Preparation of polyclonal antibody and development of a competitive ELISA for fungal (1,3)-beta-D-glucan

doi:10.3969/j.issn.1673-8640.2015.09.015

Abstract

Abstract: Objective

To develop a competitive enzyme-linked immunosorbent assay (ELISA) with high specificity and sensitivity for fungal (1,3)-beta-D-glucan.

Methods

Rabbit polyclonal antibody was produced by immunization with protein-conjugated (1,3)-beta-D-glucan and fungal extract. The highest titer and little cross-reactivity polyclonal antibody was labeled with horseradish peroxidase (HRP) and purified. Finally, a competitive ELISA was established for fungal (1,3)-beta-D-glucan.

Results

The linear range was 3.125-200 pg/mL. The recovery range for 100, 25 and 6.25 pg/mL serum antigen was 97.8%-113.6%, and the coefficient of variation for repeated test was <15%. The detection system could effectively detect low concentrations of Aspergillus fumigatus and Candida albicans and high concentration of Cryptococcus neoformans from serum samples. Meanwhile, the detection system demonstrated little interference against 5 kinds of pathogenic bacteria, including Mycobacterium tuberculosis, Escherichia coli, Salmonella, Klebsiella and Staphylococcus aureus.

Conclusions

A competitive ELISA is developed successfully for the detection of invasive fungal disease with (1,3)-beta-D-glucan used as a coated antigen and enzyme-labeled antibody HRP-Ab3B used as a detection antibody.

Key words: (1,3)-beta-D-glucan, Enzyme-linked immunosorbent assay, Invasive fungal disease

CLC Number:

R446.61

YAN Jun, YANG Wei, ZHAI Shuanzhu, XUE Zhixin, YU Peng, ZHOU Zeqi. Preparation of polyclonal antibody and development of a competitive ELISA for fungal (1,3)-beta-D-glucan[J]. Laboratory Medicine, 2015, 30(9): 931-933.

Figures/Tables 3

References 8

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菌种	血清样本中菌粉浓度(μg/mL)
菌种	10	1	0.1	0.01	0.001	0.000 1
烟曲霉	>200	>200	>200	>200	127.6±10.1	10.3±1.2
白念珠菌	>200	>200	>200	>200	88.3±8.9	9.2±0.7
隐球菌	>200	78.5±8.1	6.8±0.7	<3.125	<3.125	<3.125

菌种	血清样本中菌粉浓度(μg/mL)
菌种	10	1	0.1	0.01	0.001	0.000 1
烟曲霉	>200	>200	>200	>200	127.6±10.1	10.3±1.2
白念珠菌	>200	>200	>200	>200	88.3±8.9	9.2±0.7
隐球菌	>200	78.5±8.1	6.8±0.7	<3.125	<3.125	<3.125

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