JOURNAL OF BIOLUMINESCENCE A N D CHEMILUMINESCENCE VOL 5 179-182
(1990)
Enhanced Chemiluminescence ELISA for Listeria Specific Antigen in Cerebrospinal Fluid Using an FITC-anti-FITCSystem Dhanraj Samuel, James McLauchlin and Anthony G. Taylor Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, Colindale, London NW9 5HT, UK
An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELSA an anti-listeria monoclonal antibody, immobilized onto assay wells, was used t o capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody t o FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients. Keywords: ELISA; FITC; Listeria; monoclonal antibodies
INTRODUCTION
Listeria monocytogenes is a gram-positive bacterium which is widely distributed in nature. The disease caused by L. monocytogenes (listeriosis) affects a wide range of animals including man. L. monocytogenes can be subdivided into 13 serovariants (serovars) and the majority of infections in man are caused by L. monocytogenes serogroup 1/2a, 112b and 4b (Seeliger and Hohne, 1979). Serovar 4b accounts for 59% of all human cases in Britain (McLauchlin, 1987). Meningitis is a common clinical manifestation of listerosis (Nieman and Lorber, 1980; Seeliger and Finger, 1983), where rapid onset of symptoms and high mortality rates may occur. Early diagnosis followed by appropriate treatment with antimicro0884-3996/90/03017Y-04$05.00 01990 by John Wiley & Sons, Ltd
bial agents however may improve the poor prognosis of this disease (Robertson et a/. , 1979; Relier, 1979; Fleming et ul., 1985). Hence there is a need for rapid methods for the laboratory diagnosis of listeriosis. We previously reported a colorimetric ELISA for the detection of L. monocytogenes serogroup 4 soluble antigen in the CSF (McLauchlin et al., 1988a) and in this study a chemiluminescent method is described. In the assay an anti-Lzsreriu monoclonal antibody, CL2 (McLauchlin et a [ . , 1988b), absorbed onto microtitre wells is employed to capture antigen from CSFs. The same anti-listeria antibody labelled with FITC then reacts with captured antigen. Finally a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody (Samuel et al., 1988)
180
D. SAMUEL, J. McLAUCHLlN AND A. G. TAYLOR
reacts with the FITC-labelled antibody. The presence of the antigen is then revealed by an enhanced chemiluminescence assay for HRP. The resulting luminescence is recorded using a camera luminometer .
blood cultures or CSFs, (ii) 7 individuals where infection was not suspected, and (iii) from 18 patients with meningitis due to other species of bacteria (6 cases by Streptococcus pneumoniae, 12 by Haemophilus influenzae).
MATERIALS AND METHODS
Formamide extracted antigen
Anti-FITC-horseradish peroxidase (HRP) conjuga tes
A soluble antigen extract was prepared from L. monocytogenes strain L724 (a wild type serovar 4b strain isolated in Britain from an adult with meningitis) using a-modification of the method of Fuller (1938). This consisted of treating an acetone powder of bacterial cells (10 g dry weight) twice with formamide (BDH Chemicals) at 160°C for 15 minutes. The cellular debris was removed by centrifugation (l0,OOOg for 20 minutes), and the supernatant was extensively dialysed against phosphate buffered saline (PBS, Dulbecco A , Oxoid Ltd). The extract was then filtered through a 0.22 pm nitrocellulose filter (Sartorius) (final volume, 75 ml) .
The production and characterization of an antiFITC mouse monoclonal antibody has been previously reported (Samuel et al., 1988). An HRP (EC 1.11.1.7)-anti-FITC antibody conjugate was prepared by the method of Wilson and Nakane (1978). Anti-Listeria antibodies
We have previously reported the production of mouse monoclonal antibodies against L. monocytogenes serogroup 4 strains (McLauchlin et al., 1986). One of these antibodies, designated CL2, was used in this study and conjugated to FITC as described previously (Samuel et al., 1988). Briefly, 77pl of FITC (Smgiml in dry absolute alcohol) was added with stirring at room temperature (RT) to 8mg of purified CL2 in 0.5ml of 0.1 molil Na2C0,/NaHCO3 buffer pH9.2 containing 0.5molil NaC1. After 45min the CL2iFITC conjugate was separated from free FITC by gel filtration on a PD 10 column (Pharmacia) in Complement Fixation Buffer (CFT, Oxoid Ltd) plus 0.5 molil NaCl. The conjugate was stored in CFT buffer plus 0.5mol/l NaCl with 20% (v/v) glycerol and 0.08% (wiv) NaN, at 4°C. A CL2-HRP conjugate was prepared using the method of Wilson and Nakane (1978). Human CSF supernatant samples
Human CSFs were collected from diagnostic clinical microbiology laboratories over a period of 5 years. Samples were stored at -70"C, and repeated freezing and thawing was avoided. A total of 32 CSFs were tested. Samples were collected from: (i) 7 patients with meningitis due to L. monocytogenes serogroup 4, which were diagnosed by the isolation of this bacterium from
Substrate for the chemiluminescent assay of HRP
Sodium luminol (Sigma) before use was crystallized twice from S% NaOH as described by Stott and Kricka (1987). The substrate solution was prepared by adding 1 0 0 ~ 1of the enhancing solution (9 mg of iodephenol in 1 ml of dimethylsulphoxide) to 10 ml of luminol solution (2.5 mg of sodium luminol in 10 ml of 0.1 molil Tris buffer pH 8.6, containing 3.1 p1 of 30% v/v H 2 0 2 ) just before use essentially as described by Kricka and Thorpe (1986). Antigen capture E L S A using CL2lFITC and anti-FITC/HRP conjugates
Eight-well flat-bottom ELISA strips (Costar, Fastbinder PETG Assay Strip, Costar Europe Ltd) were treated for 72 hours with whole ascitic fluid of CL2 (Approximately 3 mgiml) diluted to 1:5000 (v:v) in PBS containing sodium azide (0.08% wh). The fluid from each well was aspirated, and 2001.11 of blocking buffer (1% (wiv) milk powder (Marvel, Cadbury's Ltd,) containing 0.08% (wiv) sodium azide in PBS) was added to each well for
ELISA FOR LlSTERlA SPECIFIC ANTIGEN
approximately 3 hours at RT. The fluid from each well was then aspirated, a further 200y1 of blocking buffer added to each well, and the strips were sealed with adhesive tape (Scotch) and stored for up to 4 weeks at 4°C. When required for use, the strips were washed six times (200 y1 per well) with wash buffer (0.1% v/v Tween 20 in PBS), and 100 yl of CSF added. The strips were then incubated for 1 hour at RT, and washed six times with wash buffer as before. CL2IFITC conjugate was added (100yI) at a dilution of 1:3000 (v/v) in ELISA buffer (5% (w/v) bovine serum albumin, Armour, 1% (w/v) milk powder, and 1% (viv) Tween-20 in PBS), and reincubated for 1 hour at 37°C. The strips were then washed six times as before, and anti-FITC/HRP conjugate added at a dilution of 1:500 (v/v) in ELISA buffer. The strips were reincubated for 1 hour at RT. Strips were then washed as before and then transferred to the camera luminometer (Dynatech) loaded with a Polaroid 612 (ASA 20,000) instant photographic film. Luminol substrate (100 $) was added to each well and the film was exposed for 30 seconds and developed for 35 seconds. RESULTS Attempts to prepare a direct HRP conjugate with CL2 antibody were unsuccessful since the conjugate precipitated on storage at 4 "C and could not be resolubilized. The FITC conjugate of CL2, unlike the CL2-HRP conjugate, retained its original solubility properties, and could be stored at 4°C for over 6 months without detectable loss in its antigen binding activity and this conjugate was used in the present study. Fig. 1 shows the results obtained. Doubling dilutions of the formamide extracted Listeria antigen in PBS was used as a positive control, starting at lil00 dilution) and the diluent alone as a negative control in the ELISA. Antigen was detected in the CSFs from 5 out of 7 patients with culture proven L. monocytogenes serogroup 4 infection, and in none of the samples from 25 other patients tested. DISCUSSION
In this paper, we have described an ELISA with a chemiluminescent signal for the detection of a L.
181
Figure 1. Photographic result of the chemiluminescent ELISA for Listeria rnonocytogenes serogroup 4 soluble antigen. Row B3 to 8 contained a serial dilution of the formamide extract of L. rnonocytogenes extract (positive control). Wells B2 and 9 were the negative controla. CSFs from 7 patients with culture-proven listeriosis were in wells D5, E4, E9, F5, F6, F7 and G5. CSFS from 25 other patients (see 'Materials and methods') were in the rcrnaining wells in rows D, E, F, and G2 to 9
monocytogenes serogroup 4 specific antigen in CSFs. The chemiluminescent method reduces the time of the ELISA compared to a similar colorimetric assays (McLauchlin et al., 1988a). Although only qualitative results are obtained using the method described here, this does not diminish the value of the method since the presence of antigen appears diagnostic for listeriosis (McLauchlin et al., 1988a). Antigen was detected in CSFs taken from 5 out of 7 patients with culture-proven serogroup 4 infections, and not in any of the other CSFs tested. Although the assay appears to be specific, and sensitive, the number of samples available for testing was highly selected and therefore the results presented here do not reflect the true sensitivity or specificity of the test. In our previous study using a similar ELISA but with a colorimetric substrate the assay was found to be 100% specific, and had a sensitivity of 27% (McLauchlin ef al., 1988b). We were unable to prepare stable CL2-HRP conjugates which could be used directly to detect the captured antigen using an ELISA. Conjugation of the CL2 antibody with FITC has the advantages over labelling with HR P with respect to both the speed of purification and stability of the conjugates. An advantage of the indirect
D. SAMUEL, J. McLAUCHLlN AND A. G. TAYLOR
182
method described here of detecting a specific antibody with a second labelled antibody is that such systems tend to be of greater sensitivity than assays employing directly labelled primary antibody. REFERENCES Fleming, D. W., Cochi, S. L., MacDonald, K. L., Brondum, J . , Hayes, P. S . , Plikaytis, B. D . , Holmes, M. B., Audurier, A , , Broome, C. V. and Reingold, A (1985). Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N . Engl. J . Med., 312, 404-407. Fuller, A. T. (1938). The formamide method for extraction of polysaceharides from haemolytic streptococci. Brit. J . Exp. Pathol., 19, 130-139. Kricka, L. J . and Thorpe, G . H. (1986). Photographic detection of chemiluminescent and bioluminescence reactions. Methods Enzymol., 133, 404420. McLauchlin, J. (1987). Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J . Appl. Bateriol., 63, 1-11. McLauchlin, J . . Beesley, J. E. and Betts, M. P. (1986). Monoclonal antibodies against Listeria monocytogenes serogroup 4 surface antigens. In Proceedings of the 9th International Symposium on the Problems of Listeriosis, Courtieu, A. L., Espaze, E. P. and Reynaud, A. E. (Eds), Universitk de Nantes, Nantes, pp. 102-105. McLauchlin, J . , Black, A , , Green, H. T., Nash, J . Q. and Taylor, A. G . (1988a). Monoclonal antibodies show Listeria monocytogenes iil necropsy tissue samples. J . Clin. Pathol., 41, 983-988. McLauchlin, J., Samuel, D. and Taylor, A. G. (1988b). The use of monoclonal antibodies to demonstrate L. monocy-
togenes in post mortem tissue using a direct immunofluorescence technique, and to detect a soluble antigen in CSF supernatant. In Proceedings of the X International Symposium on Problems of Listeriosis. Pecs, Hungary, in press. Niemen, R. E . and Lorber, B. (1980). Listeriosis in adults: a changing pattern. Report of eight cases and review of the literature, 1968-1978. Rev. Infect. Dis., 2, 207-227. Relier, J. P. (1979). (Listeriosis. J . Antimicrob. Chemother., S(Supp1 A), 51-57. Robertson, M. H., Mussalli, N. G . , Aizad, T A,, Okaro, J . M. and Banwell, G . S. (1979). Two cases of perinatal listerosis. Arch. Dis. Child, 54, 549-562. Samuel, D., Patt, R. J. and Abuknesha, R. A. (1988). A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC J . Immunol. Methods, 107, 217-224. Seeliger, H. P. R. and Finger, M. D. (1983). Listeriosis. In Infectious Diseases of the Fetus and Newborn Infant, 2nd edn, Remington, J. S. and Klein, J . 0. (eds), Saunders, Philadelphia, pp. 264-289. Seeliger, H. P. R. and Hohne, K. (1979). Serotyping of Listeria monocytogenes and related species. In Methods in Microbiology, Vol. 13, Bergan, T. and Norris, J. R. (eds), Academic Press, London, pp. 31-49. Stott, R. A. W. and Kricka, L. J . (1987). Purification of luminol for use in enhanced chemiluminescence immunoassay. In Bioluminescence and Chemiluminescence: New Perspectives, Scholmerich, J . , Andreesen, R., Kapp, A , , Ernst, M. and Woods, W G . (Eds), John Wiley, Chichester, pp. 237-240. Wilson, M. B. and Nakane, P. K. (1978). Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofkorescence and Related Staining Techniques, Knapp, W., Holubar, K. and Wick, G. (Eds), Elsevier, Amsterdam, pp. 215-224.
(1990)
Enhanced Chemiluminescence ELISA for Listeria Specific Antigen in Cerebrospinal Fluid Using an FITC-anti-FITCSystem Dhanraj Samuel, James McLauchlin and Anthony G. Taylor Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, Colindale, London NW9 5HT, UK
An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELSA an anti-listeria monoclonal antibody, immobilized onto assay wells, was used t o capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody t o FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients. Keywords: ELISA; FITC; Listeria; monoclonal antibodies
INTRODUCTION
Listeria monocytogenes is a gram-positive bacterium which is widely distributed in nature. The disease caused by L. monocytogenes (listeriosis) affects a wide range of animals including man. L. monocytogenes can be subdivided into 13 serovariants (serovars) and the majority of infections in man are caused by L. monocytogenes serogroup 1/2a, 112b and 4b (Seeliger and Hohne, 1979). Serovar 4b accounts for 59% of all human cases in Britain (McLauchlin, 1987). Meningitis is a common clinical manifestation of listerosis (Nieman and Lorber, 1980; Seeliger and Finger, 1983), where rapid onset of symptoms and high mortality rates may occur. Early diagnosis followed by appropriate treatment with antimicro0884-3996/90/03017Y-04$05.00 01990 by John Wiley & Sons, Ltd
bial agents however may improve the poor prognosis of this disease (Robertson et a/. , 1979; Relier, 1979; Fleming et ul., 1985). Hence there is a need for rapid methods for the laboratory diagnosis of listeriosis. We previously reported a colorimetric ELISA for the detection of L. monocytogenes serogroup 4 soluble antigen in the CSF (McLauchlin et al., 1988a) and in this study a chemiluminescent method is described. In the assay an anti-Lzsreriu monoclonal antibody, CL2 (McLauchlin et a [ . , 1988b), absorbed onto microtitre wells is employed to capture antigen from CSFs. The same anti-listeria antibody labelled with FITC then reacts with captured antigen. Finally a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody (Samuel et al., 1988)
180
D. SAMUEL, J. McLAUCHLlN AND A. G. TAYLOR
reacts with the FITC-labelled antibody. The presence of the antigen is then revealed by an enhanced chemiluminescence assay for HRP. The resulting luminescence is recorded using a camera luminometer .
blood cultures or CSFs, (ii) 7 individuals where infection was not suspected, and (iii) from 18 patients with meningitis due to other species of bacteria (6 cases by Streptococcus pneumoniae, 12 by Haemophilus influenzae).
MATERIALS AND METHODS
Formamide extracted antigen
Anti-FITC-horseradish peroxidase (HRP) conjuga tes
A soluble antigen extract was prepared from L. monocytogenes strain L724 (a wild type serovar 4b strain isolated in Britain from an adult with meningitis) using a-modification of the method of Fuller (1938). This consisted of treating an acetone powder of bacterial cells (10 g dry weight) twice with formamide (BDH Chemicals) at 160°C for 15 minutes. The cellular debris was removed by centrifugation (l0,OOOg for 20 minutes), and the supernatant was extensively dialysed against phosphate buffered saline (PBS, Dulbecco A , Oxoid Ltd). The extract was then filtered through a 0.22 pm nitrocellulose filter (Sartorius) (final volume, 75 ml) .
The production and characterization of an antiFITC mouse monoclonal antibody has been previously reported (Samuel et al., 1988). An HRP (EC 1.11.1.7)-anti-FITC antibody conjugate was prepared by the method of Wilson and Nakane (1978). Anti-Listeria antibodies
We have previously reported the production of mouse monoclonal antibodies against L. monocytogenes serogroup 4 strains (McLauchlin et al., 1986). One of these antibodies, designated CL2, was used in this study and conjugated to FITC as described previously (Samuel et al., 1988). Briefly, 77pl of FITC (Smgiml in dry absolute alcohol) was added with stirring at room temperature (RT) to 8mg of purified CL2 in 0.5ml of 0.1 molil Na2C0,/NaHCO3 buffer pH9.2 containing 0.5molil NaC1. After 45min the CL2iFITC conjugate was separated from free FITC by gel filtration on a PD 10 column (Pharmacia) in Complement Fixation Buffer (CFT, Oxoid Ltd) plus 0.5 molil NaCl. The conjugate was stored in CFT buffer plus 0.5mol/l NaCl with 20% (v/v) glycerol and 0.08% (wiv) NaN, at 4°C. A CL2-HRP conjugate was prepared using the method of Wilson and Nakane (1978). Human CSF supernatant samples
Human CSFs were collected from diagnostic clinical microbiology laboratories over a period of 5 years. Samples were stored at -70"C, and repeated freezing and thawing was avoided. A total of 32 CSFs were tested. Samples were collected from: (i) 7 patients with meningitis due to L. monocytogenes serogroup 4, which were diagnosed by the isolation of this bacterium from
Substrate for the chemiluminescent assay of HRP
Sodium luminol (Sigma) before use was crystallized twice from S% NaOH as described by Stott and Kricka (1987). The substrate solution was prepared by adding 1 0 0 ~ 1of the enhancing solution (9 mg of iodephenol in 1 ml of dimethylsulphoxide) to 10 ml of luminol solution (2.5 mg of sodium luminol in 10 ml of 0.1 molil Tris buffer pH 8.6, containing 3.1 p1 of 30% v/v H 2 0 2 ) just before use essentially as described by Kricka and Thorpe (1986). Antigen capture E L S A using CL2lFITC and anti-FITC/HRP conjugates
Eight-well flat-bottom ELISA strips (Costar, Fastbinder PETG Assay Strip, Costar Europe Ltd) were treated for 72 hours with whole ascitic fluid of CL2 (Approximately 3 mgiml) diluted to 1:5000 (v:v) in PBS containing sodium azide (0.08% wh). The fluid from each well was aspirated, and 2001.11 of blocking buffer (1% (wiv) milk powder (Marvel, Cadbury's Ltd,) containing 0.08% (wiv) sodium azide in PBS) was added to each well for
ELISA FOR LlSTERlA SPECIFIC ANTIGEN
approximately 3 hours at RT. The fluid from each well was then aspirated, a further 200y1 of blocking buffer added to each well, and the strips were sealed with adhesive tape (Scotch) and stored for up to 4 weeks at 4°C. When required for use, the strips were washed six times (200 y1 per well) with wash buffer (0.1% v/v Tween 20 in PBS), and 100 yl of CSF added. The strips were then incubated for 1 hour at RT, and washed six times with wash buffer as before. CL2IFITC conjugate was added (100yI) at a dilution of 1:3000 (v/v) in ELISA buffer (5% (w/v) bovine serum albumin, Armour, 1% (w/v) milk powder, and 1% (viv) Tween-20 in PBS), and reincubated for 1 hour at 37°C. The strips were then washed six times as before, and anti-FITC/HRP conjugate added at a dilution of 1:500 (v/v) in ELISA buffer. The strips were reincubated for 1 hour at RT. Strips were then washed as before and then transferred to the camera luminometer (Dynatech) loaded with a Polaroid 612 (ASA 20,000) instant photographic film. Luminol substrate (100 $) was added to each well and the film was exposed for 30 seconds and developed for 35 seconds. RESULTS Attempts to prepare a direct HRP conjugate with CL2 antibody were unsuccessful since the conjugate precipitated on storage at 4 "C and could not be resolubilized. The FITC conjugate of CL2, unlike the CL2-HRP conjugate, retained its original solubility properties, and could be stored at 4°C for over 6 months without detectable loss in its antigen binding activity and this conjugate was used in the present study. Fig. 1 shows the results obtained. Doubling dilutions of the formamide extracted Listeria antigen in PBS was used as a positive control, starting at lil00 dilution) and the diluent alone as a negative control in the ELISA. Antigen was detected in the CSFs from 5 out of 7 patients with culture proven L. monocytogenes serogroup 4 infection, and in none of the samples from 25 other patients tested. DISCUSSION
In this paper, we have described an ELISA with a chemiluminescent signal for the detection of a L.
181
Figure 1. Photographic result of the chemiluminescent ELISA for Listeria rnonocytogenes serogroup 4 soluble antigen. Row B3 to 8 contained a serial dilution of the formamide extract of L. rnonocytogenes extract (positive control). Wells B2 and 9 were the negative controla. CSFs from 7 patients with culture-proven listeriosis were in wells D5, E4, E9, F5, F6, F7 and G5. CSFS from 25 other patients (see 'Materials and methods') were in the rcrnaining wells in rows D, E, F, and G2 to 9
monocytogenes serogroup 4 specific antigen in CSFs. The chemiluminescent method reduces the time of the ELISA compared to a similar colorimetric assays (McLauchlin et al., 1988a). Although only qualitative results are obtained using the method described here, this does not diminish the value of the method since the presence of antigen appears diagnostic for listeriosis (McLauchlin et al., 1988a). Antigen was detected in CSFs taken from 5 out of 7 patients with culture-proven serogroup 4 infections, and not in any of the other CSFs tested. Although the assay appears to be specific, and sensitive, the number of samples available for testing was highly selected and therefore the results presented here do not reflect the true sensitivity or specificity of the test. In our previous study using a similar ELISA but with a colorimetric substrate the assay was found to be 100% specific, and had a sensitivity of 27% (McLauchlin ef al., 1988b). We were unable to prepare stable CL2-HRP conjugates which could be used directly to detect the captured antigen using an ELISA. Conjugation of the CL2 antibody with FITC has the advantages over labelling with HR P with respect to both the speed of purification and stability of the conjugates. An advantage of the indirect
D. SAMUEL, J. McLAUCHLlN AND A. G. TAYLOR
182
method described here of detecting a specific antibody with a second labelled antibody is that such systems tend to be of greater sensitivity than assays employing directly labelled primary antibody. REFERENCES Fleming, D. W., Cochi, S. L., MacDonald, K. L., Brondum, J . , Hayes, P. S . , Plikaytis, B. D . , Holmes, M. B., Audurier, A , , Broome, C. V. and Reingold, A (1985). Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N . Engl. J . Med., 312, 404-407. Fuller, A. T. (1938). The formamide method for extraction of polysaceharides from haemolytic streptococci. Brit. J . Exp. Pathol., 19, 130-139. Kricka, L. J . and Thorpe, G . H. (1986). Photographic detection of chemiluminescent and bioluminescence reactions. Methods Enzymol., 133, 404420. McLauchlin, J. (1987). Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J . Appl. Bateriol., 63, 1-11. McLauchlin, J . . Beesley, J. E. and Betts, M. P. (1986). Monoclonal antibodies against Listeria monocytogenes serogroup 4 surface antigens. In Proceedings of the 9th International Symposium on the Problems of Listeriosis, Courtieu, A. L., Espaze, E. P. and Reynaud, A. E. (Eds), Universitk de Nantes, Nantes, pp. 102-105. McLauchlin, J . , Black, A , , Green, H. T., Nash, J . Q. and Taylor, A. G . (1988a). Monoclonal antibodies show Listeria monocytogenes iil necropsy tissue samples. J . Clin. Pathol., 41, 983-988. McLauchlin, J., Samuel, D. and Taylor, A. G. (1988b). The use of monoclonal antibodies to demonstrate L. monocy-
togenes in post mortem tissue using a direct immunofluorescence technique, and to detect a soluble antigen in CSF supernatant. In Proceedings of the X International Symposium on Problems of Listeriosis. Pecs, Hungary, in press. Niemen, R. E . and Lorber, B. (1980). Listeriosis in adults: a changing pattern. Report of eight cases and review of the literature, 1968-1978. Rev. Infect. Dis., 2, 207-227. Relier, J. P. (1979). (Listeriosis. J . Antimicrob. Chemother., S(Supp1 A), 51-57. Robertson, M. H., Mussalli, N. G . , Aizad, T A,, Okaro, J . M. and Banwell, G . S. (1979). Two cases of perinatal listerosis. Arch. Dis. Child, 54, 549-562. Samuel, D., Patt, R. J. and Abuknesha, R. A. (1988). A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC J . Immunol. Methods, 107, 217-224. Seeliger, H. P. R. and Finger, M. D. (1983). Listeriosis. In Infectious Diseases of the Fetus and Newborn Infant, 2nd edn, Remington, J. S. and Klein, J . 0. (eds), Saunders, Philadelphia, pp. 264-289. Seeliger, H. P. R. and Hohne, K. (1979). Serotyping of Listeria monocytogenes and related species. In Methods in Microbiology, Vol. 13, Bergan, T. and Norris, J. R. (eds), Academic Press, London, pp. 31-49. Stott, R. A. W. and Kricka, L. J . (1987). Purification of luminol for use in enhanced chemiluminescence immunoassay. In Bioluminescence and Chemiluminescence: New Perspectives, Scholmerich, J . , Andreesen, R., Kapp, A , , Ernst, M. and Woods, W G . (Eds), John Wiley, Chichester, pp. 237-240. Wilson, M. B. and Nakane, P. K. (1978). Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofkorescence and Related Staining Techniques, Knapp, W., Holubar, K. and Wick, G. (Eds), Elsevier, Amsterdam, pp. 215-224.
