Académique Documents
Professionnel Documents
Culture Documents
, 2(4): 333-338
(ISSN: 2455-1716)
JULY-2016
Ph. D Scholar, Department of Molecular Biology and Genetic Engineering, G. B. Pant University of Agriculture and
Technology, Pantnagar, Uttarkhand, India
2
Associate Professor, Department of Plant Biotechnology, UAS (B), GKVK, Bangalore, India
3
Ph.D. Scholar, Department of Plant Biotechnology, UAS (B), GKVK, Bangalore, India
*
Address for Correspondence: Snigdha Tiwari, Ph. D Scholar, Department of Molecular Biology and Genetic
Engineering, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarkhand, India
Received: 20 April 2016/Revised: 19 May 2016/Accepted: 16 June 2016
ABSTRACT- Small Cardamom (Elettariacardamomum L. Maton) is one of the major spice crops of India, which were
the worlds largest producer and exporter of cardamom till 1980. There has however been a reduction in production,
mainly because of Katte disease, caused by cardamom mosaic virus (CdMV) a potyvirus. Viral diseases can be managed
effectively by early diagnosis using serological methods. In the present investigation, CdMV isolates were sampled from
Mudigere, Karnataka, ultra purified, and electron micro graphed for confirmation. Polyclonal antibodies were raised
against the virus and a direct antigen coating plate Enzyme linked immunosorbent Assay (DAC-ELISA) and Dot-ELISA
(DIBA) standardized to detect the virus in diseased and tissue cultured plants. Early diagnosis in planting material will aid
in using disease free material for better yields and hence increased profit to the farmer.
Key-words- Cardamom mosaic virus (CdMV), Electron microscopy, Direct antigen coating Enzyme linked
immunosorbent Assay (DAC-ELISA), Dot-ELISA (DIBA)
-------------------------------------------------IJLSSR-----------------------------------------------
INTRODUCTION
Website:
www.ijlssr.com
DOI:
10.21276/ijlssr.2016.2.4.5
Page 333
and
Electron
Antiserum Production
Page 334
DIBA
Disease diagnosis
Page 335
Standardization of antibody titer raised against CdMV virus for ELISA was done to know the suitable concentration of
virus and primary antibody for testing the virus. Titre fixation using different dilutions of antiserum with the different
concentration of antigen was done in ELISA plates in two runs namely ELISA 1 and ELISA 2 as mentioned earlier.
ELISA 1
The antiserum dilutions used were 1:500, 1:1000, and 1:2000. The concentrations of antigen were undiluted, 1:10, 1:50,
1:100, 1:200, 1:500 and 1:1000. Secondary Antibody dilutions were 1:1000 and 1:2000. In all combinations colour
development was observed. Optimum OD reading of 0.886 was observed in the combination of 1000 ng of antigen and
1:500 dilutions of primary antisera with 1:2000 dilutions of secondary antibodies (Table 1).
Table 1: ELISA 1 readings (OD) at 405 nm for the standardization of antibody titre raised against CdMV
Antigen Dilution
Primary
1000 ng
Antibody
Dilution
1:500
1:1000
1:2000
1:500
1:1000
1:2000
500ng
200ng
100ng
50 ng
10 ng
+ve
-ve
0.895
0.789
0.632
0.589
0.421
0.190
1.460
0.076
0.824
0.579
0.522
0.395
0.276
0.176
1.207
0.067
0.657
0.498
0.426
0.350
0.210
0.123
Secondary Antibody Dilution of 1:2000
1.060
0.087
0.886
0.635
0.512
0.492
0.376
0.312
1.346
0.082
0.683
0.539
0.483
0.384
0.306
0.216
1.060
0.087
0.762
0.642
0.502
0.448
0.346
0.293
1.207
0.067
The antiserum dilutions used were 1:500, 1:1000, and 1:2000. The concentrations of antigen were undiluted, 1:10, 1:50,
1:100, 1:200, 1:500 and 1:1000, with secondary antibody dilutions of 1:1000 and 1:2000. In all combinations colour
development was observed. Optimum OD reading of 0.960 was observed in the combination of 1000 ng of antigen and
1:500 diluted primary antisera with 1:2000 dilutions of secondary antibodies. This combination of 1:100 of antigen and
1:500 dilution of primary antiserum and 1:2000 of secondary antibody was used in further studies (Table 2).
Table 2: ELISA 2 readings (OD) at 405 nm for the standardization of antibody titre raised against CdMV
Primary Antibody Dilution
1000 ng
500ng
1:500
1:1000
1:2000
1.004
0.947
0.828
0.894
0.635
0.459
1:500
1:1000
1:2000
0.960
0.629
0.624
0.600
0.477
0.391
Antigen Dilution
200ng
100ng
50 ng
10 ng
Secondary Antibody Dilution of 1:1000
0.835
0.711
0.624
0.514
0.487
0.437
0.349
0.351
0.363
0.339
0.310
0.296
Secondary Antibody Dilution of 1:2000
0.432
0.419
0.384
0.331
0.411
0.289
0.276
0.270
0.299
0.250
0.235
0.183
+ve
-ve
1.756
1.558
1.410
0.078
0.052
0.032
1.329
1.218
1.254
0.089
0.056
0.067
Standardization of an indirect-ELISA for detecting CdMV at 5g/ml concentration of primary antibody and 1:1000 and
1:2000 dilution of secondary antibody, and a direct-ELISA at same concentration of primary antibody and 1:400 and
1:800 dilution of secondary antibody [10]. It has been reported that CdMV could be detected at 1:1000 dilution of antigen
(leaf extract) and 1:6000 dilution of primary antibody but optimum was 1:100 dilution of antigen and 1:2000 of primary
antibody [9]. The results of the present investigation are in accordance with these reports. The ELISA results also revealed
that the use of PVP and OA in blocking buffer enhanced the efficiency of ELISA. This is in accordance with the
http://ijlssr.com IJLSSR 2015 All rights are reserved
Page 336
standardized protocols for serological detection of plant viruses [13] and also the protocol used for a related virus, viz the
pepper vein banding mosaic virus [26].
Standardization of antibody titre raised against CdMV virus for Dot ELISA
Standardization of antibody titre raised against CdMV virus for ELISA was done to know the suitable concentration of
virus and primary antibody raised in rabbit. Titre fixation using different dilutions of antiserum with the different
concentration of antigen was done in nitrocellulose membrane. The antiserum dilutions used were 1:500, 1:1000 and
1:2000 with secondary antibody concentration of 1:2000 and antigen concentration of 2g/ml. In all combinations colour
development was observed in all dilutions except in negative control (containing only coating buffer) (Plate 2). Optimum
colour development observed was in 1:1000 diluted antisera. This combination of 1:1000 dilution of antiserum was used
in further studies (Table 3).
Disease diagnosis
Control
1:500
1:1000
1:2000
++++
++++
+++
Scores
Page 337
Table 4: ELISA reading (OD) at 405 for detection of CdMV in tissue culture plants
Antibody
1:500
1:1000
1:2000
-ve
0.0795
0.0724
0.0657
+ve
1.789
1.579
1.498
Infected leaves
1.480
1.257
1.043
CONCLUSION
ACKNOWLEDGMENT
REFERENCES
Sample
Tissue culture 1
0.0889
0.0860
0.0840
Tissue culture 2
0.0867
0.0880
0.0838
Healthy
0.0876
0.0879
0.0836
Page 338