2 ncm-elisa (slide set) - sweetpotato knowledge portalsteps followed in ncm-elisa a. plant sap b....
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NCM-ELISA
HEALTHY INFECTED
Washing
Negative Positive
Steps followed in NCM-ELISA
A. Plant sap
B. Blocking
C. Antibody-1
D. Antibody- 2
E. Substrate
Washing
Washing
NC-Membrane
1
2 3
Sample preparation
Cutting leaf disks Grinding leaf disks (1 ml of extraction buffer
per each leaf disk)
Extraction buffer
Sodium sulfite (Na2SO3) 0.2g (0.2%)TBS 100 ml
Tris Buffer Saline (TBS), pH 7.5 (2,000 ml)
Tris base 4.84 g (0.02M)NaCl 58.44 g (0.5 M)
Dissolve in 1,990 ml distilled water and adjust pH to 7.5 with HCl (5 N). Add distilled water to 2,000 ml final volume.
Application of samples to the nitrocellulose membrane
Method 1 Spot 17 ul sap onto each square of the nitrocellulose membrane included in the kit
Method 2Using a dot-blotting apparatus connected to a vacuum pump (at 210 mm Hg) add 30-50 ul sap
Blocking the nitrocellulose membrane
Blocking buffer solution
Milk powder (2%)Triton X-100 (2%)TBS
Incubate for 1 hour
Washing step
With TBS (one time, quickly)
Antibody-1(virus - specific)
Antibody buffer solution
Milk powder (2%)TBS (30 ml / membrane)Antibody-1 (according to instructions in bottle)
Incubate overnight
Washing step
With T-TBS (TBS containing 0.05% Tween-20)(wash four times, 3 minutes each)
Incubate for 1 hour
Antibody-2 (anti - antibody conjugated to enzyme)
Antibody buffer solution
Milk powder (2%)TBS (30 ml / membrane)Antibody-2 (according to instructions in bottle)
Washing step
With T-TBS (TBS containing 0.05% Tween-20)(wash four times, 3 minutes each)
Substrate solution
NBT BCIP
Preparation of substrate solution
+
Substrate solution (per membrane)
NBT 3.0 mgBCIP 1.5 mgSubstrate buffer 30 ml
Substrate buffer, pH 9.5
Tris base 0.1 MNaCl 0.1 MMgCl2 0.005 M (= 5 mM)
(5 Bromo-4-chloro-3-indolyl phosphate) (Nitro blue tetrazolium)
Incubate for 30 to 60 minutes
Development of the reaction
Reading results
1 9 17 25 33 41 49 57 65 73 81 89
8 16 24 32 40 48 56 64 72 80 88 96
Sample number
Negative reaction
Positive reaction
1+
2+
3+
4+
Intensityof the
reaction
Recording results
HEALTHY INFECTED
Washing
Negative Positive
Steps followed in NCM-ELISA
A. Plant sap
B. Blocking
C. Antibody-1
D. Antibody- 2
E. Substrate
Washing
Washing
NC-Membrane
NCM-ELISA
FUNDAMENTALS ON NCM - ELISA
Antibody titration
1/1,
000
1/50
1/100
1/200
1/400
1/8001/1,6001/3,2001/6,400
1/12,800
1/50
1/50
1/2,
000
1/4,
000
1/8,
000
1/16
,000
1/32
,000
1/64
,000
1/12
8,00
0
1/25
6,00
0
1/51
2,00
0
1/1,
024,
000
1/2,
048,
000
1/25
Hea
lthy
plan
t sap
Sap
dilu
tion
of in
fect
ed p
lant
Antiserum dilution
BCIP Indoxyl Ketone Indigo(blue)
NBT Diformazan(purple)
Color development reaction catalyzed by allkaline phosphatase (AP) with BCIP as substrate combined with NBT
H+
oxidation and
dimerizationAP tautomerization
reduction
Color development reaction catalyzed by allkaline phosphatase (AP) with BCIP as substrate combined with NBT
Effect of substrate components concentration on the color development
Comparison of developed membranes
Color development time
30 min 2 to 3 h 30 min 2 to 3 h