We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\time...
We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\times}$25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.
We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\times}$25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
In this study, we developed two polyclonal antibody-based ELISA system to detect conidia of the soybean sprout pathogen.
대상 데이터
The spore suspension (300 μL) prepared following the steps above, was mixed with an equal volume of Freund's Complete Adjuvant (Sigma F-5881) and emulsified by 3-WAY STOPCOCK. The antigen-adjuvant mixture was injected into the ascites of two female (6-weeks-old) BALB/C mice (200 gL/mouse). The first injection was followed by two booster injections at a three-week interval.
이론/모형
Control wells received only PBST without enzyme but were otherwise treated similarly. The influence of those treatments was monitored by using the ELISA scheme (Fig. 5).
성능/효과
In conclusion, PAbl and PAb2 are proven to be very sensitive and highly specific to the target pathogen, Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements for detecting inoculums before infection and onset of anthracnose, which would provide us with highly accurate information compared with PCR method or classical identification method (Singh et al.
참고문헌 (10)
Clark, M. F. 1981. Immunosorbent assays in plant pathology. Annu. Rev. Phytopathol. 19:83- I06
Jung, Y. K. and Kim, S. H. 2004. Isolation and identification of pathogenic and epiphytic microorganisms from soybean sprouts and control of soybean sprout rot. B.S. Thesis, Gyeongsang National University
Kang, J. H. and Song, G A. 2002. Clean soybean sprouts produced by using light and seed floating on water and its production model. Patent number: 0379839, Korean Patent Administration
Kim, D. K., Lee, S. C., Kang, J. H. and Kim, H. K. 2003. Colletotrichum Disease of Mungbean Sprout by Colletotrichum acutatum. Plant Pathol. 19:203-204
Lee, J. H., Han, K. S., Lee, S. C., Shim, C. K., Bae, D. W. and Kim, H. K. 2004. Early detection of epiphytic anthracnose inoculum on phyllosphere of Diospyros kaki var. domestica . Plant Pathol. J 20:(in press)
Meyer, U. M., Spotts, R. A. and Dewey, F. M. 2000. Detection and quantification of Botrytis cinerea by ELISA in pear stems during cold storage. Plant Dis. 84: I099- I103
Singh, O., Trevors, C. M., Boer, S. H. and lanse, J. D. 2000. Fimbrial- specific monoclonal antibody-based ELISA for Europiean potato strains of Erwinia ehrysanthemi and comparison to PCR. Plant Dis. 84:443-448
Somai, B. M. and Keinath, A. P. 2002. Development of PCRELISA for detection and differentiation ofDidymella blyoniae from related phoma species. Plant Dis. 86:7 I0-7 I6
Sundaram, S., Plasencia, J. and Banttari, E. E. 1991. Enzymelinked immunosorbent assay for detection of verticillium spp. Using antisera produced to V. dahliae from potato. Phytopathology 8I: 1485- I489
Velicheti, R. K., Lamison, C., Brill, L. M. and Sinclair, l. B. 1993. Immunodetection of Phomopsis species in asymptomatic soybean plants. Plant Dis. 77:70-73
※ AI-Helper는 부적절한 답변을 할 수 있습니다.