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Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus...
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Transcript of Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus...
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Adeola AlaPh.D Student
Virology Unit, IITA-Ibadan, Nigeria
&
Animal Physiology Unit, Department of Zoology
University of Ibadan, Nigeria
Production of Monoclonal and Polyclonal
Antibodies for Detection of Geminiviruses and
the Virus –Vector Relationships
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Outline
1. Introduction
2. Justification & Work plan
3. Production of antibodies
4. Immunization effects on animals
5. Vector transmission studies
6. Surveys for geminiviruses in Nigeria
7. Conclusions
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
1. Introduction
Geminiviruses
Family: Geminiviridae
–Genome: circular single stranded DNA 2.5–3.0 kb in length,
encapsidated in twinned (geminate) quasi-isometric particles.
–Responsible for several devastating diseases in economically
important crops of both monocotyledonous and dicotyledonous plants
worldwide (Cassava, maize, wheat, tomato, pepper, bean, cotton, etc.)
Genera:
Begomovirus: ACMV, BGMV
Mastrevirus: MSV
Curtovirus: BCTV
Topocuvirus: TPCTV
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
BEGOMOVIRUSES:CASSAVA MOSAIC DISEASE (CMD)
• CMD is the single most important production constraint to cassava in sub-Saharan Africa, including Nigeria.
• The disease results in 60-80% decrease in tuber yield, also effects the quality and impede germplasm movement (Bock, 1983)
• In Africa, several species and strains of begomoviruses have been identified in the CMD etiology.
• In Nigeria, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and East African cassava mosaic Cameroon virus(EACMCV) are most prevalent (Ariyo et al. 2005).
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
• Most economically important
virus vector (Geddes, 1990)
• It is a pest on 350 plant species,
including cassava, around the
world
Vector of CMDBemisia tabaci (Gennadius) (Homoptera: Aleyrodidae)
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Mastrevirus: Maize streak virus
• Most important virus disease of maize in Africa and the neighbouring islands of Mauritus, La Reunion and Madagascar (Rybicki and pietersen, 1998).
• Yield losses in maize due to MSV range from 0 to 100% ( Mzira 1984 and Barrow 1992)
• The virus is transmitted by Cicadulina spp, (Homoptera: cicadellidae)
C. storeyi, (=
triangula) one of the
main five species
found In West Africa
(Bosque-Perez et al.,
1990)
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
2. Justification & Work Plan
1 Specific monoclonal antibodies are needed to differentiate EACMV
from other begomoviruses.
2 Polyclonal and monoclonal antisera against MSV are needed for
virus surveys and screening germplasm.
3 Information is limited on distribution of geminiviruses in Nigeria.
4 Information on the transmission efficiency of a less predominant leaf
hopper, C .dabrowski, in MSV transmission is required to assess its
role in MSV epidemiology.
5 Effect of ACMV and MSV immunization on physiological status of
the experimental animal, Oryctolagus cuniculus (Domestic rabbit),
used routinely for antibody production. These include the effects of
the immunogens administered and the quantification of the viruses
used in immunizations. These will ensure minimal adverse effects on
health and also enable optimisation of immunisation protocols.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Aims:
• To produce antibodies for the detection and differentiation of
geminiviruses
• Determine the virus-vector interactions of MSV and leaf hoppers
Objectives:
Produce polyclonal and monoclonal antibodies against cassava
mosaic begomoviruses and MSV for detection and differentiation
of EACMV and MSV.
Conduct surveys for geminivirus distribution in Nigeria and the
food crops they infect
Compare the differences in acquisition, transmission abilities of
MSV between C. triangular and C. dabrowski
Determine the physiological status of rabbits used in routine
immunisations with ACMV and MSV
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
1. PURIFICATION OF ACMV: Method adapted from Thottappilly, 1986)
Density gradient step removed.
Method 1; From test plants, Nicotiana benthamiana
Method 2; Directly from Manihot esculenta
2. MSV PURIFICATION: Method by (Bock et al.,1974)
3. IMMUNISATIONS: Mus musculus (Mouse); Oryctolagus
cunniculus (Rabbit)
Immunized at 2 week intervals for 12 weeks
4 Bleeding
5 Storage of serum
3. Production polyclonal antibodies (PABS)
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HAT IN MEDIA,Only HGPRT +ve cells survive;- salvage pathway
TAS/ACP-ELISALIMITING DILUTION
Fig: 1
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Anim
al im
mun
ized
Viru
s
Conc
. Of
vir
us
in
purif
ied p
rep.
*(A40
5)
(mea
n va
lue:
n=2
)
Healt
hy sa
p
(mea
n va
lue:
n=2
)
Host
plan
t
Mice
Mice
4 /ACMV
3/ MSV
Grou
p A
Rabbit 1/ ACMV
0.861
1.300
0.861
0.065
0.28
0.065
N. benthamiana
Zea mays
N. benthamiana
Rabbit 1/ACMV
Mice
Mice
3 /EACMV
2 /MSV
Grou
p B
Rabbit 1/ EACMV
0.887
1.83
0.504
0.321
0.27
0.226
N.benthamiana
N. benthamiana
Zea mays
N. benthamiana
Mice 3EACMV
/ACMV
Grou
p C
Rabbits 2EACMV
/ACMV
3.498
0.143
M.esculenta
*(A405) values at 1 hr sustrate incubation
Table 1; Experimental animals used in immunisations
Group A
Group B
Group C
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ACMV/EACMV AND MSV RABBIT AND MOUSE POLYCLONAL
ANTISERA PRODUCED
RECIPROCAL ANTISERUM
TITRE IN TAS ELISA
RECIPROCAL ANTISERUM
TITRE IN ACP ELISA
ANIMAL 1hr
incubation
0/N
incubation
1hr
1nc
0/N
1nc
ACMV/EACMV
(1) ACMV/R/A1
(2) ACMV/R/B1
(3) EACMV/R/B1
(4) EA/ACMV/R/C1
(5) EA/ACMV/R/C2
(6) ACMV/M/A1-A4
(7) EACMV/M/B4
(8) EA/ACMV/M/C3
(9) EA/ACMV/M/C4
MSV
(1) MSV/R/A1
(2) MSV/M/A1
(3) MSV/M/A2
(4) MSV/M/B1
(5) MSV/M/B2
1,000
8,000 (1.95)
NDT*
256,000 (4.07)
1,600 (1.90)
1,000 (3.2)
8,000 (2.03)
256,000 (2.84)
64,000 (1.99)
–
–
32,000 (1.85)
16,000
256,000 (5.01)
128,000 (2.3)
–
–
–
–
–
64,000 (2.02)
(Unadsorbed)
16,000 (2.53)
(Adsorbed)
512,000 (1.93)
(Unadsorbed)
1,000 (1.96)
(Adsorbed)
64,000 (2.1)
(Unadsorbed)
256,000 (4.3)
(Adsorbed)
256,000 (4.73)
256,000 (3.82)
NDT
NDT
512,000 (2.51)
(Unadsorbed)
32,000 (2.38)
(Adsorbed)
1,024,000 (2.27)
(Unadsorbed)
NDT
(Adsrobed)
–
256,000 (2.5)
(Adsorbed)
–
–
250 (1.84)
4,000 (2.44)
* Not detectable titre – Not tested
( ) D/H values
ResultsTable 2
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
(1 hr incubation)
7.266.88
5.78
4.81
3.46
2.96
2.411.99
1.741.38
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1/500 1/1000 1/2000 1/4000 1/80001/160001/320001/640001/1280001/256000Antiserum Dilutions
A4
05
nm
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Tit
re p
oin
ts
HDTitre points
FIG 2: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)
ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
(O/N incubation)
4.92
6.09
6.68
5.57
3.93
3.44
2.922.72
1.811.53
0
0.5
1
1.5
2
2.5
3
3.5
4
1/500 1/1000 1/2000 1/4000 1/8000 1/160001/32000 1/640001/1280001/512000Antiserum Dilutions
A 4
05
nm
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Tit
re p
oin
ts (
A4
05
nm
)
HDTitre points
FIG 3: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)
ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
All the polyclonal antisera raised against ACMV, EACMV
and MSV are useful for virus detection by ACP-ELISA
“Compared to TAS-ELISA, ACP-ELISA for geminivirus
detection is convenient and cost-effective”
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Plate 1; Growing hybridoma cells.(a) Lower left corner: dividing cells
(b) Top right: cells forming a colony
B
A
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1st screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse
hybridomas
WELLS WITH
GROWING
HYBRIDS
WELLS +VE
BY ELISA TO
ACMV
% OF TOTAL WELLS
SECRETING SPECIFIC
ANTIBODIES AGAINST
ACMV
Plate 1 24 /96 1/24 1.04%
Plate 2 27/96 3/27 3.13%
Plate 3 49/96 4/49 4.17%
Plate 4 22/96 2/22 2.1%
Plate 5 35/96 4/35 4.2%
Plate 6 42/96 6/42 6.25%
Table 3
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
CELL LINES
ACMV R. Poly/ACMV
ANTG
ACMV R. Poly/EACM
ANTG
ACMV Ms Poly/ACMV
ANT G
ACM Ms Poly/ EACMV ANTG
DSMZ Poly/ACMV
ANTG
DSMZ poly/ EACMV ANTG
2nd
screen.
3rd
screen.
2nd
screen.
3rd
screen.
2nd screen 2nd
screen.
3rd
screen.
2nd
screen.
3rd
screen.
2nd
screen.
3rd
screen.
1H3 - - - - - - - - - - -
2C5 + - - + - - - - - -
2F6 - - - - - - - - - - -
2F61 - - - - - - - - - - -
2F62 - - - - - - - - + - -
2EE5 - - - - - - - - + - -
3A10 + - + - - + - - - - -
3A101 - + - - - - - - - - -
3B12 ++ - ++ - - - - - - + -
3F1 - - - + + - - - - + -
3F11 - - - - - - - - - - -
3B10 - - - - - - - - - - -
4F6 +++ - ++ - + - - + - + -
4F61 - - - - - - - - + - -
4A12 - - - - - - - - + - -
4A121 - - - - - - - - - - -
5B2 - - - - - ++ - - - - -
5G11 - - - - - - - - + - -
6B3 + - ++ - - ++ - - - ++ -
6B31 - + - - - - - - + - -
6B32 - + - - - - - - - -
6B33 - + - + - - - - + - -
6B34 - + - - - + - - ++ - -
6B35 - - - + + - - - + - -
TABLE 4; 2nd screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse
hybridomas
Key:
ACMV R. POLY =ACMV rabbit polyclonal antibody; ACMV MS POLY =ACMV mouse polyclonal antibody; DMSZ IGg =DMSZ rabbit immunoglobulin; ACMV
ANTG =ACMV Antigen; EACMV ANTG =EACMV Antigen
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Cell lines 1st Cloning 2nd cloning
6B3 ++ ++
3F1 +++ +++
1.5 – 1.9: + weak positive
2.0 – 3.0: ++ positive
3.0<: +++ strong positive
CLONING: EACMV/ cell lines in TAS-ELISA (O/N incubation)
Table 5
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
WELLS WITH
GROWING
HYBRIDS
WELLS +VE
BY ELISA TO
MSV
% OF TOTAL WELLS
SECRETING SPECIFIC
ANTIBODIES AGAINT
MSV
Plate 1 10/96 2/10 2.1%
Plate 2 8/96 1/8 1.04%
Plate 3 6/96 2/6 2.1
Plate 4 7/96 5/7 5.2%
Plate 5 5/96 1/5 1.04%
Plate 6 8/96 3/8 3.13%
Table 6; MSV/M/A1 AND MSV/M/A2 BALB/C (1st Screening)
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Table 7; CLONING: MSV cell lines in ACP-ELISA
Cell
lines
Ist CLONING
2ND
CLONING
Healthy sap
Diseased sap
Diseased/Healthy Healthy sap
Diseased sap
Diseased/Healthy
3F4 0.209
0.617 2.9 0.126 1.239 9.8
3E11b 0.282
2.1 7.4 0.123 0.404 3.3
1H2 0.049
0.455 9.2 0.103 0.406 3.9
5F2 0.039
0.315 8.0 0.152 0.586 3.9
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Fusions with Swiss Albino strain of mice yielded no Hybridomas
Plate 2
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MAbs 6B3 and 3F1 differentiated EACMV from ACMV
and they are useful for specific detection of EACMV.
“This is the first monoclonal antibody specific to EACMV”
Plate 3
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4. Effects of Immunisation on Rabbits
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12 Period/weeks
Te
mp
ch
an
ge
Group 1 Group 2 Group 3
(mea
n o
f tw
o r
abb
its)
Fig 4; Bi-monthly Temperature Differences In Immunized
Animals Before And After Immunisations
Group 1: Control; Distilled water alone
Group 2: Adjuvant alone
Group 3: Antigen + Adjuvant
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
PARAMETER GROUP 1 GROUP 2 GROUP 3
WEIGHT 5.248 BA 3.411 B 9.022 A
TEMPERATURE DIFFERENCE
0.0929 B 0.7143 A 0.9143 A
PCV -0.766 B 3.095 B 23.497 A
PLATELETS 39.97 A 93.38 A 41.54 A
HB 0.912 B 7.757 B 41.619 A
RBC 1.188B 9.790 B 56.563 A
MCV -1.149A -5.691BA -8.192B
MCHC 4.645 A 4.301 A 9.788 A
TOTAL WBC 3.99 B 27.21 BA 60.49 A
NEUTROPHILS 76.81 BA 108.11A 8.90 B
LYMPHOCYTES -3.97 B 19.67 B 114.23 A
EOSIN -57.8 B -63.1 B 453.50 A
MONOCYTES -33.33 B 27.78 A -20.83 B
TABLE 8 : PERCENTAGE CHANGE OF PHYSICAL AND HAEMATOLOGICAL PARAMETERS
BETWEEN GROUPS OF RABBITS IN 12 WEEKS GIVEN DIFFERENT TREATMENTS
MEANS IN THE SAME COLUMN WITH DIFFERENT LETTERS ARE SIGNIFICANTLY DIFFERENT (p<0.05).
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
5. TRANSMISSION EFFICIENCY OF MSV in
C. dabrowski and C. triangular
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
12
20 20 20 20
12
0
10
20
30
40
50
60
70
80
30 secs 15 mins 1 hr 24 hr 48 hr 96 hr
AAPs
% t
ran
sm
iss
ion
0
5
10
15
20
25
To
tal in
se
cts
us
ed
C.triangular
C.dabrowski
Total insects used
IAP (Inoculation Access Period) * = 24 hrs; AAP (Acquisition Access Period)**; No of replicates = 3
Significantly higher transmission efficiency in C.triangular at all AAPs (p<0.05)
Fig 5: Percent transmission of MSV by C. triangular and C.
dabrowski given different virus acquisition access periods
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20 20 20 20 20 20
0
10
20
30
40
50
60
30 secs 15 mins 1 hr 24 hr 48 hr 96 hr
Inoculation Access Periods (IAPs)
% t
ran
sm
issio
n
0
5
10
15
20
25
To
tal in
sects
used
C. triangular
C. dabrowski
Total insects used
Significantly higher transmission efficiency in C.triangular at all IAPs (p<0.05)
Fig 6: Percent transmission of MSV by C. triangular and
C. dabrowski given different virus inoculation access periods
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
C. triangular is an efficient vector of MSV
C. dabrowiski is relatively poor vector of MSV
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
6. SURVEY FOR GEMINIVIRUSES IN NIGERIA
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Surveyed during Oct-Nov 2002
Cassava, maize, tomato, pepper, okra, cowpea and jatropha
Fig 7: MAP OF NIGERIA SHOWING STUDY SITES FOR GEMINIVIRUSES
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Methods for virus detection
1. Serological tests (ELISA):
ACMV poly; MSV poly; SCRI Mabs; DMSZ MAbs
2. PCR:
• DNA Extraction
Extraction of DNA was by the method of Dellaporta et al., (1983).
• PCR REACTION MIXTURE AND THERMAL CYCLES –
To amplify the DNA extracted
• AGAROSE GEL ELECTROPHORESIS OF THE AMPLIFIED DNA
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
PRIMER SEQUENCES FROM PITA ET AL ( 2001a).
Primers used for PCR amplification of geminiviruses
VIRUS PRIMER SEQUENCE (5’-3’)TARGET IN DNA
ACMV ACMV -AL1/F GCG GAA TCC CTA ACA TTA TC AC1
ACMV -ARO/R GCT CGT ATG TAT CCT CTA AGG CC T G AV2
EACMV UV -AL3/F TAC ACA TGC CTC RAA TCC TG AC3
UV -AL1/R2 CTC CGC CAC AAA CTT ACG TT AC1
WHITEFLY TRANSMITTED GEMINIVIRUSES
/F
PRIMER B /R
TAA TAT TAC CKG WKG VCC
TGG ACY TTR CAW GGB CCT TCA CA
CR
CRPRIMER A
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COWPEA TOMATO PEPPER OKRA JATROPHA
STATE TSC ACMV Pab TSC ACMV Pab TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR
OYO 9 1/9 - 6 0/6 - 8 0/8 - 12 1/12 - - - -
KWARA 4 0/4 - 2 0/2 - 5 0/5 - 2 1/2 - - - -
KOGI 7 0/7 - 6 0/6 - 5 1/5 - 4 0/4 - - - -
NASSARA WA 18 1/18 - 4 0/4 - 0/3 - 3 0/3 - 2 0/2 -
BENUE 8 0/8 - 4 0/4 - 2 0/2 - 2 1/2 - - - -
ENUGU 2 0/2 - 2 0/2 - 2 0/2 - 3 0/3 - 1 0/1 -
EBONYI - - - - - - 2 0/2 - - - - - -
ONDO 4 1/4 - 5 2/5 -- 7 1/7 1/7 2 1/2 - 5 1/5 -
OGUN - - - 2 0/2 - - - - - - - - - -
NIGER 5 0/5 - 3 0/3 - - - - 3 2/3 - - - -
KADUNA 5 0/5 - 2 0/2 - 4 1/4 1/4 8 0/8 - 6 0/6 -
KANO 16 0/16 - 5 0/5 - 5 0/5 - 2 0/2 - - - -
Table 9: Occurrence of Geminiviruses in Nigeria
PAb= POLYCLONAL ANTIBODY
TSC= TOTAL SAMPLES COLLECTED
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STATE TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR
JIGAWA 5 0/5 - 2 0/2 - - 2 0/2 - - - -
BAUCHI 3 0/3 - - - - 3 0/3 - 3 0/3 - - - -
YOBE 3 0/3 - 3 0/3 - - - - - - - - - -
GOMBE 8 0/8 - - - - 4 0/4 - 3 0/3 - - - -
ADAMAWA 2 0/2 - - - - - - - 3 0/3 - - - -
TARABA 14 0/14 - 2 -- - - - - 3 0/3 - - - -
PLATEAU - - - - - - - - - 3 0/3 - - - -
EDO - - - - - - 2 0/2 - 4 0/4 - - - -
DELTA - - - 2 -- - - - 2 0/2 - - - -
IMO 5 0/5 - - - - - - - - - - -
ABIA - - - 2 - 2 0/2 - 4 0/4 - 2 0/2 -
AKWA IBOM - - - - - - 2 0/2 - 2 0/2 - - - -
CROSS RIVERS - - - - - - 4 0/4 - 3 0/3 - - - -
RIVERS - - - - - - 2 0/2 - 4 1/4 - 2 0/2 -
OSUN - - - - - - - 2 0/2 - - -
TOTAL 118 3/118 52 3/52 -- 62 3/62 --- 79 5/79 -- 18 1/18 --
COWPEA TOMATO PEPPER OKRA JATROPHA
Table 9 (Continued) : Occurrence of Geminiviruses in Nigeria
PAb= POLYCLONAL ANTIBODY
TSC= TOTAL SAMPLES COLLECTED
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Plate 4: PCR Indexing for Other Geminiviruses in Survey Samples Using
Universal Primer Pair (Broad Spectrum Detection) Uv-ali/f1/r1
•46 samples from all zones were randomly picked for analyses.
•universal primers for geminiviruses (Primer A/F; Primer B/R).
•5 samples were positive, three cassava samples (M115-lane 24; M300-lane 32, and S154-lane 42) one pepper sample (N31-
lane 10) and one tobacco sample (N22-lane 8).
•Lane 47; Negative control / healthy casava Lane 48; Positive control/ ACMV diseased cassava
M 8 10 24 32 M
M 42 M
500bp
500bp
47 48
1KB marker used
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Total positive samples detected by PCR using
universal primers for geminiviruses (Primer A/F;
Primer B/R)
93 samples analysed
•Three tomato samples (S8; S28; and S43)
•Two pepper samples (S6, N31).
•one tobacco sample (N22).
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
% incidence of ACMV/EACMV using MAbs
11
0
5
113
82
0
10
20
30
40
50
60
70
80
Arid/semi Arid Northern Guinea
savannah
Southern Guinea
savannah
Derived savannah Humid forest
Zones
% in
cid
en
ce
0
20
40
60
80
100
120
To
tal s
am
ple
s a
na
lyse
d
DSMZ 2 MAb
DSMZ 4 MAb
SCRI 17 MAb
SCRI 20 MAb
SCRI 33 MAb
SCRI 60 MAb
Total plants analysed
Fig 8: SEROLOGICAL INDEXING FOR ACMV AND EACMV IN LEAF SAMPLES
DSMZ MAb 2- REACTS WITH ACMV & EACMV DSMZ MAb 4- REACTS WITH ACMV; DOES NOT REACT WITH EACMV
IN SINGLE INFECTIONS SCRI MAb 17- REACTS WITH ACMV & EACMV SCRI MAb 20– REACTS WITH ACMV, EACMV,& ICMV SCRI MAb 33-
REACTS WITH ACMV ALONE SCRI MAb 60- REACTS WITH ICMV ALONE
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
% incidence of MSV using MSV polyclonal antibody
9
4
17
94
5
18
0
20
40
60
80
100
120
Arid/semi Arid Northern Guinea
savannah
Southern Guinea
savannah
Derived
savannah
Mid Altitude Humid forest
Zones
% i
nci
de
nc
e
0
10
20
30
40
50
60
70
80
90
100
To
tal
sam
ple
s an
alys
ed
% positive samples
Total samples analysedS
FIG 9: SEROLOGICAL INDEXING FOR MSV IN LEAF SAMPLES
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
This survey demonstrated usefulness of
antibodies produced in this study:
MAbs 6B3 and 3F1 MAbs were very specific and detected only 2
EACMV positive samples in 40 randomly selected samples from
the humid Forest and Derived Savannah Zones
Further it showed occurrence of several geminiviruses
infecting several economically important crops,
knowledge on which are scanty in Nigeria
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
7. CONCLUSIONS
• Eliminating the density gradient step in purifications of antigens is not
detrimental in the production of monoclonal antibodies.
• Each animal is unique in it’s immune response. Different antibody titres
obtained with the same type and quantity of immunising antigen
• A maximum of 6 immunisations of experimental animals with
ACMV/EACMV and MSV is adequate for high titre polyclonal antibody
production. All antibodies produced detected immunising antigen.
• Protocol used for fusions is efficient (all plates produced hybridomas)
and is recommended. EACMV specific MAb was produced.
• The Swiss albino strain of mice is unsuitable for MAb production using
X63 myeloma cell lines. Homologous fusion partners yield the highest
numbers of stable hybridomas (Maden, 1985)
• PAbs and MAbs produced efficiently detected geminiviruses in indirect
ELISA
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
C. dabrowski is inefficient as a vector of MSV and is unlikely to contribute to disease epidemics.
The antigens and adjuvant administered in study do not affect experimental animals in a clinically important or preclusive manner.
Field surveys demonstrated usefulness of diagnostic reagents and also showed occurrence of diverse geminiviruses infecting several economically important crop species.
CONCLUSIONS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
ACKNOWLEDGEMENTS
IITA, Ibadan
Scottish Crop Research Institute (SCRI), Scotland, UK
Deushe Sammlung von Microrganismen und Zellkulturen
(DSMZ), Germany