Antigen Preparation

Antigen preparation is restricted to specialized laboratories as the procedure must be performed in biohazard safety level-3 laboratory.

2.2.1. Mice, Cells, and Bacteria

2. Coxiella burnetii Nine Mile (ATCC VR 615).

3. L929 mouse fibroblasts.

2.2.2. Cell Culture

1. Eagle minimal essential medium (Biowhittaker/Cambrex) supplemented with 2 mM L-glutamine (Biowhittaker/Cambrex) and 4% fetal bovine serum (Gibco, Invitro-gen).

2. Phosphate-buffered saline (PBS), pH 7.3, supplemented with 0.1% formaldehyde.

3. PBS, pH 7.3, supplemented with 25% sucrose.

4. 150-cm2 Cell culture flask.

5. 75-cm2 Cell culture flask.

2.3. Microimmunofluorescence

1. 30-Well microscope slides (Dynatech Laboratories Ltd.).

2. 96-Well microplates for dilution (Dutscher, France).

3. Drawing pen (one for each antigen, rinsed and dried after each use).

4. Acetone.

5. PBS, pH 7.3, supplemented with 3% nonfat powdered milk.

6. Rheumatoid factor adsorbant (RF-absorbent, Behringwerke AG, Marburg, Germany).

7. PBS, pH 7.3, supplemented with Tween-20 (1/1000).

8. Fluorescein isothiocyanate-conjugated goat anti-human IgG (dilution 1:400), IgM (dilution 1:200), and IgA (dilution 1:100; Fluoline, Biomerieux, Marcy l'étoile, France). These secondary antibodies are diluted with a mixture of PBS with 3% nonfat powdered milk and a drop of Evans blue dye (Sigma, St. Quentin Fallavier, France).

9. Slides mounting reagent (Fluoprep, Biomerieux).

3. Methods

3.1. Gimenez Staining

This staining technique (7) is used during antigen preparation described in the following sections.

1. Apply diluted carbol fushin on the methanol fixed bacteria for 2 min.

2. Wash with distilled water.

3. Apply malachite green oxalate twice incubating for 9 s each time.

4. Wash with distilled water.

5. Dry and examine under 1000X magnification using an optic microscope.

3.2. Antigen Preparation

Antigen preparation is restricted to specialized laboratories because all the procedures must be performed in biohazard safety level-3 laboratory.

3.2.1. Phase IIC. burnetii

1. Grow phase II C. burnetii Nine Mile (ATCC VR 615) on confluent layers of L929 mouse fibroblasts in 150-cm2 culture flasks containing minimal essential medium supplemented with 2 mML-glutamine and 4% fetal bovine serum.

2. Examine Gimenez-stained preparations of the cells scraped from the bottoms of the flasks under light microscope to document the presence of the cells that are already infected. When 90% of the cells are infected, pellet the cells and superna-tants contained in each of the 15 flasks by centrifugation (5000^, 15 min) and resuspend in 1 mL of PBS (pH 7.3) with 0.1% formaldehyde.

3. These suspensions are pooled and kept at 4°C overnight.

4. All further steps are conducted at 4°C.

5. Fragment intact cells are by sonication and remove cellular debris by two successive centrifugations (100g, 10 min each).

6. Centrifuge the supernatants at 6000g, for 30 min in 20 mL of PBS with 25% sucrose.

7. Wash the cell pellet three times in PBS (6000gfor 10 min).

8. Resuspend the washed cell pellet is in the smallest possible volume of PBS and adjust to a concentration of 2 mg/mL as determined by spectrometer.

9. Freeze the suspension of bacteria at -20°C for further use. The bacterial suspension can be frozen and is viable for at least 1 yr.

3.2.2. Phase IC. burnetii

1. Inoculate four Balb/c mice intraperitoneally with 106 phase II C. burnetii Nine-Mile to reactivate phase I C. burnetii.

2. Ten days after inoculation, remove the spleen of each mouse aseptically, grind in 7.5 mL of minimum essential medium with 2 mM L-glutamine and 4% fetal bovine serum, and use to inoculate L929 cell monolayers taken in three 75-cm2 culture flasks.

3. Propagate the bacteria in 150-cm2 culture flasks.

4. The number of subcultures is monitored by Gimenez staining and MIA (see Note 1).

5. The sera for MIA are derived from patients with Q fever that have been tested positive only for Phase II antibodies. The cultures may be subcultured (usually three to four times) as long as 98% of the bacteria remain at Phase I.

3.3 Microimmunofluorescence Assay

1. Deposit the two antigens prepared as described in Subheadings 3.2.1. and 3.2.2. at the two poles of each well of a 30-well microscope slide using the tip of a marker.

2. Air dry the slides before fixing in acetone for 10 min. Initially, the sera are first diluted twice 1:5-fold and then serially diluted (twofold dilutions initially ranging from 1:25 to 1:3200 and more if needed) in PBS with 3% nonfat powdered milk.

3. Sera are first adsorbed with IgG rheumatoid factor adsorbant for 15 min for the determination of IgM and IgA.

Table 1

Interpretation of Serological Results for Q-Fever

Phase I antibody titer Phase II antibody titer

IgG IgM IgG Interpretation

< 100 Active Q fever improbable

>200 $50 Acute Q fever (100% predictive)

>1:800 Chronic Q fever (98% predictive)

> 1:1600 Chronic Q fever (100% predictive)

4. 100 pL of sera (dilution 1:5) and 100 pL of RF in 300 pL of PBS are reacted.

5. The 30-well microscope slides have three lines. The first, second, and third line is used or IgG, IgM IgA, respectively. A total of 30 pL of each serum sample, diluted as above, is aliquoted into each well placing the most concentrated sample at the far right corner of the plate.

6. Incubate the overlaid antigens in a moist chamber for 30 min at 37°C.

7. Wash the antigens in PBS-Tween (1/1000) followed by one wash in PBS and another in distilled water. On each occasion, the overlaid antigens are washed for 10 minutes.

8. Overlay the air dried complex with 30 pL of fluorescein isothiocyanate-conju-gated goat anti-human IgG (dilution 1:400), IgM (dilution 1:200), and IgA (dilution 1:100).

9. Incubate, wash, and dry the antigen-antibody complexes as described in the preceding steps.

10. Mount the slides with three drops of Fluoprep and examine under a fluorescence microscope (magnification X400; see Note 2).

11. Each slide incorporates the sera obtained in patients with (positive control) and without Q fever (negative control). The titre obtained for positive control must be equal to that obtained previously (see Table 1) and the negative control must be less than 1:25.

4. Notes

1. The production of phase I C. burnetii antigen is the major difficulty associated with this method. In an in vitro culture of C. burnetii, phase II antigen becomes predominant after several subcultures. However, the remaining phase I cells are sufficient to produce infection in mice (phase II cells are destroyed by the mouse immune system). Thus, after injection of phase II antigen in mice only phase I antigen is present in tissues especially the spleen. After the inoculation of crushed spleen tissue in cell culture, phase I bacteria multiply. However, because phase II bacteria grow more quickly, after four or five subcultures, Phase I bacteria are less than 98% and thus not appropriate for use as antigen for the detection of antibodies to phase I. For these reasons, after inoculation of crushed spleen tissue in cell culture, the growth of C. burnetii is monitored by Gimenez staining to evaluate the growth of the bacteria and by immunofluorescence to evaluate the contamination by phase II bacteria. This contamination must not be higher than 2% of all bacteria.

2. Compared with those of phase II samples, the interpretation of the results of MIA performed on in phase I sera is difficult due to presence of high background fluorescence. To determine the correct titer, the difference in the brightness of fluorescence must be considered rather than its extinction.

References

1. Tissot Dupont, H., Raoult, D., Brouqui, P., et al. (1992) Epidemiologic features and clinical presentation of acute Q fever in hospitalized patients: 323 French cases. Am. J. Med. 93, 427-434.

2. Brouqui, P., Tissot Dupont, H., Drancourt, M., et al. (1993) Chronic Q fever: 92 cases from France including 27 cases without endocarditis Arch Int. Med. 153, 642-648.

3. Dupuis, G., Peter, O., Peacock, M., Burgdorfer, W., and Haller, E. (1985) Immunoglobulin responses in acute Q fever. J. Clin. Microbiol. 22, 484-487.

4. Williams, J. C., Johnston, M. R., Peacock, M., Thomas, L. A., Stewart, S., and Portis, J. L. (1984) Monoclonal antibodies distinguish phase variants of Coxiella burnetii. Infect. Immun. 43, 421-428.

5. Hoover, T. A., Culp, D. W., Vodkin, M. H., Williams, J. C., and Thompson, H. A. (2002) Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, Phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile strain. Infect. Immun. 70, 6726-6733.

6. Tissot Dupont, H., Thirion, X., and Raoult, D. (1994), Q fever serology : cut off determination for microimmunofluorescence. Clin Diagn. Lab. Immunol. 1, 189196.

7. Gimenez, D. F. (1964) Staining rickettsiae in yolk-sac cultures. Stain Technol. 34, 135-140.

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