Methyl 3-(2,4- dinitrophenylamino ) propionimidate hydrochloride (DNP-N-IE), methyl 3-(2,4-dinitrophenyl-N-methylamino) propionimidate hydrochloride (DNP- NMe -IE), and methyl 3-(2,4- dinitrophenylthio ) propionimidate hydrochloride (DNP-S-IE) have been prepared. DNP-N-IE and DNP- NMe -IE both react efficiently at 0-2 degrees C and pH 7-9.5 with sheep immunoglobulin G. At least 12 DNP groups can be introduced into the antibody protein without causing any significant change in its antigen binding affinity or capacity.

Affinity Labels↗

PubMed1984

A high sensitivity immunochemiluminometric assay for human thyrotrophin.

A two-site immunochemiluminometric assay (ICMA) has been developed for the measurement of human thyrotrophin (TSH). The procedure involves reaction of serum samples with monoclonal antibodies to TSH which have been labelled with a chemiluminescent acridinium ester followed by reaction with solid-phase polyclonal antibodies to TSH. The total reaction time is 2 h and bound labelled antibody is quantified luminometrically. The assay has an absolute sensitivity of 0.004 mU/l and a linear dose-response range up to 60 mU/l. Circulating TSH concentrations in 84 normal subjects were in the range 0.4 to 4.0 mU/l and in 16 hyperthyroid patients were less than 0.03 mU/l. This assay should thus prove useful as a first line test of thyroid function.

Antibodies, Monoclonal↗

PubMed1983

Acridinium esters as high-specific-activity labels in immunoassay.

A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.

Acridines↗

PubMed1983

Two-site immunochemiluminometric assay for human alpha 1-fetoprotein.

In this two-site immunochemiluminometric assay for human alpha 1-fetoprotein, acridinium ester-labeled monoclonal antibodies are used that have an average incorporation ratio of 0.3 mol of acridinium ester per mole of antibody. The solid-phase antibody consists of sheep anti-alpha 1-fetoprotein IgG covalently coupled to a diazonium derivative of reprecipitated aminoaryl cellulose. The assay involves a 1-h incubation after the simultaneous incubation of labeled and solid-phase antibodies. The subsequent quantification time is 10 s per tube. The assay has a working range of 20 to 246 kilo-int. units/L for CVs less than or equal to 10% and a sensitivity of detection of 1.3 kilo-int. units/L. Experiments with monoclonal antibodies labeled to an average incorporation ratio of 2.8 mol of acridinium ester per mole of antibody suggest that assay sensitivity increases with increased specific activity. alpha 1-Fetoprotein concentrations in the sera of pregnant women at 14-20 weeks of gestation, as measured by the present assay, agreed with the results of a conventional radioimmunoassay.

Acridines↗

PubMed1981

Two-site assay of human alpha 1-fetoprotein using 125I-labelled monoclonal antibodies.

Monoclonal antibodies to human alpha 1-fetoprotein (AFP) have been compared with a conventionally produced antiserum using radioimmunoassay and two-site immunoradiometric techniques. A low-affinity antibody, which proved inadequate for use in a radioimmunoassay, gave a satisfactory dose-response curve in a rapid two-site assay. A higher-affinity antibody yielded a simple, rapid, and sensitive two-site assay suitable for routine measurement of serum AFP.

Animals↗

PubMed1981

Porirua Karitane Family Unit.

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Child, Preschool↗

PubMedDate not supplied

Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labelled proteins.

In order to establish optimum conditions for the chemiluminescent (CL) reaction of two acridinium ester labelled proteins (human albumin and rabbit anti-human albumin IgG), we investigated the effects of the following factors known to influence the CL emission: pH, presence of proteins, relative concentrations of components of CL reaction and presence of surfactants. Under optimal conditions of pH and hydrogen peroxide concentration, hexadecyl trimethyl ammonium chloride (CTAC) increased the intensity of the CL reaction of the acridinium ester labelled albumin by 42-fold. Triton X-100, Tween-20, 23 lauryl ether (Brij 35) and sodium dodecyl sulphate (SDS) exerted a much smaller effect. In the case of the acridinium ester labelled antibody, the greatest increase was obtained with Triton X-100 (15-fold) followed by CTAC, Brij 35 and Tween 20 (SDS decreased the emission intensity).

Acridines↗

PubMedDate not supplied

Chemiluminescent immunoassay (CLIA) for urinary albumin.

A simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester-labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol-assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1-5 mg/l, and the inter-assay CVs are less than or equal to 15%. Using 10- and 50-fold sample dilutions in assay buffer, a wide working range (1-250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 +/- 2.7 micrograms/min (mean +/- SD), range 1-15.9 micrograms/min in 36 healthy subjects (17 male, 19 female, ages 4-56 years), with day-to-day variations of 28.5 +/- 20% (mean +/- SD), range 3.3-76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf-life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.

Adolescent↗