Kit Components
|
SN |
Components |
2 × 96 T |
|
1 |
Microtiter plate |
12 strips×8 wells |
|
2 |
Wash Buffer (PBST 20×) |
30 mL |
|
3 |
Negative Control |
1tube |
|
4 |
Positive Control |
1tube |
|
5 |
Antibody Solution |
10 mL |
|
6 |
HRP enzyme |
10mL |
|
7 |
Sample Diluent |
25 mL |
|
8 |
Substrate Solution (TMB) |
20 mL |
|
9 |
Stop Solution |
20 mL |
|
10 |
BPPD (Bovine-PPD) （0.3mg/mL） |
1 |
|
11 |
APPD(Avian-PPD） （0.3mg/mL） |
1 |
Sample Preparation
(1) 5mL Blood collected by venipuncture should be allowed to clot naturally and completely.
(2) Samples are shipped to laboratory at the room temperature (18-25℃) .
(3) Samples is cultured within 30hours, the sooner, the better. Don’t put blood in the refrigerator any way.
ELISA procedure
1. Blood Cultured:
(1) Put bovine whole blood (at least 5mL) into a heparin anticoagulant tube, mix several times with gentle shaking to dissolve the heparin. Transport to the laboratory at room temperature (22°C ±3°C), and culture within 30 hours after collection.
(2) Dispense 1.5mL anticoagulant blood into each well of 24-well culture plates (Figure1), add 100μL of PBS, APPD and BPPD to corresponding well.
|
24well Cell plate |
1 |
2 |
3 |
4 |
5 |
6 |
|
A |
Bovine-1 PBS |
Bovine-1 APPD |
Bovine-1 BPPD |
Bovine-2 PBS |
Bovine-2 APPD |
Bovine-2 BPPD |
|
B |
Bovine-3 PBS |
Bovine-3 APPD |
Bovine-3 BPPD |
Bovine-4 PBS |
Bovine-4 APPD |
Bovine-4 BPPD |
|
C |
Bovine-5 PBS |
Bovine-5 APPD |
Bovine-5 BPPD |
Bovine-6 PBS |
Bovine-6 APPD |
Bovine-6 BPPD |
|
D |
Bovine-7 PBS |
Bovine-7 APPD |
Bovine-7 BPPD |
Bovine-8 PBS |
Bovine-8 APPD |
Bovine-8 BPPD |
Figure1: Incubate blood
(3) Incubate 24-well cell culture plates at 37°C for 16-24 hours.
(4) Collect plasma: centrifuge cell culture plate at 500 g/r for 10min，transfer 500μL to 96 well microplate（Figure2).
1A: Bovine-1 APPD , 1B=Bovine-1 BPPD
C7,C8,C9, and G7,G8, G9 as for Positive Control and Negative Control.
|
96 T |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
A |
1P |
1A |
1B |
2P |
2A |
2B |
3P |
3A |
3B |
4P |
4A |
4B |
|
B |
5P |
5A |
5B |
6P |
6A |
6B |
7P |
7A |
7B |
8P |
8A |
8B |
|
C |
9P |
9A |
9B |
10P |
10A |
10B |
|
|
|
11P |
11A |
11B |
|
D |
12P |
12A |
12B |
13P |
13A |
13B |
14P |
14A |
14B |
15P |
15A |
15B |
|
E |
16P |
16A |
16B |
17P |
17A |
17B |
18P |
18A |
18B |
19P |
19A |
19B |
|
F |
20P |
20A |
20B |
21P |
21A |
21B |
22P |
22A |
22B |
23P |
23A |
23B |
|
G |
24P |
24A |
24B |
25P |
25A |
25B |
|
|
|
26P |
26A |
26B |
|
H |
27P |
27A |
27B |
28P |
28A |
28B |
29P |
29A |
29B |
30P |
30A |
30B |
Figure2: Numbering samples
2. ELISA analysis
(1) Return the solution in the kit to the room temperature (18-25℃).
(2) Add 50μL Sample Diluent to each well.
(3) Add 50μL of each plasma sample, negative control, the positive control was in the corresponding well, shake slightly for 1min, seal plate and incubate at room temperature (20-25 °C) for 60 min.(Avoid sunlight and air entering the well).
(4) Discard the liquid , add 250μL washing buffer to wash plate for 6 times. Pat the hole dry the last time.
(5) Add 50μL Antibody solution to each well, then add 50μL HRP enzyme conjugate to each well,shake slightly for 1min, seal plate and incubate at room temperature (20-25 °C) for 60 min.
(6) Discard the liquid , add 250μL washing buffer to wash plate for 6 times. Pat the hole dry the last time.
(7) Add 100μL substrate respectively, incubate at 20-25℃ for 10 min.
(8) Add 100μL stop solution and read data at 450nm with 5min-10min .