Reagents
(1) Inclusion buffer (PH9.60.05M carbonate buffer):
Na2CO3 1.59 g
NaHCO3 2.93 g
Add distilled water to 1000 ml
(2) Wash buffer (PH7.4PBS): 0.15M
KH2PO40.2 g
Na2HPO4-12H2O 2.9 g
NaCl 8.0 g
KCl 0.2 g
Tween-20 0.05% 0.5 ml
Add distilled water to 1000 ml
(3) Dilution solution:
Bovine serum albumin (BSA) 0.1 g
Add washing buffer to 100 ml
Or use sheep serum, rabbit serum, and other serum with the washing solution to make 5-10%.
(4) Termination solution (3M H2SO4):
Distilled water 178.3 ml, add concentrated sulfuric acid (98%) 21.7 ml drop by drop.
(5) Substrate buffer (PH5.0 Phosphoric acid date citric acid):
0.2MNa2HPO4 (28.4 g/L) 25.7ml
0.1M citric acid (19.2 g/L) 24.3 ml
Add distilled water 50 ml.
(6) TMB (tetramethylbenzidine) using solution:
TMB (10 mg/5 ml anhydrous ethanol) 0.5 ml
Substrate buffer (PH5.5) 10 ml
0.75% H2O2 32μl
(7) ABTS using solution:
ABTS 0.5mg
Substrate buffer (PH5.5) 1ml
3% H2O2 2μl
(8) Antigens, antibodies and enzyme labeled antibodies.
(9) Normal human serum and positive control serum.
Equipment
(1) Polystyrene plastic plate (referred to as enzyme labeling plate) 40-well or 96-well, ELISA detector, 50 μl and 100 μl spiker, plastic dropper tip, a small towel, and washing bottle.
(2) Small beakers, glass rods, test tubes, pipettes and measuring cylinders.
(3) 4°C refrigerator, 37°C incubator.
Test principle
The kit is a solid-phase sandwich method enzyme-linked immunosorbent assay (ELISA). Standards of known concentration and samples of unknown concentration are added into the microtiter enzyme labeling plate for detection. The biotin-labeled antibody is first warmed at the same time. After washing, affinity-labeled HRP is added. after further warming and washing to remove unbound enzyme conjugates, substrates A and B are added, which act simultaneously with the enzyme conjugates. Color is produced. The shade of color is proportional to the concentration in the sample.
Self-prepared materials
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.
3. Oscillator and magnetic stirrer, etc.
Safety
1. Avoid direct contact with the termination liquid and substrates A and B. Once in contact with these liquids, rinse with water as soon as possible.
2. Do not eat, drink, smoke or use cosmetics during the experiment.
3. Do not aspirate any component of the kit by mouth.
4. Reagents should be stored according to label instructions and brought to room temperature before use. Diluted standards should be discarded and not stored.
5. Slabs not used in the experiment should be put back into the bag immediately and kept sealed to avoid deterioration.
6. Other reagents not in use should be packed or covered. Do not mix reagents of different lot numbers. Use before shelf life.
7. Use disposable tips to avoid cross-contamination, and avoid using a sampler with metal parts when aspirating the terminating solution and substrate A and B.
8.
8. Use clean plastic containers to configure the washing solution. Mix the components and samples in the kit well before use.
9. Wash the plate by patting it dry, and do not put absorbent paper directly into the wells to absorb water.
10. Substrate A should be volatilized, avoid opening the lid for a long time. Substrate B is sensitive to light, avoid prolonged exposure to light. Avoid contact with hands, toxic. The OD value should be read immediately after completion of the experiment.
11. The order of adding reagents should be consistent to ensure that all reaction plate wells are warmed for the same time.
12. Perform the warming operation according to the time, amount and order of addition indicated in the instructions.