, benzoylated	Sigma-Aldrich	D7884-10FT	For isolation of IgG
Illustra Microspin G-50 columns	GE-HealthCare	GE27-5330-02	For isolation of IgG
Bradford reagent	Sigma-Aldrich	B6916-500 mL	To quantify the antibody concentration
MicroBCA protein assay kit	Thermo Scientific	23235	To quantify the antibody concentration
Protein arraying buffer 2X	Whatman (Sigma Aldrich)	S00537	Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20
Tween 20	Sigma-Aldrich	P9416	Non-ionic detergent
Bovine serum albumin (BSA)	Sigma-Aldrich	A9418	Control  for printing; blocking reagent
384-wells microplate	Genetix	X6004	For antibody printing
Robot arrayer for multiple slides	MicroGrid II TAS arrayer from Digilab		For antibody printing
Epoxy substrate glass slides	Arrayit corporation	VEPO25C	Solid support for antibody printing
Alexa Fluor-647 Succinimidyl-ester	Molecular probes	A20006	Fluorochrome
DMSO	Sigma-Aldrich	D8418	Fluorochrome dissolvent
Heidolph Titramax vibrating platform shaker	Fisher Scientific		For antibody labeling
Illustra Microspin G-50 columns	Healthcare	27-5330-01	For purification of labeled antibodies
Safe seal brown 0,5 ml tubes	Sarstedt	7,27,04,001	For labeled antibodies storage
Nanodrop 1000 spectrophotometer	Thermo Scientific		To quantify antibody concentration and labeling efficiency
3 µm pore size polycarbonate 47 mm diameter filter	Millipore	TMTP04700	To concentrate cells
1M Trizma hydrochloride solution pH 8	Sigma-Aldrich	T3038	For TBSTRR preparation; to block slides
Sodium chloride	Sigma-Aldrich	S7653	For TBSTRR preparation
20 µm nylon filters	Millipore	NY2004700	For environmental extract preparation
10-12 mm filter holders	Millipore	SX0001300	For environmental extract preparation
Protease inhibitor cocktail	Sigma-Aldrich	P8340	For environmental extract storage
1M Trizma hydrochloride solution pH 9	Sigma-Aldrich	T2819	To block slides
Heidolph Duomax 1030 rocking platform shaker	VWR		To block slides; for incubation processes
VWR Galaxy miniarray microcentrifuge	VWR	C1403-VWR	To dry slides
Multi-Well microarray hybridization cassette	Arrayit corporation	AHC1X24	Cassette for 24 assays per slide
GenePix 4100A microarray scanner	Molecular Devices		Scanner for fluorescence
GenePix Pro Software	Molecular Devices		Software for image analysis and quantification