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I Rubin

Publications and source records attributed to I Rubin.

At least 37 records · Page 2Linked to original sources
PubMed1995

A reliable method to estimate the association constant for a monoclonal antibody and a protein antigen by enzyme linked immunosorbent assay, ELISA.

The association constant Ka for mouse monoclonal antibody raised against human angiotensinogen was calculated using a mathematical model, SAM I. K1 represents the equilibrium constant for the binding of antibody to the solid phase with antigen previously absorbed. K2 represents the interaction between antibody and antigen in solution (Ag + Ab = AgAb). K3 represents binding to the antigen absorbed on the solid phase by an antigen-antibody complex. K4 represents the second binding of the antigen to the antigen-antibody complex (AgAb + AgAb = (Ag)2Ab). The model unveils cooperativity for the first (K1 and K2) and second (K3 and K4) binding of antigen to antibody. The model gives the association constant in a high affinity interaction between antigen and antibody.

Angiotensinogen↗
PubMed1993

Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated nitrocellulose.

Nitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6-diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6-10 amino acid residues were dotted directly onto the matrix at 45 degrees C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase-coupled anti-rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin-layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1990

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.

Angiotensin I↗
PubMed1988

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Angiotensinogen↗
PubMed1983

A high molecular weight form of angiotensin I is generated in rat serum incubated with synthetic and native angiotensin I.

A high molecular weight form of angiotensin I, 'big angiotensin I' with m.w. 25000 was generated in rat serum incubated with angiotensin I (m.w. 1296). Angiotensin I and 'big angiotensin I' were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and extracted from the gels. Only sera with high concentrations of angiotensin I (2000-4300 ng per ml) incubated at neutral pH at 24 degrees C for several hours, contained 'big angiotensin I'. 'Big angiotensin I' was stable in sera, where angiotensin I was rapidly degraded. The proportion of 'big angiotensin I' to angiotensin I in sera, where angiotensin I was stable, was approx. 5%. In addition to 'big angiotensin I', which was generated during the incubations, a serum protein with m.w. 60000 was extracted from the SDS-PAGE. This protein had angiotensin I-like activity in the radioimmunoassay, and it was isolated from the unincubated sera in concentrations at the detection limit of the radioimmunoassay.

Angiotensin I↗
PubMed1983

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.

Angiotensin I↗
PubMed1982

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.

Angiotensin I↗
PubMed1982

Alteration of protein synthesis and induction of specific protein phosphorylation by hyperthermia.

Confluent cultures of the mouse cell line clone 1D were subjected to 1-hr hyperthermic treatments. Temperatures were increased from the control level of 37 degrees to values ranging from 38 to 45 degrees. Protein synthesis patterns were determined in fluorograms of sodium dodecyl sulfate-polyacrylamide gels labeled with [3H]leucine. Although incorporation into most proteins was either repressed or decreased by the treatment, several proteins showed an increased label of were apparently induced de novo. Among the induced proteins was a prominent band, probably a doublet, with an estimated molecular weight of 70,000 to 69,000. Crude cell lysates made from 37 degrees, 41 degrees, and 45 degrees-treated cells were tested for kinase activity at 30 degrees by a 10-min incubation with adenosine [gamma-32P]triphosphate. Several specific proteins exhibited increased phosphorylation, while phosphorylation of other proteins decreased. The most significant increase in phosphorylation was shown by a protein with molecular weight of about 37,000. We suggest that heat treatment induces or activates one or more specific phosphokinase(s) with the ability to phosphorylate proteins with approximate molecular weights of 37,000, 36,000, 23,000, and 16,000.

Animals↗
PubMed1980

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.

Animals↗
PubMed1976

A comparison of the properties of renin isolated from pig and rat kidney.

1. On isoelectric focusing, renin from rat kidneys showed three activity peaks with pI values at pH 5.0, 5.2 and 5.4 after a purification procedure involving differential centrifugation, acidification, chromatography on Sephadex G-75 and dialysis. 2. The preparation (purified 140-fold) was compared with a crude kidney extract in the absence and presence of 3 M-urea by isoelectric focusing. The pattern of activity distribution was confirmed by these experiments and the content of isoenzymes in the three groups calculated. 3. Pig renin was prepared and compared with rat renin with regard to molecular weight, acid activation, behaviour on isoelectric focusing, immunogenicity and substrate affinity. 4. Extracts of rat kidney contained multiple forms of renin with mol.wt. between 39000and 42000, whereas active pig renin had an approximate mol.wt. of 40000. Acidification of rat renal extracts did not increase the activity of renin, indicating the absence of an inactive form of renin in rat kidneys, whereas pig renin was activated by this procedure. Pig renin has isoelectric points at pH 4.6, 4.8, 5.05 and 5.2, significantly lower than for rat renin. The isoenzymes from the two species had no antigenicity in common, as shown by crossed immunoelectrophoresis or rocket immunoelectrophoresis. 5. The Michaelis constants for pig and rat renin were in the same range, 1 X 10(-6) M, when rat renin substrate was used. The relative content of rat isoenzyme with pI in the pH ranges 4.9-5.1, 5.1-5.3 and 5.3-5.5 was approx. 20, 27 and 53% respectively. Purified pig renin prepared in two different ways had isoenzymes with pI in the pH regions 4.5-4.7, 4.7-4.9, 4.9-5.05 and 5.05-5.20 in the approximate proportions 14, 24, 28 and 29%.

Animals↗