Monoclonal antibodies to human chorionic gonadotropin (HCG) and their use in two-site binding enzyme immunoassays.

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated beta-subunit (beta-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross-reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and beta-HCG, or exclusively with the non-associated beta-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated beta-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or beta-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or beta-HCG-producing tumors before and after therapy.

[Tetramethylbenzidine--a chromogenic substrate for peroxidase in enzyme immunoassay]. / Tetramethylbenzidin--ein chromogenes Substrat für Peroxidase im Enzymimmunoassay.

3,3' 5,5'-tetramethylbenzidine (TMB) is a high sensitive chromogenic substrate for horseradish-peroxidase as a marker enzyme. In an enzyme immunoassay (EIA) for alpha-1-fetoprotein and in an rapid EIA for myoglobin it reveals higher sensitivity compared to o-phenylenediamine, the increase depends on reaction time. The optimal peroxide concentration depends on reaction time of enzyme chosen in different assays. TMB used for histochemistry is also suitable for EIA if hydrochloric acid is used as stopping reagent. TMB lacks in mutagenic properties and it should preferred for peroxidase rather than all other chromogenic substrates applied up to now.

Development of an immunoenzymometric assay for alpha 1-microglobulin and measurement of its serum concentration in normal and HIV-infected persons.

alpha 1-Microglobulin was purified from urine to a purity of 97.7% in a yield of 25.8%, and was used to produce antibodies in sheep. These antibodies, purified by affinity chromatography, were used to develop a rapid one-step and a two-step immunoenzymometric assay (IEMA). The equilibrium in the reaction between solid phase-adsorbed antibodies and antigen and between the antigen and enzyme-labelled antibodies was attained within 30 and 100 min, respectively. The one-step IEMA permits a good differentiation of low alpha 1-microglobulin concentrations after 30 min reaction time. Its detection limit is 0.35 micrograms/l, and its measurement range is between 0.5 and 100 micrograms/l. The IEMA correlates well with radial immunodiffusion (r = 0.973). The mean alpha 1-microglobulin serum concentration in women is insignificantly lower (33.2 mg/l) than in men (36.1 mg/l). In both sexes the alpha 1-microglobulin concentration increases with age. HIV-infected symptomless men have a significantly lower (15.9 mg/l) alpha 1-microglobulin concentration in serum than normal persons, whereas in AIDS patients it is significantly higher (45.5 mg/l).

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

[Enzyme immunoassays for the quantification of human gamma-gamma-enolase (NSE)]. / Enzym-Immunoassays zur Quantifizierung humaner gamma-gamma-Enolase (NSE).

A direct two-site binding assay on the basis of antibodies from sheep for the quantification of human gamma-gamma enolase is described. The antibody was produced by immunization with human NSE coupled to horse spleen ferritin. The assay shows two feature: a decreased reactivity with NSE from rat and NSE from human serum in spite of 100% recovery of purified human brain NSE. The sheep antibody seems to react with epitopes less accessible on the rat NSE and on the NSE of human serum. The assay is characterized by gamma-gamma enolase specificity, a high sensitivity (2 pg) and a precision of CV = 3-7%.

Measurement of lysozyme in human body fluids: comparison of various enzyme immunoassay techniques and their diagnostic application.

Three variants of the immunoenzymometric assay of human lysozyme with HRP-labeled antibodies were compared. The highest sensitivity (with a detection limit of 0.2 micrograms lysozyme/L) was achieved by a one-step assay lasting 2 h. Between-batch precision for the techniques was 6-11%. Lysozyme reference values were determined in serum, cerebrospinal fluid and urine. In serum they are age-dependent and in urine sex-dependent when related to creatinine excretion. Serum lysozyme is increased in only 57% of the patients with active rheumatoid arthritis and is also unreliable for indicating remission. In Crohn's disease the serum lysozyme reflects activity better, but it does not exceed the diagnostic value of alpha-1-acidic glycoprotein (orosomucoid). The lysozyme quantification in cerebrospinal fluid is useful in distinguishing between viral or bacterial meningitis.

A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Down's syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

Coupling of different isoenzymes of horseradish peroxidase influences the sensitivity of enzyme immunoassay.

Labelling of IgG with various HRP isoenzymes purified by preparative isoelectric focussing influences the yield and the specific activity of the conjugates. Alkaline isoenzymes were preferably coupled by glutaraldehyde whereas application of the periodate method additionally formed relatively large quantities of conjugates with acidic isoenzymes of a high purity number, but a low specific activity. In an enzyme immunoassay for alpha fetoprotein the detection limit can be varied by a factor of 6 and even by a factor of 20 by use of the conjugates with different isoenzymes coupled by the glutaraldehyde method and the periodate method, respectively. In order to achieve enzyme immunoassays of the highest sensitivity, antibodies should be coupled to horseradish peroxidase after removing acidic isoenzymes from the enzyme preparations.

[Enzyme immunoassay for pregnancy-specific beta 1-glycoprotein (SP-1) in patients with testicular tumors]. / Enzymimmunoassay für schwangerschaftsspezifisches beta 1-Glycoprotein (SP-1) bei Patienten mit Hodentumoren.

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.

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.

[Determination of alpha-1-fetoprotein in maternal serum dried on filter paper by an enzyme immunoassay]. / Bestimmung von Alpha-1-Fetoprotein in auf Filterpapier angetrocknetem Schwangerenserum mit einem Enzymimmunoassay.

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.