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# Effect of extraction methods on recovery and affinity of immunoglobulin of bacterium Xanthomonas corylina

S.G. Borkar, Louis Gardan, M. Barzic

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S.G. Borkar, Louis Gardan, M. Barzic. Effect of extraction methods on recovery and affinity of immunoglobulin of bacterium Xanthomonas corylina. Indian Journal of Experimental Biology, 1991, 29, pp.190-191. hal-01601232

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Effect of extraction methods on recovery and affinity of immunoglobulin of bacterium Xanthomonas corylina

S G Borkar*, L Garden & M Barzic

Station de Pathologie vegetale et Phytobacteriologie, I.N.R.A,
Beaucouze 49000, Angers, France

Received 23 October 1989; revised 5 October 1990

Percent recovery of immunoglobulins from antiserum of bacterium *X. corylina* by ion-exchange chromatography method was more (up to 95%) as compared to salt-precipitation method (up to 51%) and varied with immunization period irrespective of the amount of immunoglobulin present in the serum. Immunoglobulin extracted by salt-precipitation method loses its affinity to its antigen to a greater extent as compared to immunoglobulin extracted by ion-exchange chromatography method.

Extraction of immunoglobulins from antiserum is mostly done to obtain concentrated form (as in case of salt-precipitation method) or to separate immunoglobulins of different molecular weight (as in case of ion-exchange chromatography). Immunoglobulins being sensitive protein molecules may be affected by chemicals during the process of extraction. Therefore, in the present investigation different extraction methods, viz. salt-precipitation and ion-exchange chromatography have been employed and the extracted immunoglobulins studied for their affinity to antigen *X. corylina*.

Preparation of antigen glycoprotein and production of immunoglobulin—Xanthomonas corylina strain K-100, the causative organism of bacterial leaf spot and blight of noisetier plant (*Corylus* spp) was used for the preparation of antigen glycoprotein. The production of immunoglobulins was as described earlier¹.

Extraction of immunoglobulins—Antiserum (18 ml) obtained from 2, 3, 9 and 14 weeks of immunization of rabbit (containing 35 to 39 mg protein/ml antiserum, which was dependent on the immunization) was used separately for extraction of immunoglobulins by standard salt-precipitation and ion-exchange chromatography methods.

Affinity of immunoglobulin, separation by different methods, to antigen—This was estimated by indirect method of immunofluorescence¹,². Affinity of normal, salt-precipitated and ion-exchanged immunoglobulin to the antigen was determined on the basis of end point dilution.

In immunological assay the binding ability of the immunoglobulin molecules to the antigen depends on the recognition of the molecules at the binding site and their affinity. This character can be influenced by many factor. In the present experiment, the extraction factor, viz. extraction with salt-precipitation and ion-exchange chromatography, was studied for its effect on affinity of immunoglobulin to antigen.

Extraction of immunoglobulin from antiserum by salt-precipitation and ion-exchange chromatography was affected by the age of immunoglobulin i.e. the period of immunization. Longer the immunization period, better the percent recovery of eluted immunoglobulin protein (Table 1). Percent recovery of immu-

<table><caption>Table 1 Effect of extraction methods on recovery and affinity of immunoglobulin of *X. corylina*</caption><thead><tr><th rowspan="2">Immunization Ig in antiserum<br>period (week)</th><th rowspan="2">Aflinity to X. corylina<br>(protein<br>mg ml)</th><th colspan="2">Ig eluted by<br>ion-exchange<br>chromatography<br>(protein mg ml)</th><th colspan="2">Affinity to X. corylina Ig recovered by<br>salt-precipitation<br>(protein mg ml)</th><th colspan="2">Affinity to X. corylina<br>(at dilution)</th></tr><tr><th>total</th><th>IgG</th><th>total</th><th>IgG</th><th>total</th><th>IgG</th></tr></thead><tbody><tr><td>2</td><td>35.43</td><td>1:3200</td><td>1:1600</td><td>7.0<br>(19)</td><td>1:800<br>[4]</td><td>1:400<br>[4]</td><td>13.56<br>(38)</td><td>1:400<br>[8]</td></tr><tr><td>3</td><td>35.87</td><td>1:3200</td><td>1:1600</td><td>29.94<br>(83)</td><td>1:800<br>[4]</td><td>1:800<br>[2]</td><td>14.87<br>(41)</td><td>1:400<br>[8]</td></tr><tr><td>9</td><td>39.81</td><td>1:6400</td><td>1:6400</td><td>34.03<br>(85)</td><td>1:800<br>[8]</td><td>1:800<br>[8]</td><td>20.12<br>(50)</td><td>1:400<br>[16]</td></tr><tr><td>14</td><td>38.50</td><td>1:6400</td><td>1:6400</td><td>35.58<br>(95)</td><td>1:800<br>[8]</td><td>1:800<br>[8]</td><td>17.06<br>[44]</td><td>1:400<br>[16]</td></tr></tbody></table>

Figures in round brackets represent percent recovery of Ig during extraction; Figures in square brackets represent loss (times over normal) in affinity of respective Ig.

<footnote>* Present address—Department of Plant Pathology, M.P.K.V., Agricultural Research Station, Niphad 422 303, India</footnote>
noglobulin by ion-exchange chromatography varied from 19 to 95%, which was dependent on the age of immunoglobulin. Percent recovery of immunoglobulin by salt-precipitation method was less as compared to ion-exchange method and varied from 38 to 51%, which was also dependent on the age of immunoglobulin.

Affinity studies with normal, ion-exchange and salt-precipitated immunoglobulins revealed that equal amount of immunoglobulins (3 mg protein/ml) extracted by ion-exchange and salt-precipitation method varied in their dilution end point during immunofluorescence and thus in their affinity to the antigen X. corylina. Affinity of extracted total as well as IgG was reduced (Table 1) during extraction with ion-exchange and salt-precipitation methods. The reduced loss in affinity was dependent on the method adopted for the extraction and the age of immunoglobulin i.e. the

period of immunization. These results suggest that ion-exchange is effective than salt-precipitation method to acquire the immunoglobulin (total ad IgG) having more affinity. However, immunoglobulin obtained by longer immunization period loses its affinity to greater extent during extraction with salt-precipitation and ion-exchange method. The reason seems to be the variation in sensitivity of immunoglobulin obtained at different periods of immunization, to the material used for extraction of the immunoglobulin.

Authors thank Mrs. Martin for help in immunization programme.

References

1. Borkar S G, *Indian J Exp Biol*, 27 (1989) 464.

2. Allen E & Kelman A, *Phytopathology*, 67 (1977) 1303.