Edgar Lauritzen

Covalently linked peptides for enzyme-linked immunosorbent assay

A general method is described, by which synthetic peptides are covalently linked via their carboxyl group to microtiter plates (CovaLink) for enzyme-linked immunosorbent assay (ELISA). Plates were prepared by this method with an angiotensin II peptide and with an HIV-2 peptide and attachment detected by rabbit anti-angiotensin serum and with a positive serum from an HIV-2-infected patient, respectively, using the common ELISA procedure in the last steps. The method is simple to perform, it constitutes an alternative to the common ELISA method, and eliminates the risk of inadvertent loss of peptide during the procedure. The method is highly reproducible and has a high sensitivity. It may be used for either antigen or antibody detection.

Dot immunobinding and immunoblotting of picogram and nanogram quantities of small peptides on activated nitrocellulose

Nitrocellulose was activated with divinyl sulfone, a spacer of ethylenediamine, and glutaraldehyde. The aldehyde groups on the activated nitrocellulose, Nit-CHO, were stable through one month at 4 degrees C. Peptides were attached to the membrane by reaction of the amino group with the free carbonyl, forming peptide bonds. The decapeptide angiotensin I (AI), the octapeptide angiotensin II (AII), angiotensin analogues, Met- and Leu-enkephalin (Met-E and Leu-E) were tested on the membranes with specific rabbit antibodies (sRaAb) against the peptides, and visualized by horseradish peroxidase conjugated anti-rabbit antibody (HRP-anti-RaAb). With this technique AII could be detected with a sensitivity of 20 pg/cm2 and AI by 500 pg/cm2. Substitution of Ala7 for Pro7 in AI and AII caused a marked reduced binding of anti-AI and antid-AII antisera, respectively, and it completely abolished crossreactivity of anti-AI with Ala7-AII as well as anti-AII with Ala7-AI. Peptides from the gp41 and gp36 antigens corresponding to the sequence aa596-618 of the human immunodeficiency viruses type 1 and 2, HIV-1 and HIV-2, were tested on Nit-CHO with two human sera from infected patients. The serological reactions were specific for both the HIV-1 and HIV-2 peptide, respectively. This indicated that the technique could be exploited for serological testing of humans. Separation of peptides by high performance thin layer chromatography (HPTLC) and identification by immunoblotting was demonstrated with angiotensin analogues. After separation by HPTLC on silica aluminium plates the peptides were electrotransfered by semidry electroblotting on Nit-CHO, followed by specific antibody overlays and developed as for the dot immunobinding technique. This combined method enabled us to differentiate between closely related peptide analogues and it improved the sensitivity of peptide detection 100-1000 fold as compared to visualization by quenched fluorescence on chromatography plates.

Studies on the native forms of renin in the rat kidney

This paper describes Sephadex G-100 chromatography of rat kidney extract containing various enzyme inhibitors. The high molecular weight renin (molecular weight above 50 000) constitutes about 50% of the total renin activity. Omission of the enzyme inhibitors yield solely low molecular weight renin. Upon rechromatography high molecular weight renin eluted in two peaks at lower molecular weight with a concomitant reduction of renin activity. Renin activity in the fractions from Sephadex G-100 chromatography was increased 70% by dialysis at acid as well as neutral pH through the whole molecular weight range. Cold storage of extract with low molecular weight increased renin activity about 25%. The results suggest that the fully active enzyme is not represented by the lower molecular weight forms of renin and direct connection between activation of renin and reduction of renin molecular size was not indicated.

DOT IMMUNOBINDING (DIB), ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA), AND RADIOIMMUNOASSAY (RIA) FOR DETECTING PEPTIDE ANTIGENS AND SPECIFIC ANTIBODIES

Publisher Summary This chapter reviews the techniques of dot immunobinding (DIB), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA), which are used for detecting peptide antigens and specific antibodies. These techniques allow for detection of as little as 10−18 M of specific antigens. Immunochemical assays are defined by specific binding of an antigen to the corresponding antibody and involve a signal substance that reports the reaction. This signal can be produced by an enzyme-catalyzed color reaction, which is used in the DIB and ELISA techniques, or by a radioactively labeled antigen, as presented in the RIA. The DIB of proteins on nitrocellulose membranes and polyvinylidenedifluoride (PVDF) membranes has been used for the studies of specific protein antigens. ELISA is developed from immunohistochemistry by the application of the same antibody overlay principles used for the detection of antigens in situ and is used in the laboratory detection of antibodies against rubella virus and other viruses. RIA is a technique that traditionally has been used for the detection of trace amounts—in the nanomolar and picomolar range—of peptides and other hormones in plasma and other biological fluids and extracts and is extensively used in medical clinics to determine the concentration of circulating hormones.

An angiotensin I-protein complex isolated by SDS-polyacrylamide gel electrophoresis from rat serum incubated with angiotensin I

Angiotensin I (AI) protein complex with Mr 25,000 and an angiotensin I immunoreactive protein with Mr 60,000 were isolated from rat serum. The proteins were separated from AI by sodiumdodecylsulphate polyacrylamide gel electrophoresis under denaturing and reducing conditions using SDS and 2-mercaptoethanol (SDS-PAGE). The Mr 60,000 protein was isolated from non-incubated serum. Special attention was given to the Mr 25,000 AI-protein complex, called AI-25,000, which was generated in rat serum incubated with both synthetic and native AI for 1-5 h at 24 degrees C and neutral pH. The AI-25,000 was determined in two radioimmunoassays using two anti-AI antisera and 125I-AI. The experiments showed that AI reacts with a serum protein and forms a stable complex with a high molecular mass.

Enzyme Inhibitors are not Helpful in Preserving Big Renin in the Rat Kidney

The effect of four different homogenization methods, proteolytic enzymes and enzyme inhibitors on rat renal renin was studied. Rat kidney homogenate was treated with trypsin or pepsin. Neither of these enzymes had any effect on the molecular weight pattern. Enzyme inhibitors, 10 mM N-ethylmaleimide, 5.4 mM EDTA, 1.0 mM toluenesulfonyl fluoride, 2.3 mM o-phenanthroline, 2.3 mM p-hydroxymercuribenzoate, which was added to the homogenization medium, all or some of them, as well as 1/20 to the buffer solution used throughout the experiments for dialysis and column chromatography, did not alter the molecular weight pattern. These results suggest that renins, with molecular weights from above 60 000 Dalton to 37 000 Dalton were preformed in the rat kidney and extracted in a native form.