We have developed a simple enzyme-linked immunosorbent assay (ELISA) for the direct determination of chitosan-like polysaccharide (CP) produced in the cultures of Citrobacter strains. CP in sample solutions was reacted with anti-CP rabbit polyclonal antibody immobilized in a 96-well microtiter plate and sandwiched with horseradish peroxidase-conjugated lectin, followed by color development by the enzyme activities.
Using standard chitosan solutions, a good standard curve (R²=0.977) was established between 0 and 250 μg l-¹ with the average coefficient of variation of 7.0%, and CP in the diluted culture supernatants could be directly determined. This method can apply for the direct determination of other polysaccharides in solution by selecting an inexpensive custom-made polyclonal antibody and an appropriate lectin, both of which may be commercially available.
Several microbial polysaccharides such as xanthan, levan, and curdlan have been practically produced in industrial scales and used for the production of foods, medicines, and cosmetics. To reduce the production costs, researchers have made their best efforts to improve the productivity, which has been evaluated as the wet weight or the dry weight of the target polysaccharide produced per liter after the recovery as solid from the culture supernatants.
However, the recovered materials may still contain large amounts of impurities. In addition, their biological and physiological activities do not always respond to the weight measured. Therefore, it is very useful if the major structures of polysaccharides or structures essential for their activities can be directly and easily evaluated in culture supernatant or solution.
We are studying a chitosan-like polysaccharide (CP) with the average molecular weight of >1.66 MDa produced from acetate by Citrobacter strains, which has the strong flocculating activity for suspended solids in water, and thus, can be used as a flocculant. This biopolymer can be recovered as solid by ethanol precipitation and re-solubilized with acidic solutions for the use.
Analyses of the dry weight and the sugar content of the recovered solid may provide good information on the amount of the CP in the original culture, but these analyses are not specific to the target polysaccharide as described above. In addition, they require long analytical time, laborious operational steps, and considerable amounts of the culture. Therefore, in the production of other microbial polysaccharides, more rapid and selective assays based on specific protein binding have been employed to quantify them.
Enzyme-linked immunosorbent assay (ELISA) is one of such methods and can detect a biomolecule of interest sensitively even in the presence of impurities because it is based on specific antigen-antibody reaction. ELISA is well automated with the use of 96-well microtiter plates, a plate washer, and a plate reader, and thus, it has been used in many laboratories and facilities as a conventional analytical method.
Only the bottleneck to use ELISA is a preparation of specific antibodies, which is a laborious work and very costly. Instead of antibody, lectins, which are glycan-specific binding proteins without enzyme activity, have also been used in similar assays [called enzyme-linked lectin or lectin sorbent assay (ELLA)] for the detection of polysaccharide and glycoproteins.
Lectins can be obtained from many biosources such as bacteria, plants, and animals, and many lectins are commercially available. One of the representative lectins, wheat germ agglutinin (WGA), is known to preferentially recognize N-acetyl-D-glucosamine in polysaccharides, the major constituent sugar of chitin/chitosan and the CP. Therefore, WGA has been used for the detection of chitin/ chitosan in fungal and yeast cells and environmental samples.
In this study, to construct a rapid, selective, and direct determination method for the CP in solution, we employed a simple sandwich ELISA, in which a custom-made polyclonal antibody and a horseradish peroxidase (HRP)-conjugated WGA were used as a capture antibody and instead of a detection antibody, respectively.
The established method recognized not only the CP but also authentic chitin/chitosan from shrimp shells even in a low concentration range. The CP amounts in samples could be determined as the chitosan-mg unit using the standard curve of authentic chitosan solutions.
Preparation and Purification of CP and anti-CP Rabbit IgG
Citrobacter freundii IFO13545 (equal to NBRC13545) was cultivated at 30°C and 120 rpm on a rotary shaker for 2 d in acetate medium [10.0 g CH3COONa, 1.0 g (NH4)2SO4, 1.0 g K2HPO4, 0.05 g NaCl, 0.2 g MgSO4•7H2O, 0.05 g CaCl2, 0.01 g FeCl3, and 0.1 g yeast extract in 1 l, pH 7.2]. The culture supernatant was prepared by centrifugation (8,000 x g, 4°C, 10 min). Two volumes of ethanol were added to the supernatant and the mixture was kept overnight at -20°C to precipitate crude CP, which was recovered by centrifugation (12,000 x g, 4°C, 20 min).
Standard Sandwich ELISA for the Determination of CP
In this ELISA, the exclusive solutions contained in the Protein Detector HRP Microwell Kit, Anti-Rabbit (SeraCare Life Science, Gaithersburg, MD, USA) were routinely used for convenience.
In many simple ELISAs and ELLAs, a target molecule in a sample is first coated on microtiter plate wells. This type needs enough time to immobilize the target molecule on the solid surface. For example, in the ELLA for the detection of the extracellular polysaccharides (EPSs) of Streptococcus mutans, employed a 20-h incubation time to immobilize the EPSs to the microtiter plate wells also employed a 16-h incubation time to immobilize the β-glucans of lactic acid bacteria in the ELISA.
Such long times are not suitable for the monitoring of microbial polysaccharides in growing cultures. The present method can complete the measurement in approximately 2 h, if the polyclonal antibody is immobilized to the microtiter plate wells prior to the test day.
The result shown here is only a case study, but this methodology can apply for the rapid establishment of the direct determination method for polysaccharides in solution, if an appropriate lectin specific to the target polysaccharide can be obtained.
This article first published in International Journal of BioAnalytical Methods & BioEquivalence Studies (IJBMBS) by scidoc publishers