Eur. J. Epidemiol. 0392-2990
EUROPEAN JOURNAL
July 1992, p, 568-574
Vol. 8, No. 4
OF EPIDEMIOLOGY
MONOCLONAL ANTIBODY BASED CAPTURE ELISA/ELIFA FOR DETECTION OF COXIELLA BURNETII IN CLINICAL SPECIMENS D. THIELE 1, M. KARO and H. KRAUSS lnstitut f d r Hygiene u n d Infektionskrankheiten der Tiere - Justus Liebig Universitiit Frankurter Str. 89 - D-6300 - Giessen - F.R.G.
Key words: Q fever - Coxiella burnetii - CAPTURE ELISA - Monoclonal antibody A CAPTURE ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody is described. The assay is fast, highly specific and detects a minimum dose of 2500 Coxiella (C.) burnetii particles. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories could use this assay system for investigating clinical samples of different origin for C. burnetii within a short period of time.
INTRODUCTION
Coxiella burnetii is the causative organism of Q fever in a variety of animal species and man and is recognized as a worldwide problem (2, 22). Ticks are the primary reservoir. Infected ticks transmit Q fever to cattle, sheep, goats and companion animals such as horses, dogs and cats. Within domestic animals transmission mainly occurs by infectious aerosol. Dairy cows are also a major reservoir of this zoonotic agent, shedding the organisms during parturition with placenta and birth fluids in high doses. Chronically infected cows may shed the organisms periodically with their urine and feces, and especially with milk during successive lactating periods (16). Due to its high resistance against physical as well as chemical agents C. burnetii poses a health hazard for a considerable period of time once the pathogen is disseminated in the environment. Veterinarians, abattoir workers and laboratory workers professionally exposed to the pathogen (mainly by aerosol) may be especially exposed to infection. It has been estimated that infection by one single organism can cause disease (8, 12). Ingestion of unpasteurized milk 1 Corresponding author.
containing the agent usually leads to inapparent infection. The diagnosis of Q fever is mainly based on indirect serological procedures such as complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA) (3, 15, 17, 21, 23) or immunofluorescence (IF) (6, 13). Immunoblotting as a diagnostic tool has been introduced to C. burnetii serology only recently (4, 9, 10, 20). In samples containing high numbers of the pathogen direct detection can be achieved after chemical staining, e.g., with the Gimrnez procedure. For isolation of C. burnetii in samples containing only low numbers of organisms, test animals such as guinea pigs or mice (8, 19) or more sophisticated cell culture techniques (1, 14) can be used. Both isolation techniques (animal inoculation or cell culture) are not completely satisfactory. Due to varying susceptibility of C. burnetii strains to antibiotics (24-26) they should be preferably omitted in cell culture isolation techniques. This makes it difficult to isolate the agent from contaminated samples, although infectivity of the pathogen is stable for a prolonged period of time. Using animals for isolation purposes should be avoided whenever possible despite the fact that guinea pigs or even mice are well suited for this purpose due
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to their high susceptibility. One particle of the organism may be sufficient to induce detectable antibody levels, but diagnosis by animal inoculation is time consuming (up to 3-4 weeks). In this presentation we describe a sensitive ELISA for detection of an epitope on C. burnetii lipopolysaccharide antigen, which may be used also as an enzyme-linked immunofluorescent assay (ELIFA).
MATERIALS AND METHODS
Organisms. Coxiellae used as stocks were harvested from persistently infected Buffalo-Green-Monkey (BGM) cell cultures (1) or as infected yolk sacs of embryonated hens' eggs. The following isolates were available from the collection of our institute: "Nine mile" phase (ph) II, "Frankfurt" ph I/II, "Henzerling" ph II; "Herzberg", "Mfinchen", "Balaceanu", "Boren" "Brasov", "Pop", "Stanica", and "Utvinis", all ph I. Additional isolates were kindly supplied by J. Kaz~ir, Institute of Virology, Bratislava ('Nine Mile" ph I,) and H. Becht, Institut Rir Virologie, Universit~it Giessen ("Andelfingen", "Geier", and "Soyta', all ph I). For testing possible cross-reactivity and specificity the following bacteria were used: Aehromobacter haemotysans, Acinetobacter ealcoaeeticus var. anitratus, Bordetella bronehiseptica, Chlamydia (Chl.) psittaci strain 1904, Corynebaeterium pyogenes, Escheriehia eoli, Listeria monocytogenes I and IVb, Pasteurella multocida, Salmonella typhimurium group B, Staphylococcus (S.) aureus, S. aureus Cowan I, S. aureus Wood 46, S. hyicus, Streptococcus (St.) faecalis, St. zooepidemicus, and Yersinia enterocolitica. All these bacteria were available from the collection of our institute. Additionally, Bartonella bacilliformis was obtained from J. Knobloch, Bernhard-Nocht-Institut, Hamburg, F.R.G., Legionella pneumophila from H.G. Schiefer, Inst. Rir Medizinische Mikrobiologie, Giessen, F.R.G., and Chl. trachomatis type L2 from T. Forsey, Institute of Ophthalmology, London. Partial purification of C. burnetii from cell culture. Supernatants from persistently infected BGM cell cultures containing C. burnetii were sonicated (Branson Sonifier B-12, 50 W, 10 sec; Branson Sonic Power Comp., Conn., USA). Samples were centrifuged twice at low speed (300 x g, 10 rain) to pellet cell debris. The supernatants were recentrifuged at 20,000 x g, for 30 rain. The resulting pellet containing C. burnetii was washed twice with double distilled water, diaiysed extensively against double distilled water and lyophilized. Finally, smears from resuspended material were stained with Gim6nez stain and examined for contaminating BGM material. Immunization. Female Balb/c mice 6 to 10 weeks old were infected intraperitoneally with 3.85 x 10' inclusion forming units (18) of strain Nine Mile ph I,
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harvested from infected yolk sacs of embryonated hens' eggs and resuspended in phosphate buffered saline (PBS). Mice were reinfected at day 30 and fusion was performed 4 days later. Fusion and cloning. Spleen cell suspensions containing stimulated B lymphocytes from infected Balb/c mice were fused with cells of the mouse myeloma cell line X63-Ag8.653 (7) by using 500/0 polyethylene glycol (PEG Hybri-Maxr 1300-1600, Sigma, Deisenhofen, F.R.G.). Primary cultivation of hybrids was performed using 96-well cell culture plates in the microtiter format with Rosewell Park Memorial Institute (RPMI) 1640 medium containing hypoxanthine, aminopterin and thymidine (HAT selection) supplemented with 10% fetal calf serum (FCS) in a tissue culture incubator at 370 C and 7.5% CO2. FCS was purchased as mycoplasma free (Roth, Karlsruhe, F.R.G.; Gibco, Eggenstein, F.R.G.; Seromed, Berlin, F.R.G.) and was extensively tested for the support of cells growing under the conditions of limiting dilution. Hybrids were tested for monoclonal antibody production using an ELISA system. To assure monoclonality, hybrids of interest were cloned 3 times using the limiting dilution technique with thymocytes as feeder layer (11). Results from limiting dilution were analysed using the Poisson statistical analysis for repetitive subcloning (5). ELISA for detection and characterization of antigen specific monoclonal antibodies. Antibody secreting hybridoma cells and antibody production were monitored by indirect ELISA. Briefly, wells on microtiter Immulon M129A plates (Greiner, Niirtingen, F.R.G.) were coated with Coxiella antigen Nine Mile ph I or ph II harvested and purified from persistently infected BGM cell cultures. Using Coxiella antigen propagated in cell cultures for this purpose instead of antigen propagated in yolk sacs, which was used for immunization, was advantageous for not codetecting hybrids producing antibodies against contaminating egg proteins. 100 lal antigen suspension in coating buffer (1.059 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3 dissolved in 1 1 double distilled water, adjusted to pH 9.6) per well was incubated at 37oC until the wells were dry. Remaining binding sites were blocked with 200 Ill PBS/well containing 2% bovine serum albumin (BSA), and a minimum of 50 lal hybridoma cell culture supernatant was added to the wells. For detecting bound antibody 100 lal of horseradish peroxidase labelled goat anti-mouse immunoglobulin conjugate was added (Dianova, Hamburg, F.R.G.). Bound conjugate was visualised using 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS; Serva, Heidelberg, F.R.G.) as chromogenic substrate for peroxidase (2.1 g citric acid in 100 ml double distilled water, adjusted to pH 4.2 with NaOH; add 0.011 g ABTS, store at -30°C for no longer than 6 months. Adjust temperature to 37° C before use and add 20 lal H202 [30°/0]per 100 ml). As a negative control
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wells were incubated with RPMI 1640 medium or "conditioned medium" (supernatant from propagated myeloma cells). Serum harvested from mice used for immunization and as spleen cell donors served as positive control. The same ELISA format was used to determine class, subclass and light chain type of monoclonal antibodies using the "Mouse Typer Kit" and "Mouse Typer Panel" (BioRad, Munich, F.R.G.) containing specific rabbit antisera to mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, kappa-chain and lambda chain, and goat anti-rabbit IgG (H+L) peroxidase-labelled conjugate. Hybridoma maintenance and production of antibodies. After establishing stable antibody-producing hybridomas in RPMI 1640 medium containing hypoxanthine and thymidine (omitting aminoptefin at this state) and 10% fetal calf serum, the cells were tested for the production of monoclonal antibodies under serum free RPMI medium supplemented with 2% Ultroser H Y (Gibco, Eggenstein, F.R.G.). Using this supplement culture supernatants were free of foreign immunoglobulins such as bovine immunoglobulin. Characterization of monoclonal antibodies using different test systems. To further characterize established monoclonal antibodies binding characteristics were also determined in direct and indirect immunofluorescence, dot test, indirect immunoperoxidase and immunoblot techniques. Purification of monoelonal antibodies. The monoclonal antibodies were purified on a protein A-Sepharose CL-4B column (Pharmacia, Freiburg, F.R.G.), dialysed against double distilled water and lyophilysed. Biotinylation of monoclonal antibodies. 6 mg of purified and lyophilysed monoclonal antibody were dissolved in 500 111 carbonate buffer (0.1 M NaHCO3, 0.1 M Na2CO3; pH 8.5). 50 111 of dimethylsulphoxide (DMSO) containing 1.0 mg of D-Biotinyl-eaminocaproic acid-N-hydroxysuccinimide ester (Biotin-X-NHS) were added and incubated for 12 h while shaking at room temperature (RT). The reaction mix was extensively dialysed against 1 1 of carbonate buffer for 16 h at 4° C, with 3 changes of the buffer. The dialysate was then centrifuged (8000 g, 10 min, RT), the supernatant supplemented with 2% bovine serum albumine for cryoprotection and shock-frozen in liquid nitrogen before being stored at -20 ° C. Antigen capture test. Several commercially available EL1SA microtiter plates were tested and compared. Best results could be achieved with ELISA-F plates Immulon M129A made from polystyrol with medium binding capacity for proteins (Greiner, NiJrtingen, F.R.G.). Plates were freshly coated with capture antibody, 100 lal/well (lmg/ml, diluted 1:5000 in TRIS buffered saline [TBS]: 10 mM TRIS, 100 mM NaC1,
pH 8.5), and incubated for 16 h at 4° C. Remaining binding sites were blocked with TBS containing 10 mM TRIS, 150 mM NaC1, 0.5 % Tween 20, pH 8.0, supplemented with 1% fish gelatine (Sigma, Deisenhofen, F.R.G.), for 15 rain at RT. Plates were washed three times with 250 lal washing buffer (10 mM TRIS, 150 mM NaC1, 0.5% Tween 20, pH 8.0); each washing step was 5 min and the same buffer wash always used. Samples were added in a volume of 100 lal and incubated for 2 h at RT. After three more washings the monoclonal biotinylated detection antibody wash added (100 lal, 30 rain, 1:10,000 in wash buffer). Optimal concentration for each immunoglobulin compound was initially determined with a chequerboard ELISA titration. After three more washings, streptavidin conjugated with alkaline phosphatase or peroxidase (Boehringer, Mannheim, F.R.G.) was added (100 lal, 30 min, 1:10.000 in wash buffer). Substrate was added after three final washings. For streptavidin conjugated with peroxidase 3,3'-5,5'-tetramethylbenzidine (TMB, Fluka, NeuUlna, F.R.G.) was used as substrate (stock solution I: 1 M sodium acetate, adjusted to pH 5.8 with 0.1 M citric acid, stored at 4° C; stock solution II: 10mg/ml TMB in DMSO (Sigma, Deisenhofen, F.R.G.), stored at 4o C.; working solution: 2 ml of stock solution I were mixed with 0.2 ml stock solution II and 20 ml double distilled water. 20 lal H202 (6%) were added and the solution adjusted to 370 C). The enzyme reaction proceeded for 30 rain and was stopped with 3 M H2SO4 (free of Fe ions). The optical density was measured at 450 nm in a Titertek Multiscan ELISA reader (Flow, Meckenheim, F.R.G.). For streptavidin conjugated with alkaline phosphatase 4-methylumbelliferylphosphate (4-MUP; Sigma, Deisenhofen, F.R.G.) was used as substrate (2 mg of 4-MUP were dissolved in 10 ml diethanolamine buffer, always freshly prepared and adjusted to 37° C. Diethanolamine buffer: 97.0 ml diethanolamine (Merck, Darmstadt, F.R.G.), 0.2 g NAN3,0.01 g MgCI2 x 6 H20, 800.0 ml double distilled water; adjusted to pH 9.8 with HC1, double distilled water added to 1 1, stored at 4° C and protected from light). Fluorescence was measured after 60 min using a Titertek Fluoroscan ELISA reader (Flow, Meckenheim, F.R.G.) with excitation wavelength of 355 nm and emission wavelength of 460 nm. Samples were then tested in duplicates. A standard titration of C. burnetii Nine Mile ph I (ling/ ml; 1:5000 - 1:1,280,000) was always performed in parallel. Samples containing buffer instead of antigen served as negative controls. Preparation of antigen and clinical samples. For analytical purposes a stock suspension of C burnetii Nine Mile ph I (1 mg/ml) was used. Clinical samples were obtained from 33 cases of bovine and sheep abortions and from a cow after gestation. Also ten samples of wash water from a factory processing lamb wool were investigated for Coxiella antigen.
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Class, subclass and light chain. All hybridomas tested
Analytical and clinical samples were treated with proteinase K prior to testing. Proteinase K (Sigma, Deisenhofen, F.R.G.), non self-digested, was dissolved in 10 mM TRIS with 1 mM CaC12 (addition of Ca2+ to proteinase K was carried out to degrade proteins that are commonly resistant to proteinase K) at a concentration of 400 pl/ml. To analytical and clinical samples the same volume of proteinase K was added (to give a final concentration of 200 pl/ml) and incubated for 30 rain at 56o C. For inactivation of the enzyme the reaction mixture was heated to 100° C for 15 rain.
produced antibodies of the IgG3 subclass with kappa light chains. Antibody production and purification. Three clones (2/ 15, 4/6 and 4/11) could be adapted to produce monoclonal antibodies in serum-free RPMI 1600 medium supplemented with Ultroser HY. Cell culture supernatants contained antibodies in the range from 1 to 10 pl/ml. All these antibodies could be purified on protein A-Sepharose CL-4B without considerable loss of activity. Serum-free RMPI 1640 medium supplemented with Ultroser H Y as control never yielded any detectable amount of immunoglobulins when applied to the protein A affinity chromatography column.
Isolation of C. burnetii from clinical samples. Clinical samples were sonicated in PBS till homogenization. In cases of severe bacterial contamination this homogenate was filtered through 0.45 pl filters. An equal amount of medium was added and the filtrate transferred to BGM cell cultures. BGM cells were propagated on round glass slides (12 mm diameter) in fiat bottom plastic vials (6 ml). The cell culture tubes were then centrifuged for 60 rain at 3000 g and incubated at 37° C. The presence of multiplying C. burnetii was determined by visual inspection of the cultures with an inverted microscope for typical vesicle formation, subsequent staining of coverslip cultures after Gim6nez and microscopic investigation of cells.
Statistical analysis. ELIFA results with analytical samples containing 1 mg/ml C. burnetii Nine Mile ph I antigen were used to determine several statistical parameters of the test system. The following parameters were determined: detectability (cut off) and sensitivity. Detectability of the assay was defined as the lowest concentration of antigen exceeding the zerodose precision and was estimated using the following equation: Detectabi#ty = (Absorbance zero dose + tn - 1; co; one sided • SD)
dn Sensitivity was defined as the responsiveness of the assay to changes in concentration of the substance tested. Therefore, on a sigmoid dose-response curve, sensitivity decreases at either end of the curve. Sensitivity was estimated using the following equation: Sensitivity = dR/dC. R = ELIFA units C = reciprocal dilution (log10) ~S~TS
Cloning. Five hybridoma cell lines secreting antibodies specific for C. burnetii could be established from one fusion experiment.
Binding characterization using different test systems. Preliminary binding studies using direct and indirect immunofluorescence, dot test, and the indirect immunoperoxidase test demonstrated that not all monoclonal antibodies recognized each C. burnetii isolate tested in the various test systems showing sometimes distinct recognition patterns in different systems. However, two monoclonal antibodies (4/6, 4/ 11) recognized all isolates in every test system. Immunoblotting of these two antibodies revealed reactivity with C. burnetii lipopolysaccharide. The monoclonal antibody 4/11 was used to set up the CAPTURE ELISA/ELIFA system. CAPTURE ELISA/ELIFA. Using this test system based on monoclonal antibody 4/11 as capture and detection antibody - all C. burnetii isolates were clearly recognized. All bacteria tested (20 different strains as negative controls) did not react in this assay. Using a stock solution of C. burnetii Nine Mile ph I (1 mg/ml) the assay still gave a positive signal (cutoff) when applying a dose of 100 pl of a 1:160,000 stock solution (Figs. 1, 2). This amount contained 625 pg of C. burnetii antigen, or approximately 2500 particles. The detection limit was the same when using streptavidin conjugated with peroxidase (ELISA) or strepavidin conjugated with alkaline phosphatase (ELIFA). Sensitivity defined as the responsiveness of the assay to changes in concentration of the substance tested demonstrated a maximum at a dose of 87 ng. Stock solutions of the monoclonal and biotinylated monoclonal antibody could be stored up to six months without any detectable loss of activity during that period. We used this assay system for routine diagnostic purposes in cases of abortion in sheep and cattle. Results obtained with 34 specimens (samples of placental or fetal tissues, vaginal secretion of cattle and sheep, and in one case from vaginal secretion of a cow after gestation are demonstrated in Table 1. Ten samples were positive in the CAPTURE ELISA, 8 of which could be confirmed by cell culture isolation.
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5000q
CaptureELIFA
From ten specimens of wash water from a factory processing lamb wool, eight were positive in the CAPTURE ELISA but negative using cell culture techniques. Apparently this was due to non-viable Coxiella.
~°°°i~ 3000i
~7 n
2000! !000~ 10ug
g DISCUSSION
/ / 1ug
100ng lO~ng
I ng " i00 ~g
C. burnetiiNineMileph i (totalamount)
Figure 1. - Antigen titration using the CAPTURE ELIFA, doses ranging from 10 lag - 100 pg. Data presented with standard deviation and adapted by 4-parameter logistic function. Sensitivity profile deduced from data (dR/dC) demostrating maximum sensitivity at a dose of 87 ng.
2",h
--3 ,
~ogio (reciprocaldilution) Figure 2. - Log-logit transformation of data from figure 1, including regression line and confidence interval (99%).
TABLE 1 . - Positive results achieved with antigen detection in the CAPTURE ELISA in comparison to isolation of Coxiella burnetti in BGM cell cultures. Species
n
CAPTURE ELISA
Cell culture
Cattle
19
3
3
Sheep
15
7
5
Total
34
10
8
For some time C. burnetii has been detected by time consuming isolation procedures such as the guinea pig inoculation test or propagation in embryonated eggs. In recent years, a cell culture technique of applying BGM cells and medium without antibiotics (1) has also been used in our laboratory for isolation of C. burnetii, and a similar technique with HEL cells has been described by Raoult et al. (14) for specimens collected from humans. However, contamination of the (antibioticfree) cell cultures with bacteria may occur and destroy the cells in cases of severe bacterial contamination of the sample, as is often the case with specimens from animals. The CAPTURE ELISA/ELIFA described does not have such disadvantages. Samples contaminated with bacteria can be tested without any problems and results are obtained within one working day. Even samples containing inactivated (formalin, heat, SDS/proteinase-K) C. burnetii organisms can be analysed. The aim was to select a monoclonal antibody with broad reactivity to C. burnetii, i.e., recognition of both phases and all isolates, and with no cross-reactivity to other organisms. In addition, the secreting hybridoma cell line should produce the antibody in sufficient quantities and should be able to do so in serum-free media. Other important characteristics addressed were immunoglobulin class, affinity, ease of purification and conjugation, and stability. Finally the antibody should recognize an epitope which is found repeatedly on C. burnetii for use of the antibody as capture as well as detection antibody in the antigen capture test, thus avoiding competitive effects. The monoclonal antibody applied demonstrated a broad reactivity including all C. burnetii isolates tested (ph I as well as ph II) with no cross-reactivity to LPS of a battery of gram-negative bacteria and antigenic determinants of various other organisms. Polyclonal antisera often exhibit higher sensitivity and avidity than monoclonal antibodies, whereas monoclonal antibodies generally show higher specificity. However, sensitivity of the assay presented could not be increased by use of affinity purified rabbit antiserum against C. burnetii ph I antigen as capture antibody. When applying such a serum in the CAPTURE ELISA sensitivity was actually diminished. Furthermore, standardized polyclonal antiserum usually is available in limited amounts only whereas monoclonal antibodies can be produced almost unlimitedly of uniform quality. This fact also favours the use of the monoclonal antibody-based CAPTURE ELISA/ELIFA.
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Differences between CAPTURE ELISA and ELIFA format were only minimal with a tendency of the CAPTURE ELIFA format to detect less C. burnetii particles. As ELIFA generally has been demonstrated to be superior when compared with conventional ELISA we think that possibly the CAPTURE ELIFA can be optimized further. One reason for the excellent results with the ELISA might have been the high sensitivity of the TMB substrate used in the CAPTURE ELISA format. Applying other substrates in the CAPTURE ELISA, e.g., ABTS, the ELIFA was clearly superior. Clinical samples being analysed as positive in the CAPTURE ELISA/ELIFA but negative by isolation in cell cultures may be due to non-infectious particles. This may be assumed especially for the wool washing samples which had been heated during the washing process to a minimum of 60° C. Preincubation of these samples with the non-biotinylated monoclonal antibody or rabbit antiserum specific for C. burnetii always demonstrated more than 80% neutralizing activity. Although all bacteria tested did not show any cross reactivity it can not be unambiguously ruled out that other organisms not tested may eventually cross-react. The test system described appears to be a sensitive and specific screening system for the detection o f C. burnetii in clinical specimens. It is fast, easy to perform and requires no specialized cell culture equipment or animal testing. Acknowledgements
Excellent technical assistance provided by Mrs. Rosi Frank and advice in statistical analysis by H. Willerns is gratefully acknowledged. REFERENCES
1. Arens M. (1983): Continuous multiplication of Coxiella burnetii through persisting infection in buffalo green monkey (BGM) cell cultures - J. Vet. Med. Ser. B 30: 109-116. 2. Baca O.G. and Paretsky D. (1983): Q fever and Coxiella burnetii: a model for host-parasite interactions - Microbiol. Rev. 47: 127-149. 3. Behymer D.E., Ruppanner R., Brooks D., Williams J.C. and Franti C.E. (1985): Enzyme-linked immunoassay for surveillance of Q fever - Am. J. Vet. Res. 46: 2413-2417. 4. Blondeau J.M., Williams J.C. and Marrie T.J. (1990): Phase II Coxiella burnetii antigens as measured by western blotting - Ann. N. Y. Acad. Sci. 590:187-202. 5.
Coller H.A. and Coller B.S. (1986): Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. In: Methods in Enzymology, Vol. 121; Langone, J.J. and van Vunakis, H., eds. Academic Press Inc., London 412-417. 573
6. Field P.R., Hunt J.G. and Murphy A.M. (1983): Detection and persistence of specific IgM antibody to Coxiella burnetii by enzyme-linked immunosorbent assay: a comparison with imunofluorescence and complement fLxation test - J. Infect. Dis. 148: 477-487. 7. Kearney J.E, Radbruch A., Liesegang B. and Rajewsky K. (1975): A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines J. Immunol. 123: 1548-1550. 8. Moos A. and Hackstadt T. (1987): Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model Infect. Immun. 55: 1144-1150. 9. Norlander L., Maeellaro A. and/[kesson, ,4. (1991): Humans exposed to Coxiella burnetii produce antibodies against a 67 kD Protein. In: Rickettsiae and rickettsial diseases. Kaz/ir J. and Raoult D., eds. Slovak Acad. Sci. Bratislava 334-339. 10. Novdk M., Brezina R. and Kazdtr J. (1991): Immunoblot analysis of antibody response in mice infected with Coxiella burnetii phase I. In: Rickettsiae and rickettsial diseases. Kaz~ir J. and Raoult D., eds. Slovak Acad. Sci. Bratislava 318-324. 11. Oi V.T., Jones P.P., Goding J.W. and Herzenberg L.A. (1978): Properties of monoclonal antibodies to mouse Ig aUotypes, H-2 and Ia antigens -Curr. Top. Microbiol. Immunol. 81: 115-129. 12. Ormsbee R.A., Peacock M., Gerloff R., Tallent G. and Wike D. (1978): Limits of rickettsial infectivity Infect. Immun. 19: 239-245. 13. Pbter 0., Dupuis G., Peacock G. and Burgdorfer IV. (1987): Comparison of enzyme-linked immunosorbent assay and complement fixation and indirect fluorescent-antibody test for detection of Coxiella burnetii antibody - J. Clin. Microbiol. 25: 1063-1067. 14. Raoult D., Vestris G. and Enea M. (1990): Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of the strains in HEL cells - J. Clin. Microbiol. 28: 2482-2484. 15. Rogers G. and Edlinger E. (1986): Immunoenzymatic test for Q fever - Diagn. Microbiol. Infect. Dis. 4: 125-132. 16. Sawyer L.A., Fishbein D.B. and McDade J.E. (1987): Q fever: current concepts - Rev. Infect. Dis. 9: 935-946. 17. Schmeer N., Miiller H.-P., Baumgiirtner W., Wieda J. and Krauss H. (1988): Enzyme-linked immunosorbent fluorescence assay and high-pressure liquid chromatography for analysis of humoral immune responses to Coxiella burnetii proteins - J. Clin. Microbiol. 26: 2520-2525. 18. Schneider W. (1989): Titration of Coxiella burnetii in Buffalo Green Monkey (BGM) cell cultures - Zbl. Bakt. 271: 77-84.
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19. Scott G.H., Williams Z C. and Stephenson E.H. (1987): Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii - J. Gen. Microbiol. 133: 691-700. 20. Thiele D., Willems H. and Glas M. (1991): Immunoblot analysis of IgG subclass antibody response against Coxiella burnetii in Balb/c mice. In: Rickettsiae and rickettsial diseases. Kaz/tr J and Raoult D., eds. - Slovak Acad. Sci., Bratislava 325333. 21. Werth D., Schmeer N., Miiller H.-P., Karo M. and Krauss 11. (1987): Nachweis von Antik6rpern gegen Chlamydia psittaci und Coxiella burnetii bei Hunden und Katzen: Vergleich zwischen Enzymimmuntest, Immunperoxidase-Technik, Komplementbindungsreaktion und Agargelpr/izipitationstest - J. Vet. Med. B 34: 165-176. 22. WHO (1986): Report of WHO Workshop on Q Fever, Giessen - WHO/VPH/CDS/86.68.
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23. Williams J.C., Thomas A. and Peacock M.G. (1986): Humoral immune response to Q fever: enzymelinked immunosorbent assay antibody responses to Coxiella burnetii in experimentally infected guinea pigs - J. Clin. Microbiol. 24: 935-939. 24. Yeaman M.R. and Baca O.G. (1990): Antibiotic susceptibility of Coxiella burnetii, p. 213-223. In: T. Marrie (ed.) Q fever, Vol. I: The disease. CRC Press, Boca Raton, Fla. 25. Yeaman M.R. and Baca O.G. (1991): Mechanisms that may account for differential antibiotic susceptibilities among Coxiella burnetii isolates Antimicrob. Agents Chemother. 35: 948-954. 26. Yeaman M.R, Mitscher L.A. and Bacca O.G. (1987): In vitro susceptibility of Coxiella burnetii to antibiotics, including several quinolones - Antimicrob. Agents Chemother. 31: 1079-1084.
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Vol. 8, No. 4
OF EPIDEMIOLOGY
MONOCLONAL ANTIBODY BASED CAPTURE ELISA/ELIFA FOR DETECTION OF COXIELLA BURNETII IN CLINICAL SPECIMENS D. THIELE 1, M. KARO and H. KRAUSS lnstitut f d r Hygiene u n d Infektionskrankheiten der Tiere - Justus Liebig Universitiit Frankurter Str. 89 - D-6300 - Giessen - F.R.G.
Key words: Q fever - Coxiella burnetii - CAPTURE ELISA - Monoclonal antibody A CAPTURE ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody is described. The assay is fast, highly specific and detects a minimum dose of 2500 Coxiella (C.) burnetii particles. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories could use this assay system for investigating clinical samples of different origin for C. burnetii within a short period of time.
INTRODUCTION
Coxiella burnetii is the causative organism of Q fever in a variety of animal species and man and is recognized as a worldwide problem (2, 22). Ticks are the primary reservoir. Infected ticks transmit Q fever to cattle, sheep, goats and companion animals such as horses, dogs and cats. Within domestic animals transmission mainly occurs by infectious aerosol. Dairy cows are also a major reservoir of this zoonotic agent, shedding the organisms during parturition with placenta and birth fluids in high doses. Chronically infected cows may shed the organisms periodically with their urine and feces, and especially with milk during successive lactating periods (16). Due to its high resistance against physical as well as chemical agents C. burnetii poses a health hazard for a considerable period of time once the pathogen is disseminated in the environment. Veterinarians, abattoir workers and laboratory workers professionally exposed to the pathogen (mainly by aerosol) may be especially exposed to infection. It has been estimated that infection by one single organism can cause disease (8, 12). Ingestion of unpasteurized milk 1 Corresponding author.
containing the agent usually leads to inapparent infection. The diagnosis of Q fever is mainly based on indirect serological procedures such as complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA) (3, 15, 17, 21, 23) or immunofluorescence (IF) (6, 13). Immunoblotting as a diagnostic tool has been introduced to C. burnetii serology only recently (4, 9, 10, 20). In samples containing high numbers of the pathogen direct detection can be achieved after chemical staining, e.g., with the Gimrnez procedure. For isolation of C. burnetii in samples containing only low numbers of organisms, test animals such as guinea pigs or mice (8, 19) or more sophisticated cell culture techniques (1, 14) can be used. Both isolation techniques (animal inoculation or cell culture) are not completely satisfactory. Due to varying susceptibility of C. burnetii strains to antibiotics (24-26) they should be preferably omitted in cell culture isolation techniques. This makes it difficult to isolate the agent from contaminated samples, although infectivity of the pathogen is stable for a prolonged period of time. Using animals for isolation purposes should be avoided whenever possible despite the fact that guinea pigs or even mice are well suited for this purpose due
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to their high susceptibility. One particle of the organism may be sufficient to induce detectable antibody levels, but diagnosis by animal inoculation is time consuming (up to 3-4 weeks). In this presentation we describe a sensitive ELISA for detection of an epitope on C. burnetii lipopolysaccharide antigen, which may be used also as an enzyme-linked immunofluorescent assay (ELIFA).
MATERIALS AND METHODS
Organisms. Coxiellae used as stocks were harvested from persistently infected Buffalo-Green-Monkey (BGM) cell cultures (1) or as infected yolk sacs of embryonated hens' eggs. The following isolates were available from the collection of our institute: "Nine mile" phase (ph) II, "Frankfurt" ph I/II, "Henzerling" ph II; "Herzberg", "Mfinchen", "Balaceanu", "Boren" "Brasov", "Pop", "Stanica", and "Utvinis", all ph I. Additional isolates were kindly supplied by J. Kaz~ir, Institute of Virology, Bratislava ('Nine Mile" ph I,) and H. Becht, Institut Rir Virologie, Universit~it Giessen ("Andelfingen", "Geier", and "Soyta', all ph I). For testing possible cross-reactivity and specificity the following bacteria were used: Aehromobacter haemotysans, Acinetobacter ealcoaeeticus var. anitratus, Bordetella bronehiseptica, Chlamydia (Chl.) psittaci strain 1904, Corynebaeterium pyogenes, Escheriehia eoli, Listeria monocytogenes I and IVb, Pasteurella multocida, Salmonella typhimurium group B, Staphylococcus (S.) aureus, S. aureus Cowan I, S. aureus Wood 46, S. hyicus, Streptococcus (St.) faecalis, St. zooepidemicus, and Yersinia enterocolitica. All these bacteria were available from the collection of our institute. Additionally, Bartonella bacilliformis was obtained from J. Knobloch, Bernhard-Nocht-Institut, Hamburg, F.R.G., Legionella pneumophila from H.G. Schiefer, Inst. Rir Medizinische Mikrobiologie, Giessen, F.R.G., and Chl. trachomatis type L2 from T. Forsey, Institute of Ophthalmology, London. Partial purification of C. burnetii from cell culture. Supernatants from persistently infected BGM cell cultures containing C. burnetii were sonicated (Branson Sonifier B-12, 50 W, 10 sec; Branson Sonic Power Comp., Conn., USA). Samples were centrifuged twice at low speed (300 x g, 10 rain) to pellet cell debris. The supernatants were recentrifuged at 20,000 x g, for 30 rain. The resulting pellet containing C. burnetii was washed twice with double distilled water, diaiysed extensively against double distilled water and lyophilized. Finally, smears from resuspended material were stained with Gim6nez stain and examined for contaminating BGM material. Immunization. Female Balb/c mice 6 to 10 weeks old were infected intraperitoneally with 3.85 x 10' inclusion forming units (18) of strain Nine Mile ph I,
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harvested from infected yolk sacs of embryonated hens' eggs and resuspended in phosphate buffered saline (PBS). Mice were reinfected at day 30 and fusion was performed 4 days later. Fusion and cloning. Spleen cell suspensions containing stimulated B lymphocytes from infected Balb/c mice were fused with cells of the mouse myeloma cell line X63-Ag8.653 (7) by using 500/0 polyethylene glycol (PEG Hybri-Maxr 1300-1600, Sigma, Deisenhofen, F.R.G.). Primary cultivation of hybrids was performed using 96-well cell culture plates in the microtiter format with Rosewell Park Memorial Institute (RPMI) 1640 medium containing hypoxanthine, aminopterin and thymidine (HAT selection) supplemented with 10% fetal calf serum (FCS) in a tissue culture incubator at 370 C and 7.5% CO2. FCS was purchased as mycoplasma free (Roth, Karlsruhe, F.R.G.; Gibco, Eggenstein, F.R.G.; Seromed, Berlin, F.R.G.) and was extensively tested for the support of cells growing under the conditions of limiting dilution. Hybrids were tested for monoclonal antibody production using an ELISA system. To assure monoclonality, hybrids of interest were cloned 3 times using the limiting dilution technique with thymocytes as feeder layer (11). Results from limiting dilution were analysed using the Poisson statistical analysis for repetitive subcloning (5). ELISA for detection and characterization of antigen specific monoclonal antibodies. Antibody secreting hybridoma cells and antibody production were monitored by indirect ELISA. Briefly, wells on microtiter Immulon M129A plates (Greiner, Niirtingen, F.R.G.) were coated with Coxiella antigen Nine Mile ph I or ph II harvested and purified from persistently infected BGM cell cultures. Using Coxiella antigen propagated in cell cultures for this purpose instead of antigen propagated in yolk sacs, which was used for immunization, was advantageous for not codetecting hybrids producing antibodies against contaminating egg proteins. 100 lal antigen suspension in coating buffer (1.059 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3 dissolved in 1 1 double distilled water, adjusted to pH 9.6) per well was incubated at 37oC until the wells were dry. Remaining binding sites were blocked with 200 Ill PBS/well containing 2% bovine serum albumin (BSA), and a minimum of 50 lal hybridoma cell culture supernatant was added to the wells. For detecting bound antibody 100 lal of horseradish peroxidase labelled goat anti-mouse immunoglobulin conjugate was added (Dianova, Hamburg, F.R.G.). Bound conjugate was visualised using 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS; Serva, Heidelberg, F.R.G.) as chromogenic substrate for peroxidase (2.1 g citric acid in 100 ml double distilled water, adjusted to pH 4.2 with NaOH; add 0.011 g ABTS, store at -30°C for no longer than 6 months. Adjust temperature to 37° C before use and add 20 lal H202 [30°/0]per 100 ml). As a negative control
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wells were incubated with RPMI 1640 medium or "conditioned medium" (supernatant from propagated myeloma cells). Serum harvested from mice used for immunization and as spleen cell donors served as positive control. The same ELISA format was used to determine class, subclass and light chain type of monoclonal antibodies using the "Mouse Typer Kit" and "Mouse Typer Panel" (BioRad, Munich, F.R.G.) containing specific rabbit antisera to mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, kappa-chain and lambda chain, and goat anti-rabbit IgG (H+L) peroxidase-labelled conjugate. Hybridoma maintenance and production of antibodies. After establishing stable antibody-producing hybridomas in RPMI 1640 medium containing hypoxanthine and thymidine (omitting aminoptefin at this state) and 10% fetal calf serum, the cells were tested for the production of monoclonal antibodies under serum free RPMI medium supplemented with 2% Ultroser H Y (Gibco, Eggenstein, F.R.G.). Using this supplement culture supernatants were free of foreign immunoglobulins such as bovine immunoglobulin. Characterization of monoclonal antibodies using different test systems. To further characterize established monoclonal antibodies binding characteristics were also determined in direct and indirect immunofluorescence, dot test, indirect immunoperoxidase and immunoblot techniques. Purification of monoelonal antibodies. The monoclonal antibodies were purified on a protein A-Sepharose CL-4B column (Pharmacia, Freiburg, F.R.G.), dialysed against double distilled water and lyophilysed. Biotinylation of monoclonal antibodies. 6 mg of purified and lyophilysed monoclonal antibody were dissolved in 500 111 carbonate buffer (0.1 M NaHCO3, 0.1 M Na2CO3; pH 8.5). 50 111 of dimethylsulphoxide (DMSO) containing 1.0 mg of D-Biotinyl-eaminocaproic acid-N-hydroxysuccinimide ester (Biotin-X-NHS) were added and incubated for 12 h while shaking at room temperature (RT). The reaction mix was extensively dialysed against 1 1 of carbonate buffer for 16 h at 4° C, with 3 changes of the buffer. The dialysate was then centrifuged (8000 g, 10 min, RT), the supernatant supplemented with 2% bovine serum albumine for cryoprotection and shock-frozen in liquid nitrogen before being stored at -20 ° C. Antigen capture test. Several commercially available EL1SA microtiter plates were tested and compared. Best results could be achieved with ELISA-F plates Immulon M129A made from polystyrol with medium binding capacity for proteins (Greiner, NiJrtingen, F.R.G.). Plates were freshly coated with capture antibody, 100 lal/well (lmg/ml, diluted 1:5000 in TRIS buffered saline [TBS]: 10 mM TRIS, 100 mM NaC1,
pH 8.5), and incubated for 16 h at 4° C. Remaining binding sites were blocked with TBS containing 10 mM TRIS, 150 mM NaC1, 0.5 % Tween 20, pH 8.0, supplemented with 1% fish gelatine (Sigma, Deisenhofen, F.R.G.), for 15 rain at RT. Plates were washed three times with 250 lal washing buffer (10 mM TRIS, 150 mM NaC1, 0.5% Tween 20, pH 8.0); each washing step was 5 min and the same buffer wash always used. Samples were added in a volume of 100 lal and incubated for 2 h at RT. After three more washings the monoclonal biotinylated detection antibody wash added (100 lal, 30 rain, 1:10,000 in wash buffer). Optimal concentration for each immunoglobulin compound was initially determined with a chequerboard ELISA titration. After three more washings, streptavidin conjugated with alkaline phosphatase or peroxidase (Boehringer, Mannheim, F.R.G.) was added (100 lal, 30 min, 1:10.000 in wash buffer). Substrate was added after three final washings. For streptavidin conjugated with peroxidase 3,3'-5,5'-tetramethylbenzidine (TMB, Fluka, NeuUlna, F.R.G.) was used as substrate (stock solution I: 1 M sodium acetate, adjusted to pH 5.8 with 0.1 M citric acid, stored at 4° C; stock solution II: 10mg/ml TMB in DMSO (Sigma, Deisenhofen, F.R.G.), stored at 4o C.; working solution: 2 ml of stock solution I were mixed with 0.2 ml stock solution II and 20 ml double distilled water. 20 lal H202 (6%) were added and the solution adjusted to 370 C). The enzyme reaction proceeded for 30 rain and was stopped with 3 M H2SO4 (free of Fe ions). The optical density was measured at 450 nm in a Titertek Multiscan ELISA reader (Flow, Meckenheim, F.R.G.). For streptavidin conjugated with alkaline phosphatase 4-methylumbelliferylphosphate (4-MUP; Sigma, Deisenhofen, F.R.G.) was used as substrate (2 mg of 4-MUP were dissolved in 10 ml diethanolamine buffer, always freshly prepared and adjusted to 37° C. Diethanolamine buffer: 97.0 ml diethanolamine (Merck, Darmstadt, F.R.G.), 0.2 g NAN3,0.01 g MgCI2 x 6 H20, 800.0 ml double distilled water; adjusted to pH 9.8 with HC1, double distilled water added to 1 1, stored at 4° C and protected from light). Fluorescence was measured after 60 min using a Titertek Fluoroscan ELISA reader (Flow, Meckenheim, F.R.G.) with excitation wavelength of 355 nm and emission wavelength of 460 nm. Samples were then tested in duplicates. A standard titration of C. burnetii Nine Mile ph I (ling/ ml; 1:5000 - 1:1,280,000) was always performed in parallel. Samples containing buffer instead of antigen served as negative controls. Preparation of antigen and clinical samples. For analytical purposes a stock suspension of C burnetii Nine Mile ph I (1 mg/ml) was used. Clinical samples were obtained from 33 cases of bovine and sheep abortions and from a cow after gestation. Also ten samples of wash water from a factory processing lamb wool were investigated for Coxiella antigen.
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Class, subclass and light chain. All hybridomas tested
Analytical and clinical samples were treated with proteinase K prior to testing. Proteinase K (Sigma, Deisenhofen, F.R.G.), non self-digested, was dissolved in 10 mM TRIS with 1 mM CaC12 (addition of Ca2+ to proteinase K was carried out to degrade proteins that are commonly resistant to proteinase K) at a concentration of 400 pl/ml. To analytical and clinical samples the same volume of proteinase K was added (to give a final concentration of 200 pl/ml) and incubated for 30 rain at 56o C. For inactivation of the enzyme the reaction mixture was heated to 100° C for 15 rain.
produced antibodies of the IgG3 subclass with kappa light chains. Antibody production and purification. Three clones (2/ 15, 4/6 and 4/11) could be adapted to produce monoclonal antibodies in serum-free RPMI 1600 medium supplemented with Ultroser HY. Cell culture supernatants contained antibodies in the range from 1 to 10 pl/ml. All these antibodies could be purified on protein A-Sepharose CL-4B without considerable loss of activity. Serum-free RMPI 1640 medium supplemented with Ultroser H Y as control never yielded any detectable amount of immunoglobulins when applied to the protein A affinity chromatography column.
Isolation of C. burnetii from clinical samples. Clinical samples were sonicated in PBS till homogenization. In cases of severe bacterial contamination this homogenate was filtered through 0.45 pl filters. An equal amount of medium was added and the filtrate transferred to BGM cell cultures. BGM cells were propagated on round glass slides (12 mm diameter) in fiat bottom plastic vials (6 ml). The cell culture tubes were then centrifuged for 60 rain at 3000 g and incubated at 37° C. The presence of multiplying C. burnetii was determined by visual inspection of the cultures with an inverted microscope for typical vesicle formation, subsequent staining of coverslip cultures after Gim6nez and microscopic investigation of cells.
Statistical analysis. ELIFA results with analytical samples containing 1 mg/ml C. burnetii Nine Mile ph I antigen were used to determine several statistical parameters of the test system. The following parameters were determined: detectability (cut off) and sensitivity. Detectability of the assay was defined as the lowest concentration of antigen exceeding the zerodose precision and was estimated using the following equation: Detectabi#ty = (Absorbance zero dose + tn - 1; co; one sided • SD)
dn Sensitivity was defined as the responsiveness of the assay to changes in concentration of the substance tested. Therefore, on a sigmoid dose-response curve, sensitivity decreases at either end of the curve. Sensitivity was estimated using the following equation: Sensitivity = dR/dC. R = ELIFA units C = reciprocal dilution (log10) ~S~TS
Cloning. Five hybridoma cell lines secreting antibodies specific for C. burnetii could be established from one fusion experiment.
Binding characterization using different test systems. Preliminary binding studies using direct and indirect immunofluorescence, dot test, and the indirect immunoperoxidase test demonstrated that not all monoclonal antibodies recognized each C. burnetii isolate tested in the various test systems showing sometimes distinct recognition patterns in different systems. However, two monoclonal antibodies (4/6, 4/ 11) recognized all isolates in every test system. Immunoblotting of these two antibodies revealed reactivity with C. burnetii lipopolysaccharide. The monoclonal antibody 4/11 was used to set up the CAPTURE ELISA/ELIFA system. CAPTURE ELISA/ELIFA. Using this test system based on monoclonal antibody 4/11 as capture and detection antibody - all C. burnetii isolates were clearly recognized. All bacteria tested (20 different strains as negative controls) did not react in this assay. Using a stock solution of C. burnetii Nine Mile ph I (1 mg/ml) the assay still gave a positive signal (cutoff) when applying a dose of 100 pl of a 1:160,000 stock solution (Figs. 1, 2). This amount contained 625 pg of C. burnetii antigen, or approximately 2500 particles. The detection limit was the same when using streptavidin conjugated with peroxidase (ELISA) or strepavidin conjugated with alkaline phosphatase (ELIFA). Sensitivity defined as the responsiveness of the assay to changes in concentration of the substance tested demonstrated a maximum at a dose of 87 ng. Stock solutions of the monoclonal and biotinylated monoclonal antibody could be stored up to six months without any detectable loss of activity during that period. We used this assay system for routine diagnostic purposes in cases of abortion in sheep and cattle. Results obtained with 34 specimens (samples of placental or fetal tissues, vaginal secretion of cattle and sheep, and in one case from vaginal secretion of a cow after gestation are demonstrated in Table 1. Ten samples were positive in the CAPTURE ELISA, 8 of which could be confirmed by cell culture isolation.
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5000q
CaptureELIFA
From ten specimens of wash water from a factory processing lamb wool, eight were positive in the CAPTURE ELISA but negative using cell culture techniques. Apparently this was due to non-viable Coxiella.
~°°°i~ 3000i
~7 n
2000! !000~ 10ug
g DISCUSSION
/ / 1ug
100ng lO~ng
I ng " i00 ~g
C. burnetiiNineMileph i (totalamount)
Figure 1. - Antigen titration using the CAPTURE ELIFA, doses ranging from 10 lag - 100 pg. Data presented with standard deviation and adapted by 4-parameter logistic function. Sensitivity profile deduced from data (dR/dC) demostrating maximum sensitivity at a dose of 87 ng.
2",h
--3 ,
~ogio (reciprocaldilution) Figure 2. - Log-logit transformation of data from figure 1, including regression line and confidence interval (99%).
TABLE 1 . - Positive results achieved with antigen detection in the CAPTURE ELISA in comparison to isolation of Coxiella burnetti in BGM cell cultures. Species
n
CAPTURE ELISA
Cell culture
Cattle
19
3
3
Sheep
15
7
5
Total
34
10
8
For some time C. burnetii has been detected by time consuming isolation procedures such as the guinea pig inoculation test or propagation in embryonated eggs. In recent years, a cell culture technique of applying BGM cells and medium without antibiotics (1) has also been used in our laboratory for isolation of C. burnetii, and a similar technique with HEL cells has been described by Raoult et al. (14) for specimens collected from humans. However, contamination of the (antibioticfree) cell cultures with bacteria may occur and destroy the cells in cases of severe bacterial contamination of the sample, as is often the case with specimens from animals. The CAPTURE ELISA/ELIFA described does not have such disadvantages. Samples contaminated with bacteria can be tested without any problems and results are obtained within one working day. Even samples containing inactivated (formalin, heat, SDS/proteinase-K) C. burnetii organisms can be analysed. The aim was to select a monoclonal antibody with broad reactivity to C. burnetii, i.e., recognition of both phases and all isolates, and with no cross-reactivity to other organisms. In addition, the secreting hybridoma cell line should produce the antibody in sufficient quantities and should be able to do so in serum-free media. Other important characteristics addressed were immunoglobulin class, affinity, ease of purification and conjugation, and stability. Finally the antibody should recognize an epitope which is found repeatedly on C. burnetii for use of the antibody as capture as well as detection antibody in the antigen capture test, thus avoiding competitive effects. The monoclonal antibody applied demonstrated a broad reactivity including all C. burnetii isolates tested (ph I as well as ph II) with no cross-reactivity to LPS of a battery of gram-negative bacteria and antigenic determinants of various other organisms. Polyclonal antisera often exhibit higher sensitivity and avidity than monoclonal antibodies, whereas monoclonal antibodies generally show higher specificity. However, sensitivity of the assay presented could not be increased by use of affinity purified rabbit antiserum against C. burnetii ph I antigen as capture antibody. When applying such a serum in the CAPTURE ELISA sensitivity was actually diminished. Furthermore, standardized polyclonal antiserum usually is available in limited amounts only whereas monoclonal antibodies can be produced almost unlimitedly of uniform quality. This fact also favours the use of the monoclonal antibody-based CAPTURE ELISA/ELIFA.
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Differences between CAPTURE ELISA and ELIFA format were only minimal with a tendency of the CAPTURE ELIFA format to detect less C. burnetii particles. As ELIFA generally has been demonstrated to be superior when compared with conventional ELISA we think that possibly the CAPTURE ELIFA can be optimized further. One reason for the excellent results with the ELISA might have been the high sensitivity of the TMB substrate used in the CAPTURE ELISA format. Applying other substrates in the CAPTURE ELISA, e.g., ABTS, the ELIFA was clearly superior. Clinical samples being analysed as positive in the CAPTURE ELISA/ELIFA but negative by isolation in cell cultures may be due to non-infectious particles. This may be assumed especially for the wool washing samples which had been heated during the washing process to a minimum of 60° C. Preincubation of these samples with the non-biotinylated monoclonal antibody or rabbit antiserum specific for C. burnetii always demonstrated more than 80% neutralizing activity. Although all bacteria tested did not show any cross reactivity it can not be unambiguously ruled out that other organisms not tested may eventually cross-react. The test system described appears to be a sensitive and specific screening system for the detection o f C. burnetii in clinical specimens. It is fast, easy to perform and requires no specialized cell culture equipment or animal testing. Acknowledgements
Excellent technical assistance provided by Mrs. Rosi Frank and advice in statistical analysis by H. Willerns is gratefully acknowledged. REFERENCES
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