Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 3, 1995, pp. 162-163

REPORTE CORTO / SHORT REPORT

IMMUNOENZYMATIC ASSAY FOR THE QUANTIFICATION OF RECOMBINANT HUMAN INTERLEUKIN-2

Giselle Penton, Francisco Hernandez*, Vivian Santos, Yolanda Rojas, Eduardo Penton, Pedro Lopez-Saura and Manuel Arana.

Genetic Engineering and Biotechnology Center, P.O. Box 6162, La Habana 6. C.P. 10600, Cuba.

Code Number: BA95056
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In the present paper an immunoenzymatic assay was developed for determination of recombinant human IL-2 concentrations in the range of nanomoles based upon the use of murine monoclonal antibodies (MAbs) developed and specially selected for this purpose.

INTRODUCTION

In the present paper an immunoenzymatic assay was developed for determination of recombinant human IL-2 concentrations in the range of nanomoles based upon the use of murine monoclonal antibodies (MAbs) developed and specially selected for this purpose. The system was calibrated against an international standard of IL-2 and the calculation of concentrations of samples was optimized through the use of a log-log transformation which allowed the use of the calibration curve in the concentration range as low as 1 ng/mL with a regression coefficient higher than 0.99.

EXPERIMENTAL PROCEDURES

The obtainment of MAbs was achieved according to a variant of the conventional method of Kohler and Milstein (1). Immunoglobulins were conjugated with radish peroxidase by the peryodate method (2).

The adequate concentrati<%- 1>ons and times of incubation were evaluated using polyvinyl plates coated with 1; 5; 10; 20; 50 and 100 ug/mL of the coating MAb in different conditions including: overnight incubation at 4^oC, 3 h at 37^oC and preincubation 2 h at 37^oC followed by overnight incubation at 4^oC.

The plates were incubated with the antigen, conjugated antibody and after adding substrated and stopped the reaction, the reading was performed in an ultramicroanalytical system (SUMA).

In the same way the optimal time and temperature conditions of incubation with the antigen using three times (1, 2 and 3 h) and three temperatures (25, 37 and 42^oC) were evaluated. The sensitivity of the system was determined using the absorbance values of negative controls and the three more diluted points of the standard curve of 20 determinations. Concentrations of IL-2 were calculated using a log-log transformation.

RESULTS AND DISCUSSION

In our ELISA system the optimal conditions obtained were: overnight coating at 4 C and 10 ug/mL of the MAb CBIL-2.2, polyvinyl as solid phase and incubation during 1 h at 37 C with the antigen. The coating of plates has been shown to be optimal at certain concentration values under which it is less efficient (3).

In our system 10 ug/mL was the adequate coating concentrations which does not differ significantly from the higher concentrations employed but it does from the 1 and 5 ug/mL concentrations. By the other hand the overnight coating at 4 C was enough for the binding to the solid phase of all the capture antibody. The incubation during 1 h at 37 C with the antigen resulted the best condition assayed which was evidenced by an increase of the slope of the regression line at this temperature.

The sensitivity of the system was 1 ng/mL which corresponded to 10 units of IL-2 per milliliter as determined by the demonstration of a significant difference between the absorbance values of negative controls and the point of the standard curve corresponding to 1 ng/mL of IL-2.

A log-log transformation was employed as shown in the following picture which allowed to increase the correlation coefficient from 0.90 to 0.99.

REFERENCES

1. KOHLER, G. et al. (1975). Nature 256:495

2. NAKANE, P. A. et al. (1974). J. Histochem Cytochem 22: 1084

3. MASON, D. W. et al. (1980). Biochem. J. 187:1.

Figure Copyright 1995 Sociedad Iberolatinamericana de Biotecnologia Aplicada a la Salud

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