System. Appl. Microbiol. 8, 28-31 (1986)

Enzyme-Linked Immunosorbent Assay (ELISA) for Determining Im­munological Relationships Among p-Galactosidases from Lactobacilli

MARIA ELENA NADER de MACIAS, GABRIELA PERDIGON, GUILLERMO OLIVER, and AIDA PESCE de RUIZ HOLGAD01

1 Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145,4000 Tucuman, Argentina, and Instituto de Microbiologia, Facultad de Bioquimica, Quimica y Farmacia, Universidad Nacional de Tucuman, Argentina

Received June 28, 1985

Summary

Antisera were prepared against purified inducible ~-galactosidase from Lactobacillus murinus ATCC 35020, and L. bulgaricus ATCC 11842, and were used to measure the immunological relations between the reference enzymes and ~-galactosidase from different lactobacilli. Crude extracts from eighteen species of lactobacilli were tested against these antisera, in Ouchterlony double diffussion experiments. The conventional microcomplement fixation method used to detect immunological relationships among bac­teria was compared with an enzyme-linked immunosorbent assay (ELISA) for the detection and quantifi­cation of immunological relations among ~-galactosidase from different lactobacilli. The results of these assays using both antisera supported those obtained with Ouchterlony technique. From the combined results obtained with the different methods, phylogenetic maps were derived. The maps obtained with Ouchterlony and microcomplement fixation were very similar to those obtained with Ouchterlony and ELISA. We propose the use of the sensitive and rapid ELISA test as a tool to establish immunological relations among bacteria.

Key words: ~-galactosidase - Lactobacillus - Immunological relationships - Immunodiffusion - Micro­complement fixation - ELISA

Introduction

In a previous study (Nader de Macias et al., 1985), we determined the phylogenetic relationships among lac­tobacilli by analysis of a common inducible enzyme, ~-galactosidase, using micro complement fixation (MCF) (Wasserman and Levine, 1961; Champion et al., 1975) and double immunodiffusion according to Ouchterlony (1949). Based upon the results obtained with these two methods, we made phylogenetic maps of lactobacilli.

The immunological analysis has been proposed (Lon­don et al., 1973) for the detection of structural homology among proteins, and for the determination of the degree of cross reactivity between these proteins, as has already been demonstrated by Champion et al. (1975). The im­munological comparison of the amino acid sequence of isofunctional proteins allows the construction of evolutio­nary trees (London and Kline, 1973; London et al. 1971, 1975).

1 To whom correspondence should be sent.

The development of immunological methods that use labelled antigens or antibodies allows us to consider the possibility of using the enzyme-linked immunosorbent as­say (ELISA) for the same purpose as MCF.

ELISA techniques have the advantages of specificity, rapidity and sensibility, and can be quantified when they are compared with other immunological tests. In addition, they are cheaper, usually easier to handle, and require only very simple equipment.

The present work was carried out to compare the degree of cross reaction between ~-galactosidase of eighteen strains of lactobacilli, using ELISA combined with Ouch­terlony technique, and also to confirm, by ELISA assay, the immunological relationships obtained in our first study (Nader de Macias et al., 1985), in which we used the conventional technique.

Methods

The isolation and purification of ~-galactosidases, and the pre­paration of antisera have been described previously (Nader de Macias et aI., 1983a, b, 1985).

Indirect microplate ELISA was carried out by the technique of Voller et al. (1980), with certain modifications, as follows:

- Free cell extracts were diluted in carbonate-bicarbonate buf­fer (0.06 M, pH 9.5) to a concentration of 1 mg/ml of protein (Lowry technique) and 200 III of each extract was added to an horizontal row of wells in a polystyrene microplate. The plates were incubated at 37°C for 30 min.

- Plates were washed three times with PBS-T (Phosphate-buf­fered saline, pH 7.4, 0.05 % Tween 20 [polyoxyethylene sorbitan monolaurateJ).

- 200 III of seriated antibody dilution, or control solution, were added to each vertical row of wells of the plate. The antisera dilutions were made in a range from 1/8 to 1/8192. The plates were incubated at 3rc for 30 min.

- The plates were washed three times with PBS-To - Horseradish peroxidase-labeled anti-rabbit immunoglobu-

lins (DAKO-immunoglobulins-Denmark) conjugated were di­luted to working dilution in PBS-To 200 III of this solution were added to each well and incubated at 37°C for 30 min.

- The plates were washed three times with PBS-To - Substrate solution: 200 III of 0.1 % o-phenylenediamine and

0.03 % H 20 z in methanol were added to each well and incubated at room temperature in the dark for 30 min.

- The reaction was stopped by the addition of 50 III of 8 N H2S04•

- The color change in each well was determined spec­trophotometrically at 490 nm, in a Gilford spectrophotometer. The OD4901min was taken as the quantitative expression of the reaction.

The cross reaction was expressed as the percentage of the

Microorganisms

Immunological Relationships Among Lactobacilli 29

homologous reaction using the equation of Conway-Macario et al. (1982):

CR DO/min . HR DO/min x 100 = Percent cross reactIOn

where CR and HR stand for cross and homologous reaction, respectively.

The percent of cross reaction was obtained for each antibody dilution and then averaged.

Results and Discussion

The results obtained in comparative double diffusion experiments and MCF with anti- ~-galactosidases were reported in a previous paper (Nader de Macias et ai., 1985). These data were compared with the immunoenzy­matic assay.

The ELISA assay allows a quantitative estimation of cross reaction. The degree of cross reaction among ~­galactosidases was determined with the same two antisera used in MCF and Ouchterlony techniques. These results are summarized in Table 1, and compared with the data obtained with MCF.

A critical test of ELISA efficacy is the detection and quantification of antibodies prepared against antigens from those bacterial strains which exhibit cross reactivity with closely related species, and the degree of cross reac­tion can be determined in the same assay.

The immunological specificity of ELISA is, of course, based on the specificity of the reagents in the test combina­tion. In our case, the antibodies were specific to pure ~­galactosidase: the assay gave no reaction with normal

Anti-B-galactosidase to L.bulgaricus ATCC 11842 L.murinus ATCC 35020 Isolated Immuno- % Cross Immuno- % Cross from logical Reaction logical Reaction

Distance Distance

Table 1. Quantitative estimation of antigenic homology of certain B-galac­tosidases by immunological distance and % cross reaction with anti-B­galactosidase from L. bulgaricus ATCC 11842 and L.murinus ATCC 35020

L.acidophillus ATCC I 4356 50 70 87 39 L. bulgaricus ATCC 11842 0 100 86 34 L. bulgaricus CRU 403 (a) 13 98 86 30 L. bulgaricus CRL 405 (a) 11 99 89 32 L. bulgaricus CRL 406 (a) 13 100 87 37 L. casei subsp. casei ATCC 393 43 52 95 40 L. casei subsp. casei CRL 237 (b) 46 55 94 39 L. casei subsp. pseudoplantarum CRL 203 (b) 67 42 66 52 L. casei subsp. rahmnosus CRL 207 (b) 73 46 64 49 L. cellobiosus ATCC 11739 95 45 42 63 L. (ermentum ATCC 14931 36 49 79 46 L. helveticus A TCC 15009 38 82 90 43 L.lactis A TCC 12315 27 87 87 40 L.leichmannii ATCC 7830 24 86 84 33 L.murinus ATCC 35020 (CNRZ3 313) 98 26 0 100 L.plantarum ATCC 10241 78 65 67 53 L. plantarum CRL 123 (b) 69 60 66 67 L. salivarius A TCC 11742 75 48 61 60

I American Type Culture Collection; 2 Centro de Referencia para Lactobacilos; 3 Centre Na­tional de la Recherche Zootechnique; (a) Typical Argentino cheese; (b) TaH cheese.

30 M. E. N. de Macias, G. Perdigon, G. Oliver, and A. P. de Ruiz Holgado

L. murinus Ala: 35020

L. c~lIobiosus Alec 11739

l.cas~i -var. pwudoplantarum CRl203

L. plantarum AlCC 10241

L. f~~ntum ATCC 14931

sera, and showed the same degree of reaction with pure enzyme as with cell-free extract.

One of the disadvantages with respect to MCF is that no knowledge of the exact antigen amount that reacts with each antibody dilution can be obtained: the antigens were used in a concentration of 1 mg/ml, but we could not determine the quantities bound to the plates. This was not important for our purpose, because we were interested in the percentage of cross reaction of the antibodies, using

Fig. 1. Map based on the results of ELISA experiments reported in Table 1 and of im­munodiffusion experiments with L. but­garicus ATCC 11842 anti- ~-galactosidase serum.

always the same amount of antigens contained in crude extract with similar ~-galactosidase activity.

Another disadvantage was the higher antibody concen­tration necessary to obtain a measurable reaction: the range was between 1116 and 118,000, compared to MCF where the range was between 112,500 and 1112,500. It is not significant because we work with whole serum.

In spite of these differences we obtained phylogenetic maps similar to those obtained with MCF, in which the

L.murinus ArCC 35020

L.acidophilus AlCC 4356 l.ca~i-var.ca~i CRl237

l.cas~i AlCC 393

L.c~lIobiosus AlCC 11739

l. ca~i - vat: ps~doplantarum CRl 203 l. plantarum AlCC 10241

l.ca~i - var. rhamnosus CRl 207

L. f~rm~ntum AlCC 14931

L.lactis AlCC 12315 l. bulgaricus CRL 406

L. bulgaricus ATCC 11842 l.1~ichmanni I AlCC 7830

l. bulgaricus CRl 405 l. bulgaricus CRL 403

Fig. 2. Map based on the results of ELISA experiments reported in Table 1 with L. murinus ATCC 35020 anti- ~-galactosidase serum.

different groups of lactobacilli are separated. The maps showing the phylogenetic relationships derived from the data are schematized in Figs. 1 and 2.

There is a group of strains formed by L. bulgaricus ATCC 11842 and L. bulgaricus wild types (Figs. 1 and 2). A group of other lactobacilli is related to these strains which agrees with the results of London and Kline (1973) and Gasser and Gasser (1971), who studied aldolase and lactate dehydrogenase enzyme, respectively.

There are groups of lactobacilli less closely related to these strains, as shown in Table I by a higher immunologi­cal distance and lower percentage of cross reaction in ELISA. L. casei subsp. casei collection strain and wild type are in a group different from L. casei subsp. rhamnosus and L. casei subsp. pseudoplantarum. Those findings are in agreement with those of London et al. (1971), who studied malic enzyme.

Another group did not react with either of the two anti­sera in immunodiffusion experiments, and showed the highest immunological distance and the lowest percentage of cross reaction. In the map, they are far from L. bul­garicus and from L. murinus as reference points.

As shown in Table 1, the values of immunological dis­tance from MCF and of percentage of cross reaction ob­tained with ELISA have allowed us to construct phy­logenetic trees in which the ubi cations of different groups of microorganisms are similar.

We can conclude that the ELISA test can be used for taxonomic purposes.

Acknowledgements. The author wishes to thank Dr. Ricardo Margni for his critical reading of the manuscript. - This work was supported by grants from Secreta ria de Ciencia y T ecnologia de la Republica Argentina, 1983.

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Dr. Aida Pesce de Ruiz Holgado, Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145,4000 Tucuman, Argentina