INTRODUCTION
The Pancreatitis-associated protein (PAP) is synthesized in pancreas only during pancreatic stress. In cystic fibrosis (CF) the pancreas is already diseased in utero and PAP is synthesized before birth. Four studies have shown that PAP concentration is indeed elevated in the blood of CF newborns

PAP assay on Guthrie cards might therefore help screening CF newborns.

PRINCIPLE OF THE ASSAY
The MucoPAP kit is a sandwich enzyme-linked immunoassay (ELISA) optimized to assay PAP in dried blood spots on Guthrie cards.
The wells of the microtitration plate are coated with antibodies to PAP. In a first step, the eluates of blood spots are deposited in the wells and PAP is allowed to bind to specific antibodies. All proteins not specifically bound are eliminated by washing. Then anti-PAP antibodies coupled to biotin are allowed to attach to the bound PAP. After washing, antigen-antibody complexes are detected by an avidin-peroxidase complex and are visualized by the addition of a chromogenic substrate. The intensity of the color reaction is proportional to the quantity of PAP bound in the first step.

KIT COMPOSITION

STORAGE AND STABILITY
The assay kit should be kept at 4°C until the printed expiration date. Once lyophilisates are reconstituted, they should be stored at -20°C, except for the avidin-POD solution witch remains stable at 4°C. The wash solution can be stored at -20°C. With these storage conditions, the assay kit can be used for 30 days after opening.

MATERIALS NOT SUPPLIED WITH THE KIT AND NEEDED FOR THE ASSAY
Two liters of distilled water.

Material :

• Vortex mixer
• A semi-automatic or automatic plate washer
• A plate reader for measuring absorbance at 450 nm, equipped with a 620 nm reference filter
• Disposable tip micropipettes, single- and multi-channel
• 1L bottle (washing buffer storage)
  

Disposable material :

PRECAUTIONS FOR USE

RECOMMENDATIONS

• Never allow the plate to dry since well drying may impair the quality of the results.
• Special care should be taken to the washing steps in order to avoid non-specific signal.
• Do not use reagents after their expiration date.
• Do not mix reagents from different batches.
• Avoid biological and chemical contamination of reagents and samples.
• Equilibrate every reagent at room temperature.
• Freeze-dried reagents must be reconstituted at least 10 minutes before use in order to obtain total and homogeneous dissolution.
• Shake reagents gently before use.
• Respect strictly incubation times.

SAMPLE PREPARATION
Punch in the Guthrie card a disc 3 mm in diameter, in a region with thorough blood impregnation, without overload. Put the disc in a well of a 96 U-bottom well plate. Add 150 µl PBS buffer. Allow 16 h for elution (overnight) at 4°C.

PREPARATION OF REAGENTS
The plate packaged under vacuum must be equilibrated at room temperature before removal from its wrapping.

The wash buffer (PBS/0.1% Tween) is obtained by dissolving the PBS powedr in 1L of distilled water. After complete dissolution, save 15 ml for blood elution then add to the rest of the buffer the whole content of the Tween 20 vial.

The standard range is prepared from freeze-dried rhPAP. This standard solution is reconstituted with the volume of distilled water mentioned on the vial label. This gives a solution at 4 ng/ml that is further diluted in dilution buffer to prepare the standard range.

The dilution buffer is to be reconstituted by adding to the vial 11 ml of distilled water.

Biotinylated antibodies The lyophilisate is dissolved in 11 ml of distilled water.

Avidin-POD The lyophilisate is dissolved in 11 ml of distilled water.

The substrate-chromogen solution (TMB-H2O2) is ready to use.

The Control Serum (internal control) is reconstituted with the volume of distilled water mentioned on the vial label.

ASSAY PROCEDURE
A PAP range is prepared from the reconstituted reference solution (4 ng/ml). It will include solutions with concentrations ranging from 1 to 0.06 ng/ml. The dilution buffer provided with the kit will be used to prepare successive dilutions.

Preparation of the range
Dilute 1/2 the standard PAP solution. That solution (PAP = 2 ng/ml) will be used to prepare serial delutions with the dilution buffer.

Each dilution will be assayed in duplicate (100 µl/puits). Background value will be obtained in duplicate (100 µl buffer/well)
Blood spoot eluates, the PAP range and the control sample are deposited in wells (100 µl/well) and incubated 3 h at room temperature (~21°C), after covering the plate with adhesive. Then, wells are washed 5 times with the wash buffer, as follows:

• thoroughly draw up each well,
• fill with ~300 µl wash buffer,
• repeat the first two steps 4 times,
• after the last wash, eliminate residual liquid by inverting the plate and tapping it on absorbent paper.

Note: it is recommended to use an automatic or semiautomatic plate washer.

The reconstituted solution of biotinylated anti-PAP antibodies is then immediately deposited on the plate at 100 µl/well and incubated for 30 minutes at room temperature. The plate is covered with adhesive. The plate is then washed 5 times with PBS/0.1 % Tween (as described above) and then 100 µl of the reconstituted avidin-POD solution is immediately added to each well.

After 15 minutes incubation at room temperature, the plate is washed 5 times with PBS/0.1% Tween. During the last wash, washing buffer should be left 1 min in the wells. After the plate has been tapped dry, the chromogenic substrate is added at 100 µl/well. After 10 to 15 minutes incubation in the dark, the color reaction is stopped by adding H2SO4, 100 µl/well. The absorbance of each well is read on a spectrophotometric plate reader at 450 nm, using a 630 nm filter as reference. Plates should be read immediately after the reaction is stopped

CALCULATION OF RESULTS
The concentration of PAP in each eluate is determined by extrapolation from the standard curve. This curve is constructed by plotting the mean absorbance values of each point of the range versus the theoretical concentrations. The use of a computer programme to define, from range values, the parameters of the [PAP] = f(D.O.450nm) function, and use that function to calculate PAP concentrations in eluates is recommended.

EXAMPLE OF RESULTS
NB: This is only an example. A reference curve must be obtained for each plate.

When assayed in conditions described for samples, the PAP concentration in control serum should be as indicated on the vial label.

ANALYSIS OF RESULTS
1. Calculation of PAP concentrations in blood : If the protocol given above are followed strictly and if the samples come from screening cards made from Whatmann paper ref. BFC180- B997243 (approved by the French Screening Association), raw data (PAP concentrations in eluates) have to be multiplied by a factor of 30 to obtain PAP blood concentrations. This factor must also be applied to the Control Serum.

2. Ongoing studies show that, with the present test, median blood PAP concentration in newborns is ~2.6 ng/ml. This data is only indicative.

SUMMARY OF THE TEST

1. Prepare buffers and the PAP range
2. After equilibration at room temperature, open the aluminium bag and deposit the samples and the range in the plate (100 µl/well) Incuber 3h à température ambiante
3. Draw up liquid, 5 washes
4. Distribute the biotinylated antibody (100 µl/well)
5. Incubate 30 min at room temperature
6. Draw up liquid, 5 washes
7. Distribute avidin-POD (100 µl/well)
8. Incubate 15 min at room tempearture
9. Draw up liquid, 5 washes. Dry
10. Distribute the substrate-chromogen mix (100 µl/well)
11. Incubate 10-15 min in the dark
12. Stop the reaction with H2SO4 (100 µl/well)
13. Read absorbance at 450 nm