Development of an enzyme-linked immunosorbent assay, using a monoclonal antibody against alpha2-macroglobulin, for the diagnosis of systemic lupus erythematosus.

OBJECTIVES: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum alpha2-macroglobulin (alpha2-M) from patients with systemic lupus erythematosus (SLE). DESIGN AND METHODS: Serum alpha2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using alpha2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n = 14), SLE (n = 34), rheumatoid arthritis (n = 15), Sj?gren's syndrome (n = 11), mixed connective tissue diseases (n = 12), and liver diseases (n = 11) were analyzed. RESULTS: The affinity of the mab for alpha2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA. CONCLUSIONS: The ELISA was capable of recognizing changes of glycosylation of alpha2-M in SLE and may be useful for its differential diagnosis.     

A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein

A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.     

Measurement of human G-CSF by enzyme-linked immunosorbent assay using monoclonal antibody

An IgG monoclonal antibody to recombinant human granulocyte colony-stimulating factor (G-CSF), designated HG1, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. This HG1 was capable of neutralizing the colony-stimulating activity of G-CSF in vitro, and it did not cross-react with human granulocyte/macrophage colony-stimulating factor (GM-CSF). An enzyme-linked immunosorbent assay (ELISA) for measurement of G-CSF was developed using HG1 and a polyclonal antibody against G-CSF raised in a rabbit. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml of recombinant G-CSF. This assay system therefore warrants further attention.     

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.     

Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.     

Polyclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein

This is a noncompetitive enzyme-linked immunosorbent assay for measuring low concentrations (2 to 100 micrograms/L) of human alpha 1-acid glycoprotein (AGP; orosomucoid). The method is based on a simple "sandwich" technique involving polyclonal rabbit antisera against AGP. Mean within-run and total (between-run) CVs were 6.2% and 9.7%, respectively. Analytical recovery, tested in various biological fluids, averaged 101%. The technique has been successfully applied to diluted biological fluids such as bronchoalveolar lavage, cerebrospinal and amniotic fluids, and hepatocyte-culture supernates. Because of its analytical validity and the commercial availability of the reagents, this assay is suitable for large-scale determinations of AGP concentrations in those biological fluids in which its concentration is relatively low.     

Procedure for development of an enzyme-linked immunosorbent assay. Development of an assay for human apolipoprotein A-I.

A set of criteria for selection of antibodies during the development of enzyme-linked immunosorbent assay (ELISA) is described. Using these criteria, a competitive ELISA for human apo A-I using a polyclonal goat antibody was developed. The assay recognizes apo A-I from plasma and high-density lipoprotein (HDL), as well as the pure delipidated apo A-I, equally. Intra- and inter-assay variations were 5.2% and 3.5%, respectively. Recovery rate, as determined by spiking a known quantity of pure delipidated apo A-I into a reference plasma, was determined to be 101.3%. The assay was validated by comparing the concentration of apo A-I in HDL with the dye elution method. The apo A-I ELISA to apo A-I dye elution ratio was 1.01. Apo A-I concentration in Centers for Disease Control reference material determined by this method was in agreement with the reported consensus value. Repeated freezing and thawing of the samples (three freeze-thaw cycles at -20 degrees C) as well as long-term freezing (up to 1 year at -70 degrees C) did not affect the concentration of apo A-I in the samples. The assay was applicable both to normolipidemic and dyslipidemic plasmas. No immunologic difference was noted when plasma from dyslipidemic subjects was assayed. A frequent problem of long-term storage is deamidation. The values found for apo A-I in a deamidated plasma were the same as those for the corresponding fresh plasma. Plasma apo A-I values were also positively correlated with that of HDL-cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of apolipoprotein A-I using a monoclonal antibody

A semi-automated competitive enzyme-linked immunosorbent assay for human plasma apolipoprotein (Apo) A-I has been developed which utilizes nondelipidated samples, microtiter plates, commercially available monoclonal antibodies and alkaline phosphatase conjugated second antibody. The working range of the assay is 5-100 ng of Apo A-I. The range of plasma concentrations for plasma Apo A-I was 1.21 +/- 0.34 g/l for a random sample of 40 healthy adults. Intra- and inter-assay coefficients of variation (CV) were 4 and 7%, respectively. There was a good correlation between this assay and a radial immunodiffusion assay (r = 0.96). The assay is suitable for measurement of apolipoprotein A-I in either normal or pathological plasma, lipoprotein density classes, and for cell biological and molecular biological investigations.     

Establishment of an immunoglobulin A-deficient blood donor registry with a simple in-house screening enzyme-linked immunosorbent assay

BACKGROUND: Transfusion of blood products to immunoglobulin A (IgA)-deficient patients who have developed IgA antibodies can result in serious adverse reactions. To prepare compatible blood components for these patients, blood centers usually maintain a list of IgA-deficient blood donors. An in-house enzyme-linked immunosorbent assay (ELISA) was used to identify new IgA-deficient blood donors. STUDY DESIGN AND METHODS: An in-house ELISA was used to screen blood samples. IgA-deficient samples, defined as an IgA level below 0.05 mg per dL, were sent to the American Red Cross for confirmatory testing. RESULTS: Seventy-three confirmed IgA-deficient blood donors were identified among 38,759 screened blood donor samples (frequency, 1:531). IgA antibodies were found in 39 of these 73 blood donors (53%), although only 9 donors had a history of adult IgA exposure (transfusion or pregnancy). CONCLUSIONS: With a simple in-house ELISA, 73 blood donors were identified as IgA-deficient. From this number, 34 donors, without detectable anti-IgA in their plasma, were added to our IgA-deficient blood donor panel to maximize the management of our inventory of IgA-deficient frozen blood components.     

Alpha2-plasmin inhibitor and alpha2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibody immunosorbent assay.

An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by the therapeutic infusion of urokinase was similar to that found for urokinase-activated plasma. 23 normal individuals had low levels of alpha2-plasmin inhibitor-plasmin complexes, whereas six patients with laboratory evidence for disseminated intravascular coagulation demonstrated a 16- to 35-fold increase in he concentration of these complexes. These data indicated that a useful new probe for the study of the fibrinolytic enzyme system had been developed.     

A solid-phase enzyme-linked immunosorbent assay for the quantitation of human plasma alpha 1-acid glycoprotein.

We describe a solid-phase enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in human plasma. Plasma samples are incubated with alkaline phosphatase-linked, purified alpha 1-acid glycoprotein in alpha 1-acid glycoprotein-specific antibody-coated polystyrene tubes. The alkaline phosphatase that becomes attached to the tube via an immunological reaction between the alpha 1-acid glycoprotein and the specific antibody is measured spectrophotometrically. This assay is accurate reproducible, simple, and economical. As little as 4 microgram of alpha 1-acid glycoprotein per liter can be detected. The normal range for alpha 1-acid glycoprotein in the plasma of healthy adults, as measured by this method, is 0.48-1.27 g/L; the range is significantly different, 0.29-0.73 g/L, for women who are taking oral contraceptive pills.     

Enzyme-linked immunosorbent assay for amitriptyline and other antidepressants using a monoclonal antibody

We describe and evaluate a method to measure amitriptyline and other tricyclic antidepressants by enzyme linked immunosorbent assay, using monoclonal antibody. In this assay, biological samples were first incubated with the antibody; in a second step, free remaining antibody was allowed to bind to lysozyme-nortriptyline coated immunotitration plates. The bound fraction of the monoclonal antibody was revealed with rabbit anti-mouse serum coupled to horseradish peroxidase. The optical density of the reaction product was measured with a colorimeter at 410 nm. Specificity of the antibody was investigated by means of a Farr test showing interferences in therapeutic ranges only for chlorpromazine and phenytoine. Means of intra- and inter-assay variations were 10 and 13%, respectively. The results when compared to those obtained by gas chromatography with a selective nitrogen detector gave a correlation coefficient of 0.897. Finally, the great reliability of the monoclonal antibody, the advantages of a decreased analysis time, low cost and high capacity of the procedure contribute to make this immunoassay most suitable for clinical monitoring and pharmacokinetic studies of tricyclic antidepressants.     

A competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum and urine samples

In this solid-phase competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum or urine, antiserum to human alpha 1-acid glycoprotein is incubated with solid-phase-bound alpha 1-acid glycoprotein in the presence of standard or sample. Incubation with second antibody labeled with alkaline phosphatase then follows, before development with substrate. Results obtained correlate well with a fluorescent assay involving the dye Auramine O (r = 0.953) and with radial immunodiffusion (r = 0.921). The present assay covers the range 0.2 to 5 mg/L and 16 samples take 2.5 h to complete. This assay is useful for measuring concentrations of alpha 1-acid glycoprotein in serum and also in urine, for which other assay methods are not sufficiently sensitive.     

Evaluation of an enzyme-linked immunosorbent assay for prostatic acid phosphatase

An enzyme-linked immunosorbent assay (ELISA) was developed to measure prostatic acid phosphatase in human sera. The value of this method was tested on four groups: patients with non-prostatic disease, with prostatic hypertrophy, and with untreated and treated prostatic carcinoma. The results of ELISA were also compared with those of enzyme immunoassay (EIA) and the conventional, enzymatic method. The specificity of ELISA as calculated from the hypertrophy group, was 76% against 68% for EIA and 71% for the conventional method. The sensitivity of ELISA, calculated from the untreated carcinoma group, was 57% against 60% for EIA and 70% for the conventional method. ELISA did not prove better than EIA or the conventional method in quantifying prostatic acid phosphatase in patients' sera.     

Characterization of an enzyme-linked immunosorbent assay for soluble CD163

We have recently identified a soluble plasma form of CD163 sCD163, the macrophage receptor for clearance of haptoglobin-haemoglobin complexes, and we have observed highly elevated levels of sCD163 in subgroups of haematological patients. In the present study, we describe the optimization and characterization of a sandwich ELISA for the determination of the concentration of sCD163 in plasma and serum. The optimal concentrations of antibodies were determined systematically and the assay was calibrated by CD163 purified from human spleen membranes. The minimum detection limit was below 6.25 microg/L. Recovery of CD163 added to plasma samples was 106%. The concentration of sCD163 in paired serum and plasma samples correlated well (r2=0.99); however, serum levels were 1.1 times higher than the plasma levels. The addition of haptoglobin-haemoglobin complexes did not influence the assay. A very high stability of sCD163 was measured in whole blood and in plasma subjected to different temperatures and after repeated freezing and thawing.     

Development of an enzyme-linked immunosorbent assay of apolipoprotein E-AII complex in plasma

Measurement of apolipoprotein (apo) E-AII complex in human plasma is important in determining the role of apoE in lipoprotein metabolism. In this paper, we demonstrate a new and simple method to determine apoE-All complex by using an enzyme-linked immunosorbent assay. Anti-apoE IgG (goat) was used as a capture antibody, and captured apoE-All complexes were detected by an anti-apoAll (rabbit) horseradish peroxidase-conjugated anti-rabbit IgG (goat) system. With this method, apoE-All complex was specifically determined without the interference of apoAll and was not affected by apoE monomer less than 250 mg/L. The content of the complex in reference serum, a normolipidemic serum pooled from five subjects with phenotype E3/E3, was arbitrarily defined as 100%. The coefficients of variation were 3.5%-6.3% within assay and 8.8%-11.6% between assays.