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Reply to the letter of D. Kvale and P. Brandtzaeg
Journal oflmmunologicalMethods, 103 (1987) 149-150 Elsevier
149
JIM04573
L e t t e r to the editors
Reply to the letter of D. Kvale and P. Brandtzaeg G . M . W o o d , L.K. T r e j d o s i e w i c z and M.S. L o s o w s k y Department of Medicine, St. James's Hospital, Leeds LS9 7 TF, U.K. (Received 10 July 1987)
Dear Editors, We thank Drs. Kvale and Brandtzaeg for their comments on our paper 'ELISA for measurement of secretory IgA distinct from monomeric IgA' (Wood et al., 1987). In any ELISA for secretory immunoglobulins (sIg) based on immobilised anti-secretory component (SC) as the 'capture' antibody, such as that described by Kvale and Bradtzaeg (1986) and ourselves (Wood et al., 1987), free SC and any SC-bound Ig, irrespective of isotype, will be b o u n d We have used this principle to demonstrate specificity by addition of free SC in large excess (Wood et al., 1987). We also made the point in the discussion and suggested how it might be overcome. Thus we note with interest that Dr. Kvale and colleagues have evidence for competition by serum sIgM in their assay for circulating sIgA (Kvale and Brandtzaeg, 1986; Kvale et al., 1987); we concur that sIgM may have resulted in a slight underestimation of serum sIgA in our assay (Wood et al., 1987). As in any ELISA, incomplete binding and competitive effects will occur at or near saturation of the solid-phase antibody. To avoid such artefacts, samples are usually assayed at sub-saturating concentrations by ensuring sufficient sample dilution (determined empirically). In our work, serum samples from control patients were routinely diluted 1/100 and those from patients with liver disease 1/200; the results of Kvale and Brandtzaeg (1986, Fig. 4) indicate that such dilutions are sufficient to reduce competition from sIgM to low levels. Our
Correspondence to: L.K. Trejdosiewicz, Department of Medicine, St. James's Hospital, Leeds LS9 7TF, U.K.
results for serum sIgA (mean value 6.9 m g / m l ) are comparable to theirs (median value 10 mg/1) (Kvale and Brandtzaeg, 1986), suggesting that no gross underestimation had occurred. However, it is clear from their work (Kvale and Brandtzaeg, 1986; Kvale et al., 1987) and, as they point out, from our data (Wood et al., 1987) that higher serum sample dilutions may be necessary where sIgM (or free SC) concentrations are particularly high. Our assay was primarily designed to measure secretory IgA of the intestinal mucosa, where the contribution of IgM to sIg is only about 5% of the total (Richman and Brown, 1977). Kvale and colleagues have not produced evidence to support their statement that sIgM in secretions (as opposed to serum) may interfere with sIgA estimation in such fluids. Ideally, assays for Igs in secretions should quantitate free (non-secretory) Ig, sIg and free SC. Our ELISA (Wood et al., 1987) is capable of quantifying all three: total Ig (of each isotype) can be estimated by coating with polyvalent anti-Ig and detection with class-specific conjugates; secretory Ig and free SC can be estimated by coating with anti-SC and detection with antiIgA, anti-IgM and anti-SC conjugates. References Kvale, D. and Brandtzaeg, P. (1986) An enzyme-linked immunosorbent assay for differential quantitation of secretory immunoglobulins of the A and M isotypes in human serum. J. lmmunol. Methods 86, 107. Kvale, D., Schrumpf, E., Brandtzaeg, P., Solberg, H.E., Fausa, O. and Elgjo, K. (1987) Circulating secretory immmunoglobulins of the A and M isotypes in chronic liver disease. J. Hepatol. 4, 229.
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
150 Richman, L.K. and Brown, W.R. (1977) Immunochemical characterization of IgM in h u m a n intestinal fluids. J. lmmunol. 119, 1515.
Wood, G.M., Trejdosiewicz, L.K. and Losowsky, M.S. (1987) ELISA for measurement of secretory IgA distinct from monomeric IgA. J. Immunol. Methods 97, 269.
149
JIM04573
L e t t e r to the editors
Reply to the letter of D. Kvale and P. Brandtzaeg G . M . W o o d , L.K. T r e j d o s i e w i c z and M.S. L o s o w s k y Department of Medicine, St. James's Hospital, Leeds LS9 7 TF, U.K. (Received 10 July 1987)
Dear Editors, We thank Drs. Kvale and Brandtzaeg for their comments on our paper 'ELISA for measurement of secretory IgA distinct from monomeric IgA' (Wood et al., 1987). In any ELISA for secretory immunoglobulins (sIg) based on immobilised anti-secretory component (SC) as the 'capture' antibody, such as that described by Kvale and Bradtzaeg (1986) and ourselves (Wood et al., 1987), free SC and any SC-bound Ig, irrespective of isotype, will be b o u n d We have used this principle to demonstrate specificity by addition of free SC in large excess (Wood et al., 1987). We also made the point in the discussion and suggested how it might be overcome. Thus we note with interest that Dr. Kvale and colleagues have evidence for competition by serum sIgM in their assay for circulating sIgA (Kvale and Brandtzaeg, 1986; Kvale et al., 1987); we concur that sIgM may have resulted in a slight underestimation of serum sIgA in our assay (Wood et al., 1987). As in any ELISA, incomplete binding and competitive effects will occur at or near saturation of the solid-phase antibody. To avoid such artefacts, samples are usually assayed at sub-saturating concentrations by ensuring sufficient sample dilution (determined empirically). In our work, serum samples from control patients were routinely diluted 1/100 and those from patients with liver disease 1/200; the results of Kvale and Brandtzaeg (1986, Fig. 4) indicate that such dilutions are sufficient to reduce competition from sIgM to low levels. Our
Correspondence to: L.K. Trejdosiewicz, Department of Medicine, St. James's Hospital, Leeds LS9 7TF, U.K.
results for serum sIgA (mean value 6.9 m g / m l ) are comparable to theirs (median value 10 mg/1) (Kvale and Brandtzaeg, 1986), suggesting that no gross underestimation had occurred. However, it is clear from their work (Kvale and Brandtzaeg, 1986; Kvale et al., 1987) and, as they point out, from our data (Wood et al., 1987) that higher serum sample dilutions may be necessary where sIgM (or free SC) concentrations are particularly high. Our assay was primarily designed to measure secretory IgA of the intestinal mucosa, where the contribution of IgM to sIg is only about 5% of the total (Richman and Brown, 1977). Kvale and colleagues have not produced evidence to support their statement that sIgM in secretions (as opposed to serum) may interfere with sIgA estimation in such fluids. Ideally, assays for Igs in secretions should quantitate free (non-secretory) Ig, sIg and free SC. Our ELISA (Wood et al., 1987) is capable of quantifying all three: total Ig (of each isotype) can be estimated by coating with polyvalent anti-Ig and detection with class-specific conjugates; secretory Ig and free SC can be estimated by coating with anti-SC and detection with antiIgA, anti-IgM and anti-SC conjugates. References Kvale, D. and Brandtzaeg, P. (1986) An enzyme-linked immunosorbent assay for differential quantitation of secretory immunoglobulins of the A and M isotypes in human serum. J. lmmunol. Methods 86, 107. Kvale, D., Schrumpf, E., Brandtzaeg, P., Solberg, H.E., Fausa, O. and Elgjo, K. (1987) Circulating secretory immmunoglobulins of the A and M isotypes in chronic liver disease. J. Hepatol. 4, 229.
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
150 Richman, L.K. and Brown, W.R. (1977) Immunochemical characterization of IgM in h u m a n intestinal fluids. J. lmmunol. 119, 1515.
Wood, G.M., Trejdosiewicz, L.K. and Losowsky, M.S. (1987) ELISA for measurement of secretory IgA distinct from monomeric IgA. J. Immunol. Methods 97, 269.