Introduction:
Several proteins associated with malignant transformation of cells can induce auto-antibodies which are detectable in serum. Survivin, a novel member of inhibitor of apoptosis(IAP) protein family, suggested in both control of apoptosis and cell division. Survivin is not expressed in normal adult tissue expect for the thymus and placenta, but it is abundant in fetal tissue and various human tumors, including colon, lung, gastric, breast and bladder cancer. The enzyme-linked Immunosorbent assay(ELISA) is established for measuring anti-survivin auto-antibodies in human sera and evaluate the possibility of suffering from cancer. The kit with a low false positive rate is a good test for cancer screening.
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Principle:
| ELISA format |
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Survivin recombinant protein was coated on 96 well microplate for anti-survivin auto-antibody detection in human serum. The tracer is goat anti-human IgG F(ab')2 labeled with horseradish peroxidase. After adding tetramethylbenzidine (TMB) substrate and stopping by 1N HCl, the absorbent was measured at 450 nm for each well. |
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Kit content:
1. Survivin ELISA plate: One strip holder containing 8x12 strips coated with survivin
2. Conjugate: one vial containing 11ml of HRP conjugated-goat anti-human-IgG antibodies.
3. 20x concentrated washing solution: one bottle, 20ml
4. Serum diluents: two bottles, 50ml each.
5. Positive control , 800 ul
6. Substrate (TMB): one bottle, 11ml.
7. Stop solution (1N HCl): one bottle, 11ml
8. One adhesive plate sealing film.
9. One sheet of semilog paper.
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ELISA procedures:
1. Take enough strips and put on strip holder.
2. Add 100 ul of serum diluents (blank), positive control sera and diluted serum samples to the individual well. Duplication is recommended for each control and blank.
3. Incubate at room temperature (21-25° C ) for 60 min.
4. Aspirate or shake out liquid from all wells. Add 280-300 uL of diluted 1X washing solution. Aspirate or shake out and turn plate upside down and blot on paper toweling to remove all liquid. Repeat the wash procedure 2 times (for a total of 3 washes).
5. Add 100 ul of conjugate to each well.
6. Incubate at room temperature (21-25° C ) for 60 min.
7. Wash with diluted 1X washing solution 3 times.
8. Add 100 ul of Substrate (TMB) substrate to each well.
9. Incubate at room temperature (21-25° C ) for 10 ± 2 min.
10. Add 100 ul of stop solution (1N HCl) to each well to stop the reaction. Tap the plate gently along the outsides, to mix contents of the wells.
11. Determine absorbance by reading assay plate at 450nm using 650nm as reference. The plate may be held up to 1 hour after addition of the Stop Solution before reading.
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