Serotyping and Serosubtyping of N. meningitidis with Monoclonal Antibodies (Mabs)
Differentiation and classification of bacterial strains at the sub-species level require methods that are highly reproducible, little affected by the experimental conditions, and give effective discrimination of epidemiologically unrelated strains. Further characterization of bacteria causing meningitis is especially important to document the spread of particularly pathogenic clones. Many immunoassays have been developed for serological characterization o N. meningitidis antigens.
Immunological detection of specific epitopes of two major outermembrane proteins, PorB and PorA, is the basis for the serological classification system defining, respectively, the serotypes and serosubtypes of the species 1, 3-7). A simple, low cost method for serotyping and serosubtyping meningococci, dot-blotting with Mabs, is described below.
A. Preparation of whole-cell suspensions
Whole-cell suspensions should not be used for PCR testing.
1. Grow the N. meningitidis isolate to be tested along with an appropriate reference isolate for QC on a BAP for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
2. Collect 3-5 colonies using a sterile disposable loop or sterile swab and suspend in 1 ml of PBS (pH=7.2) with 0.02% sodium-azide.
3. Inactivate the bacteria by heating the suspension at 60C for 30 minutes.
4. Adjust the optical density with PBS to 0.2 measured at 650 nm
5. Maintain whole-cell suspensions at 4C until use.
Read Also :Identification of N. meningitidis Serogroup and Performing the SASG Test
B. Performing the dot-blotting serotyping/serosubtyping test
1. Cut strips (0.5 x 10 cm) of nitrocellulose paper (pore 0.45 m). Divide each strip into 10 sections of 1 cm in length using a wax pencil and align them on a glass or hard plastic plate.
Use gloves and/or forceps when handling nitrocellulose paper.
2. Label the strips with a permanent pen on the left section.
3. Dot 2 l of the whole-cell suspension of a relevant reference isolate in the middle of the next marked section and 2 l of the test isolates successively onto each of the next sections, as shown in Figure 8.
4. Dry the strips for 15 minutes or more.
5. Add 2 ml of the blocking buffer (PBS supplemented with 3% bovine serum albumin [BSA]) into each well of an 8-well incubation tray (8-well trays with lids are commercially available).
6. Transfer the strips to be tested with the same Mab to a well (maximum 4 strips per well).
Mix gently for 30 minutes. The strips must be fully wetted by the blocking buffer.
The minimum incubation volume of the blocking buffer for 1 strip is 0.5 ml. Up to 4 strips with about 40 dotted samples can be in incubated in a total volume of 2 ml. Consider leaving an empty well in the incubation trays between each set of strips that are incubated with different Mabs.
7. Add the Mabs to the respective wells. Depending on the supplier of the Mab, the final dilution may range from 1:10 to 1:500,000.
8. Place the lid on the incubation tray and incubate with gentle mixing overnight.
9. Carefully remove the antibody solutions from the strips using a pipet tip connected to a suction pump or similar device. Rinse the tip with water between each set of strips that were incubated with different Mabs to prevent transfer of the Mabs.
10. Rinse the strips 3 times with no more than 2 ml PBS directly in the wells. Remove the PBS in the same manner in which the antibody solutions were removed.
11. Add 2 ml of PBS supplemented with 3% BSA with horseradish peroxidase-labelled rabbit anti mouse antibody (typically used at a 1:2,000 dilution) and incubate for 2 hours with gentle mixing. Be sure that the strips are fully wetted.
12. Rinse the strips in 2 ml of PBS for 5 minutes twice as in step 9.
13. Wash the strips for several minutes in 0.05 M sodium acetate buffer, pH 5.0, and then discard
the solution.
14. Put the strips in a plastic box and add 10 ml of sodium acetate buffer with 0.4 ml of 3-amino- 9-ethylcarbazole (AEC) in dimethylformamide (DMF). Mix for 2-3 minutes.
15. Add 10 l of H2O2 at 30% and stain each dot until distinct red dots are observed for the reference isolate (2-10 minutes).
Reading the dot-blotting serotyping/serosubtyping test results
1. Rinse the strips under running water, align them on a glass plate, and absorb the excess water with filter paper or tilt the plate slightly to dry the strips at room temperature (25°C).
2. Grade the staining intensity of each dot (positive, weak, or negative) visually relative to the reference strain.
3. When dried, the strips can be taped onto a sheet of paper and kept in a plastic pocket protected from light.
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