Sandwich ELISA Using a Mouse/Human Chimeric CSLEX-1 Antibody.

Abstract:

BACKGROUND:An assay using a mouse antisialyl Lewis X (sLeX) antibody (CSLEX-1) is used clinically for screening and monitoring patients with breast cancer in Japan. However, the IgM isoform of CSLEX-1 is not preferred for the assay because the bulkiness of IgM generally causes poor accessibility to the antigen. To solve this problem, we developed an antisLeX mouse/human chimeric IgG antibody, CH-CSLEX-1, using transgenic silkworms. The performance of a homologous sandwich ELISA of CH-CSLEX1 was then evaluated. METHODS:To generate CH-CSLEX-1, we used a GAL4/UAS binary gene expression system in transgenic silkworms. The reactivities of CSLEX-1 and CH-CSLEX-1 were determined in a Biacore analysis. To confirm antigen specificity, 3 antigens [sLeX, sLeA, and Lewis Y (LeY)] were used. RESULTS:CH-CSLEX-1 formed correctly as an IgG class of immunoglobulin molecule with an isoelectric point close to the predicted value. The best combination for capturing and probing in a sandwich ELISA was determined as a homologous combination of CH-CSLEX-1. The CH-CSLEX-1 assay specifically detected sLeX, but not sLeA and LeY. A correlation analysis with 107 human samples showed good concordance between the conventional CSLEX-1 assay (homologous sandwich ELISA using CSLEX-1) and the CH-CSLEX-1 assay (r = 0.98). Moreover, the CH-CSLEX-1 assay was not affected by either human antimouse IgG antibodies (HAMA IgG) or HAMA IgM. CONCLUSIONS:The mouse/human chimeric antibody CH-CSLEX-1 allowed the establishment of a highly specific sandwich ELISA for sLeX that was not affected by HAMA.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Yamashita J,Kobayashi I,Tatematsu K,Sezutsu H,Noda K,Ishihara H

doi

10.1373/clinchem.2016.260968

subject

Has Abstract

pub_date

2016-11-01 00:00:00

pages

1516-1523

issue

11

eissn

0009-9147

issn

1530-8561

pii

clinchem.2016.260968

journal_volume

62

pub_type

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