LEAP Testing Service

LATEX FINGERPRINT ASSAY (L001)

I.  Scope
This immunoblot procedure qualitatively assesses the transfer of antigenic protein by the glove sample in comparison to control gloves that contain high and low antigenic protein levels by pressing the glove (inside and/or outside) to nitrocellulose and then developing the blot using anti-latex antibody.
 

II.  Procedure

1. Cut a piece of nitrocellulose 10 cm by 2 cm for every required lane, without touching the nitrocellulose. (e.g., a 10 cm by 8 cm sheet is required to test two samples with the positive and negative controls).Use forceps to handle nitrocellulose throughout the procedure to avoid adding unwanted proteins.
   
2. Wet membrane in carbonate buffer pH 9.6 and place it on a clean glass plate with only a slight film of liquid remaining on the membrane.
   
3. Thoroughly wash hands and don the glove to be tested, pulling it tight to the finger.
   
4. Press the finger down on the nitrocellulose for 3 seconds at ~5 pounds of pressure.
   Start the fingerprints at the bottom and work to the top, making a total of four imprints with the same finger for each of the samples and controls.  Keep membrane moist during imprinting.
   
5. After all imprints have been made, rinse membrane with a squirt bottle (top to bottom so that particles or proteins do not cross lanes) one time with T-PBS.
   
6. Block membrane in tray face-up by rocking for 1 hour in blocking solution
   (5% non-fat dry milk protein) at room temperature. Avoid polystyrene since it binds protein.
   
7. Wash membrane (in tray) 2-3 times with T-PBS.
   
8. Incubate membrane in rabbit anti-latex protein (1/1000 of pooled anti-sera in PBS) overnight at room temperature or a 1/500 dilution for 2 hours at 37°C with continuous rocking.
   
9. Wash membrane 2-3 times with T-PBS.
   
10. Incubate membrane in goat anti-rabbit-AP (1/5000 in PBS) for 1 hour at room temperature.
    
11. Wash membrane 5 times with T-PBS and 1 time with a small amount of AP buffer.
    
12. Developing membrane:  Mix NBT/BCIP in AP buffer pH 9.5  (66 ul of NBT and 33 ul of BCIP for ever 10 ml of AP buffer). Make fresh before use.  Incubate membrane in developer for 20 minutes until development is adequate with respect to controls and background.  To stop reaction, rinse membrane with water and allow membrane to air dry before mounting.