Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Summary

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Abstract

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-μm filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 μm) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Introduction

The detection and monitoring of microorganisms in complex natural microbial communities are crucial in many fields, including biomedicine, environmental ecology, and astrobiology. Cyanobacteria are prokaryotic microorganisms well-known for their ability to form blooms (excessive proliferation) of cells in fresh water. They are ubiquitous, and many species are able to produce toxins, leading not only to a potential risk for human health, but also to an ecological impact. In this regard, it is essential to develop rapid and sensitive methods for the early detection of cyanobacteria and/or their toxins in soil and water. For this purpose, a multiplex fl....

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 Vilasouto reservoir (northern Spain).
2. Quantify the number of cells using a cell counti....

Results

This work describes a multiplex immunoassay test for the simultaneous identification of the most relevant freshwater cyanobacterial species (Table 1) using the CYANOCHIP antibody microarray. The microarray can be a 3 x 8 microarray format printed onto microscope slides. Each microarray is made up of a set of 17 antibodies printed in a triplicate spot pattern, their corresponding pre-immune antibodies and BSA as negative controls. The microarrays also include a fluorescent.......

Discussion

Here, a multiplex fluorescent sandwich immunoassay using the CYANOCHIP, a 17-antibody microarray for the detection and identification of a wide range of cyanobacterial genera, is described²². These cyanobacteria represent the most frequent benthic and planktonic genera in freshwater habitats, some of them being toxin producers. Recently, the fluorescent sandwich immunoassay format has been used to identify microorganisms and/or bioanalytes in environmental applications^26.......

Materials

List of materials used in this article

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)	Thermo Fisher 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	72,704,001	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
1 M 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
1 M 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