Hideaki Tanimori

A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support.

A method was devised for enzyme labeling goat antibody to rabbit immunoglobulin G with beta-D-galactosidase (EC 3.2.1.23) using a heterobifunctional cross-linker, N-(gamma-maleimidobutyryloxy)-succinimide. Labeling of the purified antibody was by a continuous 2-step process and including a chromatographic purification procedure could be completed within one day. The partially purified anti-rabbit IgG was coated on Amino-Dylark cylinders, a new solid support, using glutaraldehyde as the coupling reagent. With enzyme-labeled antibody and the solid-phase anti-rabbit IgG, a sandwich enzyme immunoassay for rabbit IgG was developed with a lower limit of detection at 3.5 pM (0.1 ng/tube). The specificity of the assay was excellent and all 4 types of IgG tested showed 0.0001% or less cross-reactivity with rabbit IgG.

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using β-d-galactosidase coupled to a monoclonal antibody of IgM isotype

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

Enzyme immunoassay with high sensitivity and accuracy for specific antibody to neocarzinostatin

A quantitative enzyme immunoassay (EIA) for specific antibody to neocarzinostatin (NCS) is described which uses enzyme-labeled anti-rabbit IgG antibody, solid-phase NCS and standard purified specific antibody to NCS. The dose of the standard was determined by sandwich EIA for rabbit IgG. The lower detection limit was 3 ng of the specific antibody per tube. The accuracy of the assay was excellent and a comparative study with the sandwich EIA for rabbit IgG showed good correlation. The antiserum to NCS of the highest titer was found to contain 0.6 mg and 40 mg per ml of specific antibody to NCS and of normal IgG, respectively. The accuracy of the assay results and the purity of the standard was established by 2 recovery tests for anti-NCS antibody.

Detection and Quantitative Assessment of a Vibrio cholerae O1 Species in Several Foods by a Novel Enzyme Immunoassay

A selected antibody enzyme immunoassay (SAEIA) for the general detection of Vibrio cholerae O1 species has been developed using the immunological reagents of a rabbit antiserum specific for V. cholerae O1 classical Inaba 569B and immobilized cell fragments of V. cholerae O1 E1 Tor 85P6, and β‐D‐galactosidase‐labeled goat anti‐rabbit immunoglobulin G as tracer. The SAEIA was specific for V. cholerae O1 species and showed low cross‐reaction values to other microorganism species tested including Vibrio parahaemolyticus. The detection limit of the SAEIA was 4,500 cells per assay for all the 13 strains of V. cholerae O1 examined. Quantitative comparison on the growth of the E1 Tor 85P4 in several foods cultured for 24 hr were studied using the SAEIA. Preceding the experiments, little inhibition of every food homogenate for the measurement of the SAEIA was first demonstrated and then the homogenate was directly used for an assay sample. The interaction of the growth of Escherichia coli to that of V. cholerae O1 in a food was also found to be little under the mixed culturing of both bacteria using the SAEIA.

A Novel Enzyme Immunoassay Commonly Applied for Ten Strains of Pyricularia oryzae

Antiserum against a strain of the rice blast fungus Pyricularia oryzae was elicited in rabbits immunized with its cell fragments emulsified with incomplete Freunds adjuvant. The fragments were also used as solid‐phase antigens. A highly sensitive, competitive type enzyme‐linked immunosorbent assay for P. oryzae was developed by using these two preparations as the immune reagents together with the use of β‐d‐galactosidase‐labeled anti‐rabbit IgG as the tracer. Cross‐reactivity of nine different strains of P. oryzae were measured by the assay. Sensitivity and accuracy of the assay was improved by choosing the cell fragments of the least cross‐reactive strain as the solid‐phase antigen. The improved method was successfully applied for sensitive and accurate assay of all ten strains of P. oryzae with the common measuring range between 1 and 100 ng per tube. Other species of microorganisms had little reactivity in this immunoassay indicating that the assay is specific to P. oryzae group microorganisms.

A new solid support for sandwich enzyme immunoassays of human immunoglobulin G.

Two antibodies were prepared for use in a sandwich enzyme immunoassay of human IgG. Completely purified guinea pig anti-human IgG was labelled with beta-D-galactosidase (EC 3.2.1.23), using a heterobifunctional cross-linker named GMBS. Partially purified anti-human IgG was immobilized on a new solid support: Amino-Dylark balls. Optimal conditions for immobilizing the antibody, using glutaraldehyde as the coupling reagent, were studied in detail. With the enzyme-labelled antibody and the solid-phase anti-human IgG, a sandwich enzyme immunoassay of human IgG with a lower limit of detection at 10.5 pM (0.3 ng/tube) was developed. A comparative study of the EIA method and a laser nephelometric method showed a good correlation. The specificity of the assay was excellent: all 4 types of IgG tested showed the maximum 0.0001%; human IgA, IgM and albumin possessed the maximum 0.54% in their cross-reactivity values with human IgG.

A new liquid-phase enzyme immunoassay for rabbit IgM antibodies and its application for comparing the specificity of IgM and IgG antibodies contained in the same antiserum

A new liquid-phase enzyme immunoassay (EIA) has been developed to compare the specificities of IgM and IgG antibodies when they are both present in the same serum sample and directed towards a simple hapten. The hapten viomycin (VM) was used as the model antigen and antibodies to VM were raised in rabbits. In the immunoassay VM, labelled with the enzyme galactosidase as marker (VM-GAL), was mixed with rabbit anti-VM serum. First IgM anti-VM antibodies bound to VM-GAL were precipitated with a guinea pig anti-rabbit IgM serum and galactosidase activity was measured in the precipitate. Then IgG anti-VM antibodies bound to VM-GAL were precipitated from the supernatant with a goat anti-rabbit IgG serum and enzyme activity was measured in this precipitate. The guinea pig anti-rabbit IgM and goat anti-rabbit IgG were specific for IgM and IgG respectively and did not appear to cross-react. Nine analogues of VM were used as inhibitors in this immunoassay to compare the specificities of IgM and IgG antibodies for determinants on VM. The results suggest that recognition of the fine structure of VM by IgM is less strict than recognition by IgG.