Elaine M. Bailyes

The distinction of bone and liver isoenzymes of alkaline phosphatase in serum using a monoclonal antibody

Out of 31 mouse monoclonal antibodies to human liver and bone ALP, one antibody coded BAP 1/9, showing a preference for binding the bone isoenzyme, and one antibody coded LAP 1/5, showing no preference, were selected to study binding of the serum forms of ALP. Using a plate capture assay format BAP 1/9 showed a 1.8-fold preference for binding purified bone versus liver ALP in the presence of 10% ALP-free serum compared with 2.1- to 2.25-fold preference in buffered salt solution. BAP 1/9 showed a preference for binding ALP from serum samples containing predominantly bone ALP compared to those containing predominantly liver ALP. Using predefined mixtures of serum forms of bone and liver ALP a linear relationship between ALP bound to BAP 1/9 and the fraction of bone ALP was obtained.

A monoclonal antibody inhibiting rat liver 5'-nucleotidase.

5’-Nucleotidase (EC 3.1.3.5) is an intrinsic glycoprotein of the plasma membrane. Inhibitory antisera have been used in defining it as an ectoenzyme [I], to investigate its rno~~en~ent during endocytosis and membrane re-cycling [2], and to assess its physiological function [3]_ The enzyme has been extensively purified [4,5], but not to homogen~it~~. Antisera raised to S’nucleotidase preparations therefore contain antibodies to other prateins and this has hindered their use for immunological localisation and other studies. We have prepared a monoclonal antibody which avoids such difikulties, and used this in the characterization of highly purified, solubilized 5’-nucleotidase. The results indicate that this antibody should be of value in purifying the enzyme further, and in studying its subcellular distribution and circulation.

The Properties, Structure, Function, Intracellular Localisation and Movement of Hepatic 5′-Nucleotidase

5′-Nucleotidase (EC 3.1.3.5) was first described 50 years ago (Reis 1934) and was subsequently shown to be widely distributed in animal tissues (Reis 1951) as an intrinsic plasma membrane enzyme (Essner et al. 1958; Segal and Brenner 1960; Song and Bodansky 1967). Its ease of assay and stability made it a convenient and popular marker for the plasma membrane in subcellular fractionation (Evans 1978) and its measurement has also been found useful over many years as an indicator of cholestasis when raised levels of 5′-nucleotidase activity are present in patients’ sera (Ellis et al. 1978). The discovery that 5′-nucleotidase was an ectoenzyme in several cell types (De Pierre and Karnovsky 1974a, b; Trams and Lauter 1974; Newby et al. 1975) and the subsequent observation of its internalisation from the cell surface and re-cycling in the absence of protein synthesis (Stanley et al. 1980; Widnell et al. 1982) has led to more active investigation of its properties. This has been fuelled by a growing interest in its function which remains enigmatic (Stanley et al. 1982).

An enzyme-amplified monoclonal immunoenzymometric assay for prostatic acid phosphatase.

An immunoassay for prostatic acid phosphatase is described in which a high degree of specificity for the prostatic isoenzyme, obtained by the use of monoclonal antibodies, is combined with great sensitivity, made possible by enzyme-amplified measurement of the combination of the isoenzyme with its antibody. The increase in sensitivity thus achieved is of the order of 170 times that of conventional methods of measurement. The advantages of the enzyme-amplified method have been shown to be particularly useful in detecting and monitoring small abnormalities of prostatic acid phosphatase levels in patients with prostatic cancer.

The presence and measurement of secretory component in human bile and blood

Five monoclonal antibodies which recognized three separate epitopes on the free secretory component molecule were produced using free secretory component obtained from human colostrum. Two-site immunoradiometric assays were developed to measure free secretory component and secretory IgA. Monoclonal antibody M9 was used on coated plates as the capture antibody. Monoclonal antibody M7 was used as the labelled signal antibody for the assay of free secretory component and a commercially available monoclonal anti-IgA antibody was used as the labelled signal antibody for the assay of secretory IgA. Free secretory component was found in human serum and bile. In serum, its concentration was raised in patients with high serum alkaline phosphatase due to liver disorders but not in patients with high serum alkaline phosphatase due to non-liver disorders. In bile from bile duct drains collected during the first week after liver transplantation, free secretory component was found in concentrations of up to 33 mg/l, in vast excess of that found in bile from gallstone patients (up to 0.3 mg/l). Bile from gallstone patients but not from liver transplant patients produced proteolytic degradation of free secretory component when incubated in vitro. The finding of large amounts of free secretory component, the free cleaved fragment of the polymeric IgA receptor in human bile, further supports the existence of the blood to bile transhepatocytic pathway in humans.

MONOCLONAL ANTIBODIES RECOGNIZE 2300 YEARS AGED ALKALINE PHOSPHATASE

It was attempted to monitor the immunological response of monoclonal antibodies directed to human alkaline phosphatase in ancient Egyptian bones from the ptolemeic period. The intactness of the respective epitopes of the bone enzyme was successfully demonstrated in an ELISA. Fortunately, the mummified bone was not contaminated by fungi and bacteria due to the fungicidal and bactericidal reactivity of the ancient pretreatment employing resins of pistachio for mummification. The enzyme was enriched using gel chromatography, anion exchange and affinity chromatography to yield 310 +/- 7 mU/mg. The enzymically active fractions of the wheat-germ lectin affinity chromatography were subjected to ELISA. The best binding affinity was detected using the monoclonal antibody BAP A while the reactions of all the other four antibodies BAP B, BAP G, BAP 4A5 and BAP 5D4 were substantially diminished.

A monoclonal antibody capture assay for intestinal alkaline phosphatase and the measurement of this isoenzyme in pregnancy

Intestinal ALP was purified from meconium to a specific activity of 1,100 U/mg protein and used as antigen in the preparation of 7 monoclonal antibodies. Two of these antibodies were specific for intestinal ALP and reacted with different epitopes. Both bound adult intestinal ALP better than fetal intestinal ALP. One of these antibodies was used to establish a capture assay for intestinal ALP in human serum over the range 0.5-16 U/l. Reference ranges of serum intestinal ALP concentrations were established in relation to blood groups. Measurement of intestinal ALP in the serum of pregnant women showed no correlation with pre-term fetal passage of meconium.

Characterization of the Unusual Insulin of Psammomys obesus, a Rodent With Nutrition-Induced NIDDM-Like Syndrome

Psammomys obesus fed a high-calorie diet develops a NIDDM-like syndrome. The use of reverse-phase highperformance liquid chromatography (HPLC) to study Psammomys insulin biosynthesis and release revealed a very delayed elution time for the Psammomys insulin peak appearing near the position of human proinsulin. This unusual peak was initially thought to represent partially processed insulin on the basis of its molecular size and susceptibility to trimming by carboxypeptidase B (CpB). However, the findings of an active carboxypeptidase E (CpE) enzyme and the normal amidated forms of gastrin and cholecystokinin octapeptide (CCK-8) in Psammomys tissues were inconsistent with CpE-related aberrant processing of insulin. Moreover, amino acid sequencing of the delayed peak of Psammomys insulin revealed fully processed insulin with amino acid sequence as predicted by the cDNA. The unique presence of a B-30 phenylalanine residue, resulting in an increased hydrophobicity of the insulin molecule, probably underlies the marked delay in elution time on HPLC. The unusual structure of Psammomys insulin does not appear to contribute to the proinsulinemia observed in diabetic Psammomys since the HPLC-purified molecule did not inhibit PCI and PC2 convertase activities in an in vitro assay.

Approaches to Isolating Minor Membrane Components Using Monoclonal Antibodies, as in Purifying 5′-Nucleotidase

Isolation of a minor membrane protein requires optimal solubilization, then a protocol, with suitable affinity steps, that gives an active product in good purity and yield. Amphi-pathic detergents of diverse molecular structure are commonly used, aiming at “the smallest and least polydisperse active protein” [1] by criteria including non-sedimentability at high g forces and Polyacrylamide gel electrophoresis (PAGE) which also indicates the solubilized active protein’s molecular size. Immuno-affinity purification, very effective and of wide use because monoclonal antibodies can be prepared for a specific protein available only in partially purified form, entails immunization, production and purification of monoclonal antibody, preparation of immunoadsorbent, and adsorption and elution of antigen.