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.

Two-colour combination enzyme-linked immunosorbent assay for the simultaneous detection of HBV and HIV infection.

To reduce the cost, time and waste in screening for HIV and HBV infections a combined assay for HIV-1 and -2 antibodies and HBsAg has been developed. Monoclonal anti-HBs antibodies were co-immobilized with synthetic peptides representing immunodominant regions of HIV-1 and -2. The presence of anti-HIV antibodies in the samples was detected with alkaline phosphatase-labelled anti-human IgG and of HBsAg with horseradish peroxidase-labelled monoclonal anti-HBs antibodies by a sequential substrate reaction. In this assay, HBsAg was detectable in a concentration range between 0.25 and 0.30 U/ml and the results were available within 3 h. The specificity, tested on 5000 serum samples from blood donors after confirmation, was 99.8% for HBsAg and 99.5% for anti-HIV antibody detection. All serum samples taken from 600 HIV-1- and 115 HIV-2-infected individuals were correctly classified as reactive. The two-colour HBsAg-anti-HIV-1/-2 combination ELISA meets all the requirements of single parameter assays with regard to precision, stability and robustness.

Creatine kinase isoenzyme BB in serum of healthy adults and children.

The activity of the creatine kinase isoenzyme BB was determined in the serum of 26 healthy adults and 31 children. The isoenzyme BB could be proved as a normal component in the human serum. In the adults examined, an activity of 0.56 +/- 0.16 I.U./l (x +/- S.D.) was determined. The activity of creatine kinase isoenzyme BB in the serum does not depend on sex but is subject, however, to a strong age dependence. Only at an age of more than 18 years, isoenzyme BB activities adjust to those of adults.

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).

Temperature dependent rise in activity of horseradish peroxidase caused by non-ionic detergents and its use in enzyme-immunoassay.

Non-ionic detergents such as polyoxyethylene-octylphenol or -sorbitolester were found to increase activity of horseradish peroxidase due to delay of inactivation in the course of substrate reaction. This rise in activity was investigated using different chromogens and was highest with o-dianisidine. An increasing stability of the enzyme to higher reaction temperatures was observed when detergents were added to the substrate solution, and the action of detergents also is enhanced with increasing reaction temperature and time. Different degrees of activation were found when comparing substrate conversion with and without detergents using free peroxidase (2.7-fold) and conjugated peroxidase bound to the solid phase by antigen antibody reaction (1.8-fold). In enzyme-immunoassay, detection limit and analytical sensitivity can be doubled.

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.

Abnormalities in the composition of serum high density lipoprotein in renal transplant recipients.

Levels of HDL-cholesterol, HDL-triglyceride and apolipoprotein A were determined in the sera of 26 female and 44 male renal transplant recipients. In comparison with controls, no significant abnormalities in HDL-cholesterol values could be found, but apolipoprotein A and HDL-triglyceride concentrations, were increased significantly. The increased apolipoprotein A/HDL-cholesterol ratio and the decreased apolipoprotein A/HDL-triglyceride and HDL-cholesterol/HDL-triglyceride ratios in these patients demonstrate abnormalities in HDL-composition. These results permit the conclusion that, in renal transplant recipients, the levels of HDL-cholesterol and particularly the changes in HDL-composition should be taken into account when considering the reason for the high incidence of cardiovascular disease in these patients.

[Thromboelastography and inhibitors of aggregation]. / Thrombelastogramm und Aggregationshemmer

The effect of maintained treatment with salicylo-acetic acid (1.5 g/day) on the thrombelastogram was examined in the venous blood from 18 accidental patients. In the prevention of thrombembolia, resulting values of the thrombelastograph are not sufficient to show functional changes of platelets. Some of the patients revealed prolonged times for reaction and formation of coagula, thus discreet influencing the thrombelastogram might be supposed.

Comparison of chromogens for the determination of horseradish peroxidase as a marker in enzyme immunoassay.

o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), o-dianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene-sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of o-dianisidine.

Lecithin:cholesterol acyltransferase activity and HDL composition in serum of patients with kidney transplants.

In comparison with a control group, lecithin:cholesterol acyltransferase activities and HDL-cholesterol concentrations in serum of patients with kidney transplants were unchanged, while apolipoprotein A and HDL-triglyceride concentrations were increased. This constellation shows that the cholesterol metabolism in patients with kidney transplants differs from that of patients undergoing chronic haemodialysis.

[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.

Quantitative determination of CD4/CD8 molecules by a cell marker ELISA.

Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.

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.

Lecithin: cholesterol acyltransferase activity, HDL-cholesterol and apolipoprotein A in serum of patients undergoing chronic haemodialysis.

In comparison with a control group, significantly lower lecithin:cholesterol acyltransferase activities and HDL-cholesterol values were found in the serum of patients undergoing chronic haemodialysis, while apolipoprotein A concentration remained unchanged. Reduced activity of lecithin:cholesterol acyltransferase is an indication of the abnormal cholesterol metabolism in haemodialysed patients and could be, in addition to other factors, an explanation for the high incidence of cardiovascular diseases in this patient group.

Increased creatine kinase BB activity in rat plasma induced by hypoxia.

Rats show an elevated creatine kinase (CK) activity in plasma after a 20-hour exposure to hypoxia. The increase of CK activity is essentially caused by CK-BB activity attaining its maximum at the 3rd day after exposure to hypoxia. The possibility of establishing a potential brain damage by determining CK-BB activity in plasma is discussed.

Quantitation of a pregnancy associated globulin in the rat by two-site binding enzyme immunoassay.

An indirect two-site binding enzyme immunoassay has been developed to quantify the pregnancy associated globulin (PAG) in rats. Anti-PAG from guinea pig is adsorbed to solid phase and binds serum-PAG, which then reacts with its remaining free determinants with anti-PAG from rabbit. Sheep anti-rabbit IgG, labeled with horse radish peroxidase (HRP), binds to the rabbit anti-PAG antibody and the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The optical density is directly related to the quantity of PAG to be measured. Reproducible results are obtained in the range from 5 to 200 micrograms/l. The test detects 12 fmoles of PAG. Female rats show a 1000-fold higher serum level of PAG than male rats. Strain differences of the distribution of the PAG serum concentration were only found between male rats.

Effect of IgG-horseradish peroxidase conjugates purified on Con A-Sepharose upon sensitivity of enzyme immunoassay.

Antibodies coupled to horseradish peroxidase (HRP) were purified by gel filtration and by combination of affinity chromatography and gel filtration. Binding kinetics to solid phase bound antigens were studied of the antibody-enzyme conjugates in the presence and in the absence of unlabelled IgG. The detection limit in enzyme immunoassay was found to be lowered by factor 16 after elimination of unlabelled IgG from the conjugate.

The rat female protein, a pentraxin with lectinic properties.

The C-reactive protein is the major acute phase protein (APP) in humans which binds lectin-like to different membraneous structures and exerts an important function in non-specific defense. Because of a pentameric molecular symmetry CRP as well as serum amyloid P component (SAP) and hamster female protein (FP) was merged into a special protein family named pentraxins. In rats a protein was found referred to as rat FP which was close related to hamster FP with respect to hormonal regulation and APP nature as well. Based on this conformity the molecular structure of rat FP was analyzed and as the results a pentameric structure could be demonstrated for rat FP, too. Furthermore, the response of rat CRP and FP on injection of adrenal hormones, agents being involved in acute phase reaction, was investigated. Epinephrine administration led to an increase in CRP and a decrease in FP serum concentration. Dexamethasone has the same effect in case of FP and changed the CRP concentration in a biphasic way with a maximum at about 0.01 mg/kg, a minimum at 0.6 mg/kg and a return to control values at 1.8 mg/kg. Thus, the results indicate a neuroendocrine control of CRP and FP but probably in a different way. Using FITC-labelled lectin the exposition of galactose-containing membraneous structures could be demonstrated in carbon tetrachloride-injured liver tissue in contrast to controls. These binding sites are in accordance with increased FP-binding shown by immunofluorescence histochemistry. Thus, lectin-like properties may be ascribed to rat FP comparable to CRP and SAP activity. The results are discussed with respect to findings from literature that also the acetylcholine receptor seems to have a pentameric structure.

HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein.

Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.

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).