From: International course on Laboratory methods for the diagnosis of leptospirosis
KIT Biomedical Research, Amsterdam, The Netherlands
Over the period 1987-1993 seven hundred and twenty eight (728) Leptospira isolates were identified to serovar level mainly by means of monoclonal antibodies or otherwise by cross agglutinin absorption tests (CAAT) or several DNA based techniques. Approximately 76 % of these 728 Leptospira isolates belong to the following 14 serovars (Serogroup Serovar Numbers Percentages):
Autumnalis, Bim, 48, 6.6%;
Ballum, Arborea, 36, 4.9%;
Canicola, Canicola, 16, 2.2%;
Canicola, Dukou, 10, 1.4%;
Grippotyphosa, Grippotyphosa, 118, 16.2%;
Grippotyphosa, Grippo-like, 10, 1.4%;
Icterohaemorrhagiae, Copenhageni, 128, 17.6%;
Icterohaemorrhagiae, Icterohaemorrhagiae, 13, 1.8%;
Icterohaemorrhagiae, Lai, 14, 1.9%;
Pomona, Mozdok, 27, 3.7%;
Pomona, Pomona, 21, 2.9%;
Sejroe, Hardjo, 47, 6.5%;
Sejroe, Saxkoebing, 42, 5.8%;
Tarassovi, Tarassovi, 23, 3.2%.
The remaining 24 % of the Leptospira isolates belong to 44 other serovars, some of them being new ones. Isolates belonging to the 14 serovars mentioned in the table can easily be typed by the use of our panels of monoclonal antibodies in a simple microscopic agglutination test. In some cases a comparison is made between a leptospiral isolate and the reference strain of the serovar to which this isolate belongs (histograms). At the point of writing this, from a total of 205 reference strains belonging to their resp. serovars, 122 reference strains can be identified to serovar level and 75 to subgroup level.
Production of monoclonal antibodies
Our monoclonal antibodies were produced as follows: BALB/c mice were immunized against various reference strains by injecting leptospires from a culture (EMJH) and/or by injecting leptospires mixed with Freund’s incomplete adjuvant. Four days after the booster (second) injection, the spleen was removed and spleen cells were fused with mouse myeloma cells SP2/O-Ag-14 according to standard techniques. The resulting hybridomas were screened initially by the ELISA and in later fusions by a modified microscopic agglutination test (MAT). Hybridomas secreting antibodies of the desired specificity were cloned three times by limiting dilution and eventually cultured in the peritoneal cavity of pristane primed BALB/c mice. Ascites containing large amounts of monoclonal antibodies were harvested from these mice.
The aim of the selection procedure in the various fusions was to obtain a set of monoclonal antibodies which could recognize as many different serovars of a particular group as possible. Using combinations of positively reacting monoclonal antibodies, characteristic patterns of agglutination were observed for each serovar according to the specific pattern of antigenic determinants of that serovar. Characterization with monoclonal antibodies is related to conventional typing and based on the recognition of characteristic antigen patterns of serovars by panels of mAbs. The specificity of mAbs is limited among other things by the antigenic structure of the immunizing strain and the immunological repertoire of the mouse (Terpstra, 1991). A strain may be characterized by a specific combination of traits (mosaic of epitopes). One of the advantages of working with mAbs is that a large number of strains can be typed in a short time. Mislabelled strains can be traced accurately and more easily than with the use of ordinary rabbit antisera. Differences in agglutination profiles obtained with a panel of mAbs may be indicative for new serovars and even subserovar differences may be observed (Terpstra et al., 1985). Peripheral laboratories can type quickly and easily when equipped with panels of mAbs, adjusted to the locally circulating strains.
mAbs allow a precise definition of antigens, which gives them an important place in the identification of microorganisms and therefore also in the quality control of culture collections. This is an improvement in comparison to conventional rabbit antisera.
Characteristic antigenic pattern (histograms)
Serovars can be identified by their characteristic antigenic pattern recognised by a set of mAbs. If the serovars that circulate in a given area are well known, panels of mAbs can be composed that allow on a routine basis the typing of isolates. Some examples are shown on the next pages.
Cross-reactivity with serovars of other groups
Widely different patterns of cross-agglutination can be observed with certain mAbs. Apparently mAb reacts with different antigenic determinants shared by various groups of serovars.
Strains used for the production of mAbs
(Fusionnumber, Serovar, Strain, Reacting serogroups).
12, Copenhageni, Wijnberg, Icterohaemorrhagiae/Sarmin;
13, Hardjo type Prajitno, Hardjoprajitno, Sejroe / Hebdomadis;
16, Hardjo type Prajitno, Hardjoprajitno, Sejroe / Hebdomadis/Mini;
20, Icterohaemorrhagiae, Ictero I, Icterohaemorrhagiae/Javanica/Sarmin;
21, Hardjo type Prajitno, Hardjoprajitno, Sejroe / Hebdomadis;
22, Hardjo type Prajitno, Hardjoprajitno, Sejroe;
28, Hardjo type Bovis, Hardjobovis, Sejroe;
35, Polonica, 493 Poland, Sejroe;
38, Hardjo type Prajitno, Hardjoprajitno, Sejroe/Hebdomadis;
43, Pomona, Pomona, Pomona;
46, Proechimys, 1161 U, Pomona;
48, Tropica, CZ 299, Pomona;
50, Saxkoebing, Mus 24, Sejroe;
52, Mankarso, Mankarso, Icterohaemorrhagiae;
58, Mozdok, 5621, Pomona;
61, Mozdok, 5621, Pomona;
64, Bim, 1051, Autumnalis / Djasiman / Cynopteri / Louisiana;
65, Bim, 1051, Autumnalis;
69, Fortbragg, Fort Bragg, Autumnalis / Djasiman / Cynopteri / Louisiana;
70, Copenhageni, M 20, Icterohaemorrhagiae / Sarmin;
71, Grippotyphosa, Moskva V, Grippotyphosa / Pomona;
74, Castellonis, Castellon 3, Ballum;
81, Bratislava, Jez Bratislava, Australis / Panama;
82, Lai, Lai, Icterohaemorrhagiae / Canicola / Pyrogenes;
89, Ndambari, Ndambari, Icterohaemorrhagiae;
90, Peruviana, LT 941, Australis;
98, Sorexjalna, Sorex Jalna, Javanica / Sarmin / Celledoni;
106, Beye, 1537 U, Sejroe / Hebdomadis / Mini;
129, Losbanos, LT 101-69, Bataviae / Tarassovi;
132, Bajan, Toad 67, Australis;
134, Pyrogenes, Salinem, Pyrogenes;
151, Tarassovi, Perepelitsin, Tarassovi / Shermani;
152, Canicola, Hond Utrecht IV, Canicola / Ranarum;
164, Vanderhoedeni, Kipod 179, Grippotyphosa;
165, Grippotyphosa, Dutch M., Grippotyphosa.
mAbs in the way they are prepared in our laboratory are not particularly suitable for the recognition of new and previously unrecorded strains. These new strains may catch the attention in a typing experiment by presenting an unfamiliar agglutination profile with the panel of mAbs. In the present serovar conception the recognized reference strain stands as a representative for what is often a cluster of closely related strains with a slightly different antigenic make-up. This cluster is delineated by falling within the boundaries of a 10% residual titre after repeated absorptions according to the serovar definition by the cross-agglutinin absorption using conventional rabbit sera. This test is insufficiently sensitive to clearly reveal the differences between strains. Serovars of certain groups (for instance Pomona and Grippotyphosa groups) are antigenically closely related and can be classified only with difficulty by conventional typing methods. Clusters of antigenically interrelated pomona or mozdok-like and grippotyphosa-like strains are characterized by differences in agglutination titres with several mAbs. Those differences may be due to true, possibly quantitative, antigenic variation between strains. Even though mAbs producing hybridomas are selected with reference strains it is still possible in some cases to differentiate between strains on subserovar level, in other words strains belonging to the same serovar may react differently to some mAbs.
Microscopic agglutination test for the detection of antibodies to leptospires
The performance and interpretation of the MAT for typing with mAbs is the same as MAT with patient’s or rabbit sera, except that dilution of mAbs instead of sera are being added.
Mouse monoclonal antisera (mAb): Add distilled water as indicated on the ampoules for reconstitution of the freeze-dried mAb.
Dispense in small aliquots of 50 ul and store indefinitely at -20 degrees C or maximum for 8 weeks at +4 degrees C.
Prozone effect: Some monoclonal antibodies, usually IgM, show a prozone effect in their reaction in the lower dilutions. Reading of (for instance) the contents of the wells of columns 2, 5, 8 and 11 is needed.
- Alex JC, Korver H, Ratnam S, Venkatesan RA. Leptospira isolates of Madras City. Indian J Med Microbiol,17-4, 269-273, 1993.
- Cinco M, Banfi E, Stornello C, Campo F, Korver H. First human isolate of Leptospira interrogans as serovar bratislava in Italy. FEMS Microbiol Immunol 47, 499-504, 1989.
- Desmecht M, Korver H, Terpstra WJ. Isolation of Leptospira interrogans serovars saxkoebing, grippotyphosa and copenhageni from muskrats in Belgium (in Dutch) Vlaams Diergeneeskd Tijdschr, 60,59-63, 1991.
- Feresu SB, Bolin CA, Korver H. A new leptospiral serovar in the Icterohaemorrhagiae serogroup isolated from an ox in Zimbabwe. Int J Syst Bact, 43, 179-182, 1993.
- Gravekamp C, Korver H, Montgomery J, Everard COR, Carrington DG, Ellis WA, Terpstra WJ. Leptospires isolated from toads and frogs on the island of Barbados. Zbl Bakt 275, 403-411, 1991.
- Herrmann JL, Bakoss P, Korver H, Bulu AA, Bellenger E, Terpstra WJ, Saint Girons I, Baranton G. A new serovar in the Grippotyphosa serogroup comprising leptospiral isolates from different regions. Int J Syst Bacteriol, 44, 362-364, 1994.
- Korver H, Kolk AHJ, Vingerhoed J, Leeuwen J van,Terpstra WJ. Classification of serovars of the Icterohaemorrhagiae serogroup by monoclonal antibodies. Isr J Vet Med, 44-1, 15-18, 1988.
- Korver H, Ellis WA, Kim MJ, Terpstra WJ. Antigenic and genetic analysis of Korean Leptospira isolates. In: Leptospirosis, Proceedings of the Leptospirosis Research Conference 1990; Ed.Kobayashi; Publ.Hokusen-Sha Publishing Co., 235-270, 1991.
- Pereira MM, Korver H, Mazzonelli JM, Andrade J, Moraes G. Search for leptospires and specific antibodies in wild animals trapped in a periurban area of Rio de Janeiro, Brazil. In: Leptospirosis, Proceedings of the Leptospirosis Research Conference 1990; Ed.Kobayashi; Publ.Hokusen-Sha Publishing Co., 42-52, 1991.
- Subcommittee on the Taxonomy of Leptospira (TSCL). Int J Syst Bacteriol 37, 472-473, 1987.
- Terpstra WJ, Korver H, Leeuwen J van, Klatser PR, Kolk AHJ. The classification of Sejroe group serovars of Leptospira interrogans with monoclonal antibodies. Zbl Bakt Hyg A 259, 498-506, 1985.
- Terpstra WJ, Korver H, Leeuwen J van, Kolk AHJ, Schoone GJ. Alternative methods for the classification of leptospiral serovars. In: The present state of leptospirosis diagnosis and control. Eds.WA Ellis and TWA Little, Publ.Nijhoff, 1986.
- Terpstra WJ, Korver H, Schoone GJ, Leeuwen J van, Schonemann CE, Jonge-Aglibut S de, Kolk AHJ. Comparative classification of Leptospira serovars of the Pomona group by monoclonal antibodies and restriction-endonuclease analysis. Zbl Bakt Hyg A 266, 412-421, 1987.