Protocol 121 Basic realtime immunoPCR protocol

Chemicals:

Equipment:

Adsorption buffer: 0.2 M NaH2PO4

0.005. Tween 20, and 0.1% Germall Blocking/incubation buffer: 0.5% BSA, 0.05% Tween 20 in phosphate buffered saline pH 7.3 (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4)

Capture antibody

Detection antibody/DNA conjugate Antigen standard PCR chemicals

For washing: use either a multichannel pipette or Nunc® immunowash. Other automatic washing devices that handle V-shaped PCR-tubes can also be used.

Real-time PCR instrument with 96-well plate format.

Precision pipettes PCR microtiter plate Adhesive seal

Vortex

Microcentrifuge

1. Adsorption of capture antibody. Dilute capture antibody to desired concentration in 0.2 M NaH2PO4. A volume of 2,500 |jl is needed per 96-well plate. Add 25 |l of the capture antibody solution to each well and cover the plate with adhesive seal. Incubate at 4 °C overnight (see Figure 12.4)

2. Blocking. Wash the antibody coated wells three times with wash buffer. Add 200 |l blocking buffer to each well. Cover with adhesive seal and incubate at 37°C for 1 h or at 4°C overnight

3. Prepare analyte and detection antibody/DNA conjugate solution. Prepare an analyte dilution series by diluting stock

Step 1

Step 1

Figure 12.4

Steps in basic real-time immuno-PCR.

antigen solution in incubation buffer. The standard concentrations should cover the concentrations of the samples that will be analyzed. Never attempt to quantify antigen at concentrations outside the range of the standards. Prepare also an antibody/DNA conjugate solution of suitable concentration in incubation buffer. For duplicate samples mix 11 ^l of either the analyte standard or the test sample with 44 ^l of diluted conjugate solution in a clean microtiter plate. Prepare BC samples by mixing 11 ^l of incubation buffer with 44 ^l of the conjugate solution in a separate tube. Incubate all samples for 1 h at room temperature

4. Add samples to the wells. Wash the blocked wells ones with wash buffer and add 25 ^l of the incubated samples to each well. Seal with adhesive seal and incubate at room temperature for 1 h

5. Wash the wells six times with wash buffer and ten times with milli-Q water

6. Tap the microtiter plate upside down on paper towels to remove as much of the water as possible

7. Run real-time PCR. Add 25 ^l PCR mastermix to each well. Prepare also an NTC by adding PCR mastermix to a clean well. Seal the microtiter plate with an adhesive seal that is compatible with the real-time PCR instrument. Start the PCR. Use the same cycling protocol as for the regular real-time PCR

Data analysis Construct a standard curve from the standard samples by plotting Ct versus the logarithm of analyte concentration. Fit a line by linear regression to the data points that follow linear response. Frequently the lowest and highest concentrations deviate from linearity. To include those one may use more advanced models, such as 3rd degree polynomial to fit the data points. To determine the sensitivity of the assay, run replicates of the BC together with the standard curve samples (Plate scheme in Figure 12.5). Estimate the apparent concentrations the BC signals correspond to using the standard curve, and calculate the standard deviation (SD) and mean. The limit of detection is defined as the mean of the apparent concentrations of the BC samples plus three times the SD (ACS Committee on Environmental Improvement, 1980). The reproducibility of the assay is determined from at least eight replicates of standard samples with high, medium, and low analyte concentration that are run together with a complete standard curve (Figure 12.5). The sample concentrations should be within the linear range of the standard curve. Estimate the concentrations from the repeat samples from the standard curve and calculate their mean and the SD. Finally calculate the relative standard deviation at the three concentrations as the SD/mean. Typically the relative standard deviation decreases with increasing analyte concentration.

bc

bc

bc

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1

1

bc

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2

2

bc

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3

3

bc

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4

4

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6

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7

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Microtiter plate set-up to estimate the sensitivity and reproducibility of the realtime immuno-PCR. Numbers 1 to 7 represent standard antigen samples of different concentrations. BC is the background control. Repeats of standard samples with low (L), medium (M), and high (H) concentrations of antigen.

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