3 2 1. One-Step Immunoassay Detection
All assays should be performed in duplicate or triplicate.
1. Coat multiwell stnps overnight at room temperature with 50 pL/well of 10 pg/mL anti-CAT or anti-hGH capture antibody, diluted in IX PBS (see Note 2)
2. Discard coating solution, and block wells for 2 h at 37°C with 250 pL/well of blocking buffer.
3 Wash wells 3X with wash buffer (see Note 3)
All following incubations are performed at room temperature with shaking at 150 rpm.
4. Incubate wells for 1 h with 100 pL of purified CAT enzyme (5-10,000 pg/mL, see Note 4) or purified hGH (5-50,000 pg/mL; see Note 5), serially diluted in blocking buffer, or 100 pL of cell extract (CAT) or culture medium (hGH) diluted in blocking buffer.
6 Incubate wells for 1 h with 100 pL of anti-CAT-AP, diluted 1T000 m blocking buffer, or 100 pL of anti-hGH-AP, diluted 1.500 in blocking buffer (see Note 6)
7 Wash wells 3X with wash buffer
8 Prepare required volume of CSPD/Sapphire-II solution (10% Sapphire-II, 17 pL CSPD per 1 mL, diluted in assay buffer).
9. Wash wells IX with assay buffer
10. Add 100 pL of CSPD/Sapphire-II solution to each well and incubate at room temperature for 10 mm.
11 Measure light emission at 10 min intervals (see Note 7)
3.2.2 Two-Step CAT Immunoassay Detection Perform Section 3.2 1. through step 5
6a Incubate wells for 1 h with 100 pL biotinylated anti-CAT, diluted 1 3000 in blocking buffer
6b. Wash wells 3X with wash buffer.
6c Incubate wells for 45 min with 100 pL streptavidin-AP conjugate, diluted 1:10,000 in blocking buffer.
1 Extracts should be used immediately or stored at —70°C Extracts should be frozen in dry ice/ethanol before transfer to -70°C to avoid degradation of CAT protein Prolonged storage at 4°C is not recommended Protease inhibitors may be added to stabilize extracts. 0.2 mMPMSF, 5 mM DTT, and 5 pg/mL aprotinin
2. Capture antibodies can be monoclonal or polyclonal and should be used for coating at a concentration of 0 2-10 pg/mL (14). The performance and optimal coating concentration for different antibody preparations should be determined.
3 To wash wells, solution is squirted vigorously into each well with a plastic wash bottle. Wash solution is then flicked out of wells. Following each final wash, the multiwell strips are blotted onto a clean piece of absorbent material to remove remaining wash solution Alternatively, an automated plate washer could be used
4 The one-step detection (Section 3.2 1 ) can be shortened by simultaneous incubation of CAT enzyme and anti-CAT alkaline phosphatase conjugate in wells. In this case, 50 pL of CAT enzyme dilution and 50 pL of conjugate (diluted 1 500) are both added to the well together and incubated for 1 h The CAT enzyme dilutions and the conjugate dilution are prepared as 2X such that the final concentrations of CAT and conjugate are identical for both the sequential and simultaneous incubations.
5. The detection can be shortened by simultaneous incubation of hGH and anti-hGH alkaline phosphatase conjugate in wells In this case, 50 pL of hGH dilution and 50 pL of conjugate (diluted 1'250) are both added to the well together and incubated for 1 h The hGH dilutions and the conjugate dilution are prepared as 2X such that the final concentrations of hGH and conjugate are identical for both the sequential and simultaneous incubations. Simultaneous hGH capture and detector conjugate incubations result in higher sensitivity compared to sequential incubations when the assay is performed with purified protein
6. The dilution of a particular antibody-alkaline phosphatase conjugate should be optimized for maximal signal/noise.
7 Maximum light emission is reached 10-20 min following addition of CSPD Measure light emission at 10 min intervals until maximum light output is achieved
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13 Boehringer Mannheim CAT ELISA Protocol, Cat No 1363 727
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