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Biomedical subjects

B Porstmann

Publications and source records attributed to B Porstmann.

At least 37 records · Page 2Linked to original sources
PubMed1985

Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase?

Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying chromogens the optimum temperature for beta Gal is 37 degrees C and depends for HRP and AP on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes--both related to mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too--especially for beta Gal-IgG by a factor of 333 compared to the colorimetric procedure. In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes. The HRP-IgG conjugates warranted lowest detection limits for AFP (0.5-1 microgram/1), highest analytical sensitivity (slope of standard curves) at shortest periods of substrate reaction compared to the other enzymes.

Alkaline Phosphatase↗
PubMed1985

Stabilization of the substrate reaction of horseradish peroxidase with o-phenylenediamine in the enzyme immunoassay.

When the horseradish peroxidase reaction is stopped with acid, the decay of unconverted hydrogen peroxide is responsible for the further oxidation of o-phenylenediamine. This leads to a time-dependent flattening of the standard curve in the enzyme immunoassay, after the reaction has been stopped. Addition of reducing agents, such as sulphite ions, to the stopping solution, prevents the further oxidation of o-phenylenediamine by completely reducing the remaining hydrogen peroxide. The developed colour is then stabilized.

Chemical Phenomena↗
PubMed1985

[Enzyme immunoassay for pregnancy-specific beta 1-glycoprotein (SP-1) in patients with testicular tumors].

The use of a two-side-binding-enzyme-immunoassay for pregnancy-specific beta 1-glycoprotein (SP-1) in tumours of the testicles is described. The lower limit of evidence is with 4 ng/ml near to the physiological region. In 17 of 41 non-seminomatous germinal tumours of the testicles (41%) initially increased SP-1-serum concentrations were present, the other measuring values correlated with the course of the disease. In all tumours of the testicles with initially increased SP-1-titres the use of the SP-1-test gives a further possibility of the regulation of therapy and control of the course.

Adolescent↗
PubMed1984

Characterization by Scatchard plots of monoclonal antibody enzyme conjugates directed against alpha-1-foetoprotein.

A radioimmunoassay was used to investigate the affinity of 2 monoclonal antibodies against human alpha-1-foetoprotein before and after conjugation with horseradish peroxidase. The equilibrium constant of the high-affinity antibody was reduced 10-fold whereas it remained unaffected in the low-affinity antibody. With the Scatchard diagram, quantification of unlabelled antibodies in the unpurified conjugate mixture is also possible if antibody affinity is changed by the coupling procedure.

Antibodies, Monoclonal↗
PubMed1984

An antibody chimera technique applied to enzyme immunoassay for human alpha-1-fetoprotein with monoclonal and polyclonal antibodies.

A 2-step enzyme immunoassay (EIA) for human alpha-1-fetoprotein (AFP) is proposed, which uses covalently coupled anti-AFP IgG and anti-horseradish peroxidase (HRP) IgG (antibody chimera) binding HRP as the marker enzyme immunologically. The use of polyclonal and monoclonal anti-AFP linked to anti-HRP antibodies was compared with a conventional 2-site binding EIA with HRP covalently bound to anti-AFP IgG. The sensitivity of the conventional EIA is increased by the use of an antibody chimera comprising a molar ratio of anti-AFP IgG: anti-HRP IgG of 1:8, especially if monoclonal antibodies are employed. This improved sensitivity may be achieved by a very simple coupling procedure without purification of conjugate and with very crude HRP preparations.

Animals↗
PubMed1984

Application of antibody chimera in enzyme and erythro immunoassay.

Covalent linkage of antibodies directed against marker substances and antigen specific antibodies resulted in antibody chimera. Their usefulness was proved in an enzyme and erythro immunoassay for alpha-1-fetoprotein. Whereas the enzyme immunoassay showed the same sensitivity, precision and practicability when compared with assays using covalently linked enzyme antibody conjugates, the antibody chimera technique enables the application of crude enzyme preparations. The erythro immunoassay is well suited as screening procedure and allows also the quantitation of the antigen determining the pseudo peroxidase activity of bound erythrocytes.

Antibodies↗
PubMed1984

[Determination of alpha-1-fetoprotein in maternal serum dried on filter paper by an enzyme immunoassay].

The suitability of different carrier materials for absorption of serum was investigated with respect to a centralised screening for AFP serum levels in pregnant women, which needs a transmission of the samples. AFP was quantified in an indirect two-site binding enzyme immunoassay. AFP concentrations of native serum and of the eluate from the samples dried on filter paper were in a good agreement with a coefficient of correlation of 0.902. But storage of dried serum samples should not exceed one week because a two week storage diminished the coefficient of correlation to 0.766 and AFP concentrations of paper dried samples were overestimated.

Female↗
PubMed1983

Comparison of monoclonal and polyclonal antibodies in a two-site binding enzyme immunoassay for alphafetoprotein (AFP).

A two-site binding enzyme immunoassay for the detection of alphafetoprotein (AFP) was developed by using either a combination of two monoclonal antibodies or of one monoclonal antibody and polyclonal antibodies. The conjugation of the monoclonal antibodies to peroxidase by the periodate method yielded a somewhat higher sensitivity in the enzyme immunoassay (EIA), when compared to conjugates produced by the glutaraldehyde method. The detection limit was 10 micrograms AFP per litre when using only monoclonal antibodies in the assay. Simultaneous incubation of the sample and the monoclonal labelled antibody should be avoided unless using at least two different dilutions of the sample for investigation.

Animals↗
PubMed1983

Application of the peroxidase anti-peroxidase system as an universal reagent for the two-site binding enzyme immunoassay.

An indirect two-site binding enzyme immunoassay (EIA) is described, in which horse-radish peroxidase (HRP) is bound immunologically to anti-HRP IgG in the form of peroxidase anti-peroxidase complexes (PAP-complex). In this EIA both purified HRP and crude HRP had the same sensitivity due to the selective reaction of the monospecific anti-HRP IgG with the highly active HRP isoenzymes in both preparations. To obtain similar results the amount of crude HRP needed is 18 times higher than that of purified HRP. A comparison of differently composed PAP-complexes showed that only those formed in an excess of HRP yielded a highly sensitive EIA. Urea splitting of the PAP-complexes did not raise the specific activity of the enzyme. The PAP-complexes were used in an assay for quantification of the pregnancy-associated alpha 2-glycoprotein and compared with an indirect two-site binding EIA, in which purified HRP was covalently bound to IgG. Both test variants resulted in the same sensitivity and showed similar precision.

Animals↗
PubMed1983

[Enzyme immunoassay of alpha 1-fetoproteins in patients with testicular tumors].

The use of a newly developed immunoassay for alpha-1-protein in testicular tumours is described. With 2 ng AFP/ml serum the lower detection group of the test is in the physiological range. Of 39 non-seminomatous testicular tumours, 59% had a high AFP-serum level post-operatively, of 51 on initial diagnosis 54%; 12 seminomas remained AFP-negative. Checking the AFP level ist valuable for diagnosis, staging and prognosis of all testicular tumours.

Adult↗
PubMed1983

[Demonstration of circulating immune complexes using a C1q-solid phase enzyme immunoassay].

An enzyme-immunoassay for the quantitative determination of circulating immune complexes in human serum is described. The technique uses alkaline phosphatase-conjugated anti-human IgG to detect immune complexes bound to solid phase C1q. This assay is sensitive detecting 4 ng/ml to 60 micrograms/ml aggregated IgG. First data from normal individuals and patients with systemic lupus erythematosus show that this method is specific and sensitive for detection of circulating immune complexes.

Antigen-Antibody Complex↗
PubMed1982

Comparison of direct and indirect two-site binding enzyme immunoassay.

Rabbit IgG, directed against HBsAg, was purified by positive and by negative affinity chromatography and applied in horseradish peroxidase labelled as well as in unlabelled form in the direct and indirect two-site binding enzyme immunoassay (EIA). Comparing direct and indirect assay the latter is more sensitive and less conjugate consuming. In contrast to the indirect assay in which antibodies, purified by positive affinity chromatography, do not alter detection limit, a 4- to 8-fold higher sensitivity was achieved in the direct EIA in contrast to antibodies, purified by negative affinity chromatography. In the indirect EIA unlabelled second and labelled third antibodies were incubated successively as well as simultaneously. The latter procedure shortened the assay time but needed antibodies purified by positive affinity chromatography and a 10-fold higher conjugate concentration. Greatest sensitivity was obtained in the indirect EIA by the use of labelled second and labelled third antibodies (20-30 ng/l HBsAg).

Chromatography, Affinity↗