2 NCM-ELISA (slide set) - Sweetpotato Knowledge PortalSteps followed in NCM-ELISA A. Plant sap B....

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