Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent

peer reviewedConjugates of alkaline phosphatase (AP) and mouse monoclonal immunoglobulins G (IgG) were prepared by means of the heterobifunctional linker, N-succinimidyl 3-(2-pyridyldithio)-propionate. The efficiency of such conjugates can be improved by optimizing the degree of substitution of IgG and AP. We have determined conditions yielding better performing conjugates than those synthesized by methods described previously. Moreover, the results obtained with the technique presented here are quite reproducible with all four monoclonal antibodies tested

Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates

Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation procedures. The conjugates were tested by enzyme immunoassay and their efficiency was characterized by the antibody and enzyme concentrations needed to obtain an arbitrary OD value. The suitability of antibody for conjugation through NH2 groups was tested by fluorodinitrobenzene (FDNB). HRP conjugates were produced by two variants of the sodium periodate procedure and two variants of the glutaraldehyde method, as well as by the heterobifunctional linker N-succinimidyl 3-(2-pyridyldithio)pro-pionate (SPDP). Two of the four antibodies were coupled by a third variant of the periodate method, through their carbohydrate moieties. The periodate-mediated conjugations, using sugar moieties on the enzyme, provided the most efficient HRP conjugates, regardless of the antibody subclass or isoelectric point. The glutaraldehyde procedures consistently gave the worst results. AP conjugates were prepared using the same methods. The most efficient and reproducible AP conjugates with all four monoclonal antibodies were obtained using the SPDP procedure. The efficiency of the other methods differed from one antibody to another

Assessment of the new immunological test Hemoblot for detecting occult blood in faeces

Hemoblot, a new immunological faecal occult blood test, produced by Gamma, Angleur, Belgium, was characterized and compared with another immunological test (HemeSelect, SmithKline Diagnostics, USA) and with a guaiac test (Hemoccult II, SmithKline Diagnostics). The analytical sensitivity of Hemoblot is 0.15 mg haemoglobin/g faeces and the test is specific for human haemoglobin. In addition, 135 symptomatic patients who had to undergo a colonoscopy were tested using the three tests. Two criteria were considered for the analysis: (1) the blood criterion: any pathology likely to cause colorectal or other bleeding; and (2) the precancerous-cancerous criterion: the pathology being either a colorectal polyp > 0.5 cm or a colorectal cancer. Considering both criteria, the sensitivity of Hemoblot was significantly higher than the sensitivity of Hemoccult: 38% and 23%, respectively, for the blood criterion; and 54% and 29% for the precancerous-cancerous criterion. Sensitivity and specificity did not differ statistically between Hemoblot and HemeSelect but Hemoblot was faster and simpler to perform. It could be widely used in mass screening

Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent

peer reviewedConjugates of alkaline phosphatase (AP) and mouse monoclonal immunoglobulins G (IgG) were prepared by means of the heterobifunctional linker, N-succinimidyl 3-(2-pyridyldithio)-propionate. The efficiency of such conjugates can be improved by optimizing the degree of substitution of IgG and AP. We have determined conditions yielding better performing conjugates than those synthesized by methods described previously. Moreover, the results obtained with the technique presented here are quite reproducible with all four monoclonal antibodies tested