Direct two-side binding enzyme immunoassay for human alpha-1-fetoprotein. Part 2: Investigation of criteria determining the quality of the assay parameters.
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Biomedical subjects
Publications and source records attributed to T Porstmann.
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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.
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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.
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.
Immunological cross-reaction and antigenic identity between pregnancy-associated alpha 2-glycoprotein (alpha 2PAG) and alpha 2PAG analogues in the mouse and the rat are demonstrated. The proteins have been characterized independently in 2 different laboratories and the physicochemical and biological properties of the rat and the mouse alpha 2PAG analogues and human alpha 2PAG are compared and discussed.
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.
A solid phase radioimmunoassay was developed for the epitope analysis of human alpha-fetoprotein (AFP) using hybridoma culture fluids. The following incubation sequence was performed: anti-mouse Ig, hybridoma culture fluid, normal mouse serum, and a mixture of 125I-labeled AFP and hybridoma culture fluid. Seven epitopes were detected by monoclonal antibodies produced by 15 presumably independent hybridoma clones.
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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.
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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.
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.
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.
A solid phase radioimmunoassay using the incubation sequence: mouse immunoglobulin (Ig) rabbit anti-mouse Ig, monoclonal antibody and 125I-labeled antigen was performed to determine the class and subclass of seven murine monoclonal antibodies against human alpha-fetoprotein. All antibodies belonged to the IgG class. The subclass type was IgG 2a for four antibodies, IgG 1 for two antibodies and IgG 2b for one antibody.
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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).