The ability of counter immunoelectrophoresis (CIE) to detectBacteroides fragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection ofB. fragilis endotoxin in amniotic fluid in a concentration of 40 ng/ml or more. The sensitivity threshold was reduced to 2 ng/ml by using a highly reactive IgG-fraction isolated from rabbit anti-B. fragilis IPL E 323 antiserum

Beckmann, I. (Ilse)

Graaff, K. (K.) de

Meisel-Mikolajczyk, F.

Wallenburg, H.C.S. (Henk)

Erasmus University Digital Repository

Antonie van Leeuwenhoek 66: 333-336, 1994. 333 © 1994 Kluwer Academic Publishers. Printed in the Netherlands. Detection of Bacteroidesfragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis I. Beckmann 1, K. de Graaf f  1, F. Me ise l -M ikota jczyk  2 & H.C.S. Wal lenburg 11 Department of Obstetrics and Gynecology, Erasmus University Medical School, Rotterdam, The Netherlands 2 Department of Bacteriology and Immunology, Medical Academy, Warsaw, Poland Accepted 2 May 1994 Key words." B. fragilis, endotoxin, counterimmunoelectrophoresis, ultrafiltration, amniotic fluid Abstract The ability of counter immunoelectrophoresis (CIE) to detect Bacteroidesfragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection of B. fragiIis endotoxin i  amniotic fluid in a concentration f 40 ng/ml or more. The sensitivity threshold was reduced to 2 ng/ml by using a highly reactive IgG-fraction isolated from rabbit anti-B, fragilis IPL E 323 antiserum. Introduction Counterimmunoelectrophoresis (CIE) has been suc- cessfully employed for the detection of polysaccha- ride antigens isolated from various species of the genus Streptococcus, Pneumococcus, Haemophilus and Neisseria in serum (Shackelford et al. 1974; Ingrain et al. 1972; Granoff et al. 1977; Shetty et al. 1985), liquor cerebrospinalis (Shackelford et al. 1974; Ingram et al. 1972; Granoff et al. 1977), urine (Shack- elford et al. 1974), sputum (E1-Refaie & Dulake 1975; Ericsson et al. 1986) and tears (Powell et al. 1988). Streptococcus B polysaccharide has also been detect- ed in amniotic fluid (Jacobs et al. 1981). The specific detection of lipopolysaccharides (LPS), the endotoxins of gram negative bacteria, by CIE in amniotic fluid has not yet been described. During our study of the maternal and fetal immuneresponse to B. fragilis endotoxin in pregnant guinea pigs (Beckmann et al. 1991) it became nec- essary to assess in amniotic fluid the presence of B. fragilis endotoxin in low concentrations. This present study describes a modified system of CIE for the specif- ic detection of B. fragilis endotoxin i  small quantities in amniotic fluid. Material and methods B. fragilis endotoxin Bacteria (B. fragilis IPL E 323) were extracted with phenol/water and the lipopolysaccharide isolated as described by Beckmann et al. (1989). Antisera Rabbit antisera gainst B. fragilis IPL E 323 bacte- ria were prepared as described by Meisel-Mikotajczyk et al. (1981). The antisera E28 and E29 were from different rabbits immunized with the same bacterial suspension. Isolation of lgG from B. fragilis antiserum According to the method of McKinney & Parkinson (1987) rabbit anti B. fragilis antiserum was precipitat- ed with n-caprylic acid, cooled to 40 C and fraction- ated with ammonium sulfate (45% saturation). After stirring for 30 min the precipitated IgG was collected by centrifugation at 2000 g for 20 rain. The precipitate was dissolved in a small volume of phosphate buffered saline (PBS) and dialysed at 4 o C over night against PBS. After heating for 20 min at 55 o C the protein 334 content of the solution was determined by absorption spectrometry at 260 nm. IgGl was isolated from antiserum E2(83), IgG2 from antiserum B. fragilis E2(8). The sera were obtained by immunization of rabbits with different preparations of bacterial suspensions. Amniotic fluid Amniotic fluid from healthy pregnant guinea pigs was isolated on day 61 of the gestation, centrifuged at 1500 rpm, and the clear supernatant s ored at - 200 C until assay. Table 1. Minimal detectable concentration ofB. fragilis LPS in CIE depending onantibody source. Antibody source Number oftests Detectable concentrations LPS (/zg/ml) Antiserum E28 15 0.31-0.63 Antiserum E2 9 3 0.31-0.63 IgG-fraction (1) 3 0.02-0.03 IgG-fraction (2) 3 0.75-1.56 Ultrafilters Ultrafilters UFC3 LCC00 (Molecular weight (MW) limit MW < 5000 D) and UFC3 LGCOO (MW limit MW < 10000 D) were from Millipore B.V., Etten-Leur, The Netherlands. fold. The volumes of remaining and filtered fluids were measured and kept at - 20 ° C until further testing. Results Chemicals All chemicals were from Sigma, St. Louis, U.S.A. and Merck, Darmstadt, Germany. Counterimmunoelectrophoresis ( CIE)CIE was performed on microscopic slides covered with 2 ml of agarose gel (1% agarose in Laurell buffer (pH 8.6). After electrophoresis the slides were incu- bated over night in a humid chamber at 4 o C in PBS and stained according to the method of Weeke (Weeke 1973) with Coomassie Brilliant Blue G 225. All results were assessed blinded, by an independent investiga- tor. Grabar-immunoelectrophoresis The one-dimensional immunoelectrophoresis onmicroscopic slides was performed as described by Beckmann et al. (1985). Ultrafiltration of amniotic fluid LPS dissolved in amniotic fluid was kept for 3 min at 100 ° C and 400 #1 of the solution were applied to an ultrafilter and centrifuged for 60 min at 2000 g and 200 C. New solution was added, until the original solution remaining in the filter was concentrated ten- The optimum sensitivity threshold for the detection of B. fragilis LPS with CIE was determined by varying the conditions (quantity of antibody, distance between antigen and antibody reservoirs, field strength and duration of electrophoresis). The final procedure was as follows: On agarose-covered microscopic slides two rows of holes (diameter 3.5 mm) with a distance of 3 mm between rows were applied. B. fragilis LPS, dissolved in PBS (pH 7.4), was diluted 1:2 in amniot- ic fluid, and 6 #1 of each dilution were placed into the holes directed towards the cathode; 8#1 antiserum were put into the holes directed towards the anode. Controls for amniotic fluid and PBS were included. After elec- trophoresis for 45 min at a field strength of 3.2 V/cm and 200 C, the immunoprecipitates were processed as described above. Under these conditions 310-630 ng of B. fragilis LPS could be detected. The detection limit depends on the concentration fantibodies within the antibody source. Table 1 shows the detectable LPS-concentrations after application of different sources of antibodies in the CIE test. The ultrafiltration of amniotic fluid, containing B. fragilis IPL E 323 LPS, was performed with two types of ultrafilters (5000 and 10000 D), permitting the pas- sage of substances with a molecular weight < 5000 D or < 10000 D. The permeability ofboth filters for the LPS of B. fragilis was investigated by Grabar immunoelec- trophoresis and CIE of residues and filtrates after ultra- filtration of 500 #g LPS dissolved in 1 ml amniotic flu- id. Figure 1 shows the results of Grabar immunoelec- \ ,J Fig. 1. Immunoelectrophoresis of B. fragilis lipopolysaccharide (LPS) before and after ultrafiltration. Left: LPS before ultra filtration, right: LPS in the residue after ultrafiltration through a 5000 D filter, middle: B. fragilis antiserum. trophoresis of the residue after ultrafiltration through the 5000 D filter, and of the original LPS-solution. The residue still contains all components ofthe het- erogenous LPS-fraction, and the filtrate did not react with a visible precipitate. CIE with this filtrate was 335 also negative, in contrast to the reaction of the filtrate obtained through the 10000 D filter, in which a small quantity of LPS could be detected. Filtration of an LPS-solution in amniotic fluid through a 5000 D-filter gave quantitative r sults as shown for one example in Table 2. The LPS-concentration in the residue was calculat- ed from the final titer in a CIE-test, compared with the titer of the original, unfiltered solution. The sensitivity threshold f CIE, after tenfold con- centration of the antigen solution by ultrafiltration, was determined as 40 ng LPS/ml amniotic fluid (anti- body source rabbit-anti B. fragi l is antiserum) and 2 ng LPS/ml (antibody source IgG). Discussion Detection of endotoxins in amniotic fluid by CIE has not yet been described. In connection with intrauter- ine infections, endotoxins were mainly detected by the limulus amebocyte lysate assay, which is very sensi- tive, but does not permit identification of the endotoxin. The sensitivity reached by combining ultrafiltration of lipopolysaccharides with CIE is of the same order of magnitude as that described for the detection of cap- sular polysaccharides byCIE in other body fluids, but lower than the sensitivity of the limulus assay. For the ultrafiltration f a lipopolysaccharide the molecular weight of the concentrated substance must be known and the pore size of the filter membrane should be carefully adjusted. Although the molecular weight of B. fragiI is LPS of 12000 D (Kasper & Seiler 1975) was above the indicated limit of the 10000 D filter, antigenic omponents were present in the filtrate, which made the use of a filter with a smaller pore size necessary. The CIE results obtained with different batches of anti B. fragi l is antiserum and IgG indicate that the sensitivity depends heavily on the antibody source. With a highly reactive IgG-preparation, the sensitivity was almost enfold increased and, in combination with ultrafiltration, reached adetection limit of 2 ng/ml. The method as described in this report is useful for the specific detection of small quantities of endotoxins in amniotic fluid. 336 Table 2. Concentration fB. fragilis LPS in amniotic fluid by nltrafiltration through a5000D-filter. Applied in Filtrate Residue Theoretic oncentration in residue Detected by CIE ~zg LPS /zl /zl /zl t~g/ml tzg/ml (%) 1.25 1000 920 60 21 20 95 References Beckmann I, Meisel-Mikotajczyk F, Leszczynski P & Wallenburg HCS (1991) Immune response to endotoxin isolated from Bac- teroidesfragilis in the pregnant guinea pig. Int Arch Allergy Appl Immuno142:222-226 Beckmann I, van Eijk HG, Meisel-Mikotajczyk F & Wailenburg HCS (1989) Detection of 2-Keto-3-Deoxyoctonatein endotoxins isolated from six reference strains of the Bacteroides fragilis group. Int J Biochem 21:661-666 Beckmann I, Paelinck J, Zuijderduijn J, Sawicka-Grzelak A & Meisel-Mikolajczyk F (1985) lmmunochemical investigations of antigens isolated from Bacteroides ovatus strain ATCC 8483. Antonie van Leeuwenhoek 51 : 263-273 El-Refaie M & Dulake C (1975) Counter-current immunoelec- trophoresis for the diagnosis of pneumococcal chest infection. J Clin Path 28:801-806 Ericsson CH, Hallander HO, Rosen A, Sj6gren AM & Sj6gren I (1986) Routine use of counterimmunoelectrophoresis for the detection of pneumococcal ntigen in sputum. Med Microbiol Immuno1175: 241-249 Granoff DM, Congeni B, Baker R, Ogra P & Nankervis GA (1977) Countercurrent immunoelectrophoresis in the diagnosis of Haemophilus influenzae type b infection: Relationship of detec- tion of capsular antigen to age, antibody response, and therapy. Am J Dis Child 131:1357-1362 lngram DL, Anderson P & Smith DH (1972) Countercurrent immu- noelectrophoresis in the diagnosis of systemic diseases caused by Haemophilus influenzae type b. J Pediatr 81: 1156-1159 Jacobs RE Yamauchi T & Eisenach KD (1981) Detection of Strep- tococcal antigen by counterimmunoelectrophoresis. Am J Clin Patho175:203-208 Kasper DL & Seiler MW (1975) Immunochemical haracterization of the outer membrane complex of Bacteroidesfragilis subspecies fragilis. J lnf Dis 132:440-450 McKinney MM & Parkinson A (1987) A simple, non- chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Meth 96:271-278 Meisel-Mikolajczyk F & Grzelak-Puczyhska I (1981) Rapid serotyp- ing of bacteria of the Bacteroides fragilis group by direct immunofluorescence serotype specific conjugates. J Appl Bac- terio151:393-398 Powell KR, Kaplan SB, Hall CB, Nasello MA & Roghmann KJ (1988) Periorbital cellulitis: Clinical and laboratory findings in 146 episodes, including tear countercurrent immunoelec- trophoresis in 89 episodes. Am J Dis Child 142:853-857 Shackelford PG, Campbell J & Feigin RD (1974) Countercurrent immunoelectrophoresis in the evaluation ofchildhood infections. J Pediatr 85:405-411 Shetty NR Srinivasa H & Bhat P (1985) Coagglutination a d counter immunoelectrophoresis in the rapid diagnosis of typhoid fever. Am J Clin Pathol 84:80-84 Weeke B (1973) General remarks on principles, equipment, reagents and procedures. In: Axelsen NH, Kroll J & Weeke B (Eds) A Manual of Quantitative Immunoelectrophoresis, Methods and Applications (pp 15-35) Universitetsforlaget, Oslo 

Detection of Bacteroides fragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis

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