Vol. 30, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1457-1461
0095-1137/92/061457-05$02.00/0 Copyright
)
1992, American Society for Microbiology
Detection of Borreliacidal Antibodies in Hamsters by Using Flow Cytometry K. F. SACHSENMEIER,"12 K. SCHELL,3 L. W. MORRISSEY,3 D. R. PENNELL,, R. M. WEST,4 STEVEN M. CALLISTER,4 AND R. F. SCHELL' 2* Wisconsin State Laboratory of Hygiene, 1 Department of Medical Microbiology and Immunology, 2 and University of Wisconsin Comprehensive Cancer Center Flow Cytometry Unit,3 University of Wisconsin, Madison, Wisconsin 53706, and the Gundersen Medical Foundation, La Crosse, Wisconsin 546014
Received 26 November 1991/Accepted 5 March 1992
Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease. Lyme disease, a complex, multisystem disorder caused by Borrelia burgdorferi, is transmitted to humans by ticks of the Lodes ricinus complex (3, 23). Lyme disease is now the most common tickborne illness in the United States (5). Its most characteristic clinical feature is an expanding skin lesion called erythema migrans, yet patients with the disease may present with arthritic, cardiac, or neurological symptoms without a skin rash (22). Clinical pictures vary and are, at times, not distinctive, especially in the absence of erythema migrans. Determination of antibody levels to B. burgdorferi is the most frequently used laboratory method for the diagnosis of Lyme disease. Established procedures include the indirect fluorescent-antibody test (18, 25), formats of the enzymelinked immunosorbent assay (7, 18) including the antibody capture assay (2), and the quantitative fluorescence immunoassay (11, 17). These procedures, however, have demonstrated a lack of sensitivity, especially in the early stages of disease, and cross-reactivity with other spirochetal or autoimmune diseases (7, 11, 17, 18, 25). Attempts to improve these tests have focused on the use of antigenic fractions, such as the purified 41-kDa flagellar protein (6, 8-10); incorporation of serum-absorbing agents (12, 16, 25); and separate detection and quantitation of immunoglobulin M (IgM) and IgG antibodies (2, 15). Multiple laboratories have attempted to improve the serodiagnosis of Lyme disease by use of other methods (2, 6, 15). Recently, we developed an in vitro borreliacidal assay that accurately reflects the levels of protective antibody as determined by passive transfer of immunity (14, 19, 20). When immune serum was incubated with B. burgdorferi, spirochetes were killed within hours. Peak borreliacidal activity was detected in sera obtained from hamsters infected for 3 weeks with the Lyme disease spirochete. Similar studies demonstrated that sera from patients with Lyme disease, especially those with arthritis, killed B. burgdorferi organisms (4). However, the detection of borreliacidal activity of immune serum required 3 to 5 days of incubation after the interaction of immune serum with B. burgdorferi organisms. In the present report, we present evidence that flow *
cytometry can be used to determine borreliacidal activity. This assay can be performed within 3 h of mixing immune serum with Lyme disease spirochetes.
MATERIALS AND METHODS Medium. Barbour-Stoenner-Kelly medium (BSK) was prepared by adding 3 g of N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid buffer (Sigma Chemical Co., St. Louis, Mo.), 2.5 g of neopeptone (Difco Laboratories, Detroit, Mich.), 0.35 g of sodium citrate, 2.5 g of glucose, 1.1 g of sodium bicarbonate, 1.27 g of TC yeastolate (Difco), 0.4 g of sodium pyruvate (Sigma), and 0.2 g of N-acetyl-D-glucosamine (Sigma) to a 1-liter flask which contained 450 ml of distilled water. A 25-g amount of bovine serum albumin (fraction V; Sigma) was then added. The suspension was slowly stirred (speed set at 1) for 5 h at room temperature on a hot plate (model PC 351; Corning Glass Works, Corning, N.Y.). After stirring, the medium was adjusted to pH 7.5 with 1 N NaOH and was sterilized by filtration through a 0.22-p,m-pore-size filter (Millipore Corp., Bedford, Mass.). After sterilization, 50 ml of sterile CMRL 1066 liquid medium (10x) without glutamine (GIBCO Laboratories, Grand Island, N.Y.) was added. The medium was divided into aliquots (10 ml) and supplemented with 6% sterile rabbit serum (Pelfreeze Biologicals, Inc., Rogers, Ariz), which was previously shown to support growth of B. burgdorferi. BSK was filtered (0.22-,um-pore-size filter; Millipore) before use in the borreliacidal assay. Organisms. B. burgdorferi isolates 297, G25, and PBi were obtained from Russell C. Johnson (University of Minnesota, Minneapolis). Isolates 297 and PBi were isolated from human spinal fluid (23, 24), and strain G25 was isolated from L ricinus. The isolates were maintained by passage in hamsters and then grown in BSK at 32°C for 5 days. After examination by dark-field microscopy, the suspensions of B. burgdorferi were adjusted with fresh BSK so that they contained approximately 107 organisms per ml. One-milliliter aliquots were then dispensed in vials, which were sealed and stored in liquid nitrogen until use. Preparation of hamster serum. A group of five or more immunocompetent LSH hamsters were injected subcutaneously in each hind paw with 0.2 ml of BSK containing 106
Corresponding author. 1457
1458
SACHSENMEIER ET AL.
J. CLIN. MICROBIOL.
IV
B
A 10
3.
10
3 ,,,
,
~
JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1457-1461
0095-1137/92/061457-05$02.00/0 Copyright
)
1992, American Society for Microbiology
Detection of Borreliacidal Antibodies in Hamsters by Using Flow Cytometry K. F. SACHSENMEIER,"12 K. SCHELL,3 L. W. MORRISSEY,3 D. R. PENNELL,, R. M. WEST,4 STEVEN M. CALLISTER,4 AND R. F. SCHELL' 2* Wisconsin State Laboratory of Hygiene, 1 Department of Medical Microbiology and Immunology, 2 and University of Wisconsin Comprehensive Cancer Center Flow Cytometry Unit,3 University of Wisconsin, Madison, Wisconsin 53706, and the Gundersen Medical Foundation, La Crosse, Wisconsin 546014
Received 26 November 1991/Accepted 5 March 1992
Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease. Lyme disease, a complex, multisystem disorder caused by Borrelia burgdorferi, is transmitted to humans by ticks of the Lodes ricinus complex (3, 23). Lyme disease is now the most common tickborne illness in the United States (5). Its most characteristic clinical feature is an expanding skin lesion called erythema migrans, yet patients with the disease may present with arthritic, cardiac, or neurological symptoms without a skin rash (22). Clinical pictures vary and are, at times, not distinctive, especially in the absence of erythema migrans. Determination of antibody levels to B. burgdorferi is the most frequently used laboratory method for the diagnosis of Lyme disease. Established procedures include the indirect fluorescent-antibody test (18, 25), formats of the enzymelinked immunosorbent assay (7, 18) including the antibody capture assay (2), and the quantitative fluorescence immunoassay (11, 17). These procedures, however, have demonstrated a lack of sensitivity, especially in the early stages of disease, and cross-reactivity with other spirochetal or autoimmune diseases (7, 11, 17, 18, 25). Attempts to improve these tests have focused on the use of antigenic fractions, such as the purified 41-kDa flagellar protein (6, 8-10); incorporation of serum-absorbing agents (12, 16, 25); and separate detection and quantitation of immunoglobulin M (IgM) and IgG antibodies (2, 15). Multiple laboratories have attempted to improve the serodiagnosis of Lyme disease by use of other methods (2, 6, 15). Recently, we developed an in vitro borreliacidal assay that accurately reflects the levels of protective antibody as determined by passive transfer of immunity (14, 19, 20). When immune serum was incubated with B. burgdorferi, spirochetes were killed within hours. Peak borreliacidal activity was detected in sera obtained from hamsters infected for 3 weeks with the Lyme disease spirochete. Similar studies demonstrated that sera from patients with Lyme disease, especially those with arthritis, killed B. burgdorferi organisms (4). However, the detection of borreliacidal activity of immune serum required 3 to 5 days of incubation after the interaction of immune serum with B. burgdorferi organisms. In the present report, we present evidence that flow *
cytometry can be used to determine borreliacidal activity. This assay can be performed within 3 h of mixing immune serum with Lyme disease spirochetes.
MATERIALS AND METHODS Medium. Barbour-Stoenner-Kelly medium (BSK) was prepared by adding 3 g of N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid buffer (Sigma Chemical Co., St. Louis, Mo.), 2.5 g of neopeptone (Difco Laboratories, Detroit, Mich.), 0.35 g of sodium citrate, 2.5 g of glucose, 1.1 g of sodium bicarbonate, 1.27 g of TC yeastolate (Difco), 0.4 g of sodium pyruvate (Sigma), and 0.2 g of N-acetyl-D-glucosamine (Sigma) to a 1-liter flask which contained 450 ml of distilled water. A 25-g amount of bovine serum albumin (fraction V; Sigma) was then added. The suspension was slowly stirred (speed set at 1) for 5 h at room temperature on a hot plate (model PC 351; Corning Glass Works, Corning, N.Y.). After stirring, the medium was adjusted to pH 7.5 with 1 N NaOH and was sterilized by filtration through a 0.22-p,m-pore-size filter (Millipore Corp., Bedford, Mass.). After sterilization, 50 ml of sterile CMRL 1066 liquid medium (10x) without glutamine (GIBCO Laboratories, Grand Island, N.Y.) was added. The medium was divided into aliquots (10 ml) and supplemented with 6% sterile rabbit serum (Pelfreeze Biologicals, Inc., Rogers, Ariz), which was previously shown to support growth of B. burgdorferi. BSK was filtered (0.22-,um-pore-size filter; Millipore) before use in the borreliacidal assay. Organisms. B. burgdorferi isolates 297, G25, and PBi were obtained from Russell C. Johnson (University of Minnesota, Minneapolis). Isolates 297 and PBi were isolated from human spinal fluid (23, 24), and strain G25 was isolated from L ricinus. The isolates were maintained by passage in hamsters and then grown in BSK at 32°C for 5 days. After examination by dark-field microscopy, the suspensions of B. burgdorferi were adjusted with fresh BSK so that they contained approximately 107 organisms per ml. One-milliliter aliquots were then dispensed in vials, which were sealed and stored in liquid nitrogen until use. Preparation of hamster serum. A group of five or more immunocompetent LSH hamsters were injected subcutaneously in each hind paw with 0.2 ml of BSK containing 106
Corresponding author. 1457
1458
SACHSENMEIER ET AL.
J. CLIN. MICROBIOL.
IV
B
A 10
3.
10
3 ,,,
,
~
