Enzyme immunoassay for the quantitation of immunoglobulin M class antibodies to Salmonella minnesota R595 and Escherichia coli J5 lipopolysaccharides

The level of human immunoglobulin (IgM) in plasma specific for the lipopolysaccharide of Salmonella minnesota R595 (R595 LPS) and Escherichia coli J5 (J5 LPS) was quantitated by an enzyme immunoassay (EIA) in which purified antigen is adsorbed directly onto polystyrene-acrylic copolymer cuvettes. Highly purified anti-J5 and R595 LPS specific IgM prepared by ion-exchange resin, gel filtration, and affinity resin chromatography were used as standards. The levels of specific IgM were determined in 200 plasma samples obtained from normal donors. Anti-R595 IgM levels varied from less than 30 micrograms/ml (91%), from 30 to 100 micrograms/ml (8.5%), and greater than 100 micrograms/ml (0.5%). Anti-J5 IgM levels in 68% of the donor plasmas were less than or equal to 5 micrograms/ml. The levels in 30.5% of the donor plasmas ranged from 6 to 100 micrograms/ml; the remaining 1.5% had greater than 100 micrograms/ml anti-J5 IgM. Specific IgM levels in four lots of normal pooled plasma each consisting of about 10,000 L averaged 12.7 micrograms/ml and 13.3 micrograms/ml for R595 and J5, respectively. The assay was modified to quantitate rabbit plasma as well. For this purpose, the EIA has been performed on microtiter plates, and the core LPS was fixed onto the wells by chemical treatment with glutaraldehyde which results in higher stability and retention of the antigen in the wells. Specificity of the EIA was demonstrated by the absence of significant cross reactivity between R595 IgM and J5 LPS and between J5 IgM and R595 LPS, furthermore, we only observed partial adsorption (approximately 25%-33%) of the R595 and J5 IgM by Pseudomonas aeruginosa LPS, a wild type endotoxin. The described quantitative assay is useful for both scientific studies and clinical investigations.     

A sensitive sandwich enzyme immunoassay for human myoglobin using Fab'-horseradish peroxidase conjugate: methods and results in normal subjects and patients with various diseases

A sensitive sandwich enzyme immunoassay (EIA) for human myoglobin (Mb) was developed. Serum Mb was assayed by incubation with an anti-Mb rabbit IgG-coated polystyrene ball and then with affinity-purified anti-Mb Fab'-horseradish peroxidase (HRP) conjugate. The HRP activity bound to the polystyrene ball was assayed fluorimetrically. The sensitivity was 3.1 pg/tube and serum Mb levels of 0.4-1000 micrograms/l could be determined. The recoveries of Mb added to human sera at 3 concentrations were 92.8-99.8%. The within- and between-assay coefficients of variations (CV values) were both below 10%. The regression equation of values determined by EIA and radioimmunoassay (RIA) was y(EIA) = 0.964x (RIA)--2.30 and the correlation coefficient was 0.984. The mean normal serum concentration of Mb was 21.5 +/- 6.0 micrograms/l (mean +/- SD) in males and 16.9 +/- 5.8 micrograms/l in females. The significance of serum Mb determination by the EIA in various diseases was confirmed. The present EIA for Mb is more sensitive and economical than RIA and should be useful for determining Mb in biological materials.     

Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants

Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG(4)lambda) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity.The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.     

A sensitive and specific sandwich enzyme immunoassay for human thyroid-stimulating hormone

A sensitive and specific sandwich enzyme immunoassay (EIA) for human thyroid-stimulating hormone (hTSH) has been developed. hTSH is incubated with anti-hTSH IgG-coated polystyrene balls, and after washing they are further incubated with anti-hTSH Fab'-β-d-galactosidase conjugate. The β-d-galactosidase activity bound to the polystyrene balls is proportional to the amount of hTSH to be assayed. Polystyrene balls are coated with rabbit anti-hTSH IgG which had been affinity-purified and treated with human chorionic gonadotropin-Sepharose 4B to remove antibodies cross-reacting with structurally related hormones. Rabbit anti-hTSH Fab', which had been affinity-purified was conjugated with β-d-galactosidase from Escherichia coli using N,N′-o-phenylenedimaleimide.In the specific sandwich enzyme immunoassay developed, 1 nU (1 × 10^?9 U) of hTSH per tube can be measured and the sensitivity for serum hTSH level is 0.1 μU/ml when 10 μl of serum is used. No significant interference was observed in the presence of 1.3 mU hLH/tube, 0.5 mU hFSH/tube or 0.5 U hCG/tube. Recoveries of hTSH added to human sera were 95.3–104% with a standard deviation of 12.0–14.9%. The coefficients of within-assay and between-assay variations were 6.0–7.5% and 4.9–8.7%, respectively. The regression equation and coefficient for correlation to radioimmunoassay (RIA) were y (RIA) = 0.95x (EIA) + 3.2 and 0.97, respectively.Serum levels of hTSH in normal male and female adults were 2.4 ± 1.0 (SD) (n = 41) and 2.9 ± 1.3 (n = 46) μU/ml, respectively; those in hyperthyroidism and hypothyroidism were 0.28 ± 0.06 (n = 20) and 49.6 ± 24.7 (n = 22) μU/ml, respectively; and those in pregnant and postmenopausal women were 2.5 ± 1.2 (n = 7) and 2.7 ± 1.0 (n = 35) μU/ml, respectively, indicating that high serum levels of hCG or hLH and hFSH under these conditions did not significantly interfere with the present assay of hTSH at normal levels.     

Novel enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay) for anti-thyroglobulin IgG in human serum

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in human serum is described. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. After eliminating nonspecific IgG in test serum by washing, the complex was eluted from the polystyrene ball with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG 7-chain) Fab'-horse-radish peroxidase conjugate. The present enzyme immunoassay was 1,000–3,000-fold more sensitive than the conventional enzyme immunoassay, in which a thyroglobulin-coated polystyrene ball was incubated with serum containing anti-thyroglobulin IgG and subsequently with rabbit (anti-human IgG 7–chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in all patients with Graves' disease and chronic thyroiditis.     

Measurement of anti-thyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay

Anti-thyroglobulin IgG in human serum was measured by a novel and sensitive immune complex transfer enzyme immunoassay. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between human anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. The polystyrene ball was washed to eliminate most nonspecific IgG in test serum, and the complex was eluted from the polystyrene ball, to which nonspecific IgG had been adsorbed, with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay was 1,000 to 3,000-fold more sensitive than the conventional enzyme immunoassay, in which a human thyroglobulin-coated polystyrene ball was incubated with serum containing human anti-thyroglobulin IgG and, after washing, with rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in about 10% of healthy subjects and in all patients with Graves' disease and chronic thyroiditis. The principle of the present method may make it possible to sensitively measure other autoantibodies including anti-thyroid peroxidase and anti-thyrotropin receptor antibodies to aid diagnosis of thyroid diseases.     

Immunoreactive tissue kallikrein in human serum

Human urinary kallikrein and an antiserum to it raised in the rabbit were used to detect and quantitate immunoreactive tissue kallikrein in human serum. Both 125I-labeled kallikrein and the unlabeled purified enzyme appear complexed to higher molecular weight entities in serum, but specific binding between radiolabeled enzyme and antiserum was unaffected by the presence of serum or plasma. Parallelism to standard displacement curves was always seen with radioimmunoassay of normal sera as well as with human mixed saliva or pancreatic extracts. Assay sensitivity is 160 pg/ml of serum, or 16 pg per tube. Purified plasma kallikrein or prekallikrein in concentrations up to 10 micrograms/ml showed no displacement. Acetone-kaolin activation of plasma produced the expected 30-fold increase in Tos-Arg-OMe esterase activity but no change in immunoreactive tissue kallikrein levels. Serum concentrations were 3.8 +/- 0.7 (mean +/- SE) ng/ml in 21 normal volunteers, and were similar in patients with Fletcher trait or Hageman factor deficiency. Significantly increased serum concentrations were seen with long-term low dietary sodium intake or acute forms of pancreatitis. Although the relation of this immunoreactive material to any active tissue kallikrein within the circulation remains to be determined, our studies provide a new parameter for the assessment of a system repeatedly suggested to have some role in regulation of vascular resistance.     

Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with β-d-galactosidase from Escherichia coli

A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with β-d-galactosidase from Escherichia coli. Using the antiferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 μ1 of serum was 0.23–4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 μl of serum. The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9–8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92X(EIA) + 3.0 and 0.99 (n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of ¹²⁵I-labelled anti-ferritin IgG used was not sufficiently high.     

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.     

Membrane-bound lactoferrin alters the surface properties of polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) aggregate and avidly attach to endothelium in response to chemotactic agents. This response may be related in part to the release of the specific granule constituent lactoferrin (LF). We found by using immunohistology and biochemical and biophysical techniques that LF binds to the membrane and alters the surface properties of the PMN. Upon exposure of PMN treated with 5 micrograms/ml cytochalasin B to 2 x 10(-7) M formyl-methionine-leucine-phenylalanine for 5 min, the PMN mobilized LF to their surface as observed by immunoperoxidase staining for LF. At added LF levels ranging from 4 to 15 micrograms/10(7) PMN there was a dose-dependent reduction in PMN surface charge reaching 4 mV, when the partitioning into the membrane of a charged amphipathic nitroxide spin label was measured by electron spin resonance spectroscopy, whereas transferrin was without effect. When 125I-FeLF was added to human PMN in increasing amounts and the results corrected for the residual amount of free LF contaminating the cells, the PMN were saturated with LF at concentrations between 100 and 200 nM in the medium. Human PMN bound 1.35 x 10(6) molecules per cell and the calculated value for the association constant for these receptors was 5.2 x 10(6) M-1. Additionally, 6 micrograms/ml LF served as an opsonin for rabbit MN to promote PMN uptake by rabbit macrophages, when assessed by electron microscopy, but lysozyme did not. These studies indicate that LF can bind to the surface of the PMN and reduce its surface charge. This correlates with enhanced "stickiness" leading to a variety of cell-cell interactions.     

Characterization of a monoclonal antibody specific for prostatic secretory protein of 94 amino acids (PSP94) and development of a two-site binding enzyme immunoassay for PSP94

Purified prostatic secretory protein of 94 amino acids (PSP94) was used to generate polyclonal rabbit anti-PSP94 IgG and a murine monoclonal antibody 6B6 (mAB6B6). Both antibodies were highly specific for PSP94 by immunoblotting. Immunohistochemical studies demonstrated the specificity of mAB6B6 for human prostatic epithelial tissue. A two-site binding enzyme immunoassay for the detection of PSP94 was developed using a combination of the antibodies. The sandwich-ELISA yielded satisfactory results when mAB6B6 complexed to peroxidase conjugated goat anti-mouse-IgG (Fc) was incubated simultaneously with the sample. The assay has a dynamic range of 2-30 micrograms/l. This immunoassay was employed to measure PSP94 in male human sera (8 +/- 4 micrograms/l), female human sera (5.7 +/- 3.4 micrograms/l), follicular fluid (3.9 +/- 2.9 micrograms/l) and human seminal plasma (1.02 +/- 0.8 g/l).     

Determination of tissue plasminogen activator in plasma samples by means of a radioimmunoassay

A solid phase double antibody radioimmunoassay for measuring tissue plasminogen activator (t-PA) in human plasma is described using purified human melanoma cell t-PA and specific goat anti-t-PA IgG. The concentration of t-PA antigen in citrated human plasma sampled at rest was 4.4 +/- 1.3 micrograms/l (mean +/- SD) for healthy individuals (n = 20). After 10 min of venous occlusion the level of t-PA antigen was increased to 13.9 +/- 6.2 micrograms/l. The accuracy was monitored by recovery experiments using plasma from nine healthy individuals and nine patients to which was added low (5 micrograms/l) and high (25 micrograms/l) levels of purified t-PA. The detection limit of the method was estimated as 0.08 micrograms/l. An excellent correlation was found on comparing the t-PA antigen increase with the t-PA activity increase obtained during venous occlusion. In plasma collected prior to venous stasis, the total concentration of detectable t-PA antigen is found mainly in an inactive form. In contrast the t-PA antigen released upon venous occlusion seems to be largely in an active form.     

More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum

A more sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum and its use for the measurement of antithyroglobulin IgG in healthy subjects and patients with thyroid diseases are described. Antithyroglobulin IgG in test serum was reacted simultaneously with dinitrophenylthyroglobulin and thyroglobulin-beta-D-galactosidase conjugate. The complex formed of antithyroglobulin IgG, dinitrophenyl thyroglobulin, and thyroglobulin-beta-D-galactosidase conjugate was trapped onto two polystyrene balls coated with affinity-purified rabbit (antidinitrophenyl bovine serum albumin) IgG. The polystyrene balls were washed to eliminate nonspecific IgG in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to two polystyrene balls coated with affinity-purified rabbit (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the polystyrene balls was assayed by fluorimetry using 4-methylumbelliferyl-beta-D-galactoside as substrate. As compared with the previous immune complex transfer enzyme immunoassay, the procedure was simplified by reducing one incubation step and one washing step, and the detection limit of antithyroglobulin IgG in serum (0.1 microgram/liter) was lowered 100-fold. More careful and extensive examination than in a previous study revealed the presence of antithyroglobulin IgG in a large proportion of healthy subjects and in all patients with Graves' disease and chronic thyroiditis.     

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.     

Human anti-murine antibody interference in measurement of carcinoembryonic antigen assessed with a double-antibody enzyme immunoassay

Fifty-eight plasma specimens from 30 patients who had undergone presurgical radioimmunoscintigraphy with 111In-labeled anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (Mab) and who had no clinical evidence of disease after surgical resection showed increased concentrations of CEA (greater than or equal to 5 micrograms/L) in plasma when studied with the previously available commercial CEA enzyme immunoassay (EIA) from Roche. The possible role of anti-murine antibody (HAMA) interference was addressed by adding mouse IgG (mIgG) to the plasma (2 g/L) before assay. Fifteen specimens (26%) showed no change in CEA (reflecting a true increase as shown by the original results), 22 (38%) showed a decrease in CEA of greater than 15% but remained positive (reflecting an artefactual increase), and 21 (36%) became CEA-negative (less than or equal to 5 micrograms/L; reflecting a false increase). Subsequently, we assayed the same samples with a modified version of this CEA EIA kit and 47 specimens remained CEA positive (greater than 5 micrograms/L): 25 (53%) were truly increased, 12 (26%) remained artefactually increased, and 10 (21%) continued to show a false increase. The degree of interference in the original EIA kit correlated with the plasma concentration of HAMA (P less than 0.005). All artefactually and falsely increased CEA values observed in both kits were corrected by addition of polyclonal mIgG or of a mixture of IgG1, IgG2a, and IgG2b Mabs before assay. This correction is important in the follow-up of patients who receive murine Mabs for treatment or diagnosis.     

Ultrasensitive immune complex transfer enzyme immunoassay of HIV-1 p24 antigen with less serum interference using 2,4-dinitrophenyl-anti-HIV-1 p24 IgG and indirectly immobilized (anti-2,4-dinitrophenyl group) Fab..

In the immune complex transfer enzyme immunoassay previously reported, the immune complex consisting of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-HIV-1 p24 Fab' conjugate, HIV-1 p24 antigen and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate was trapped on polystyrene beads coated directly with affinity-purified (anti-2,4-dinitrophenyl group) IgG and was transferred to polystyrene beads coated with biotinyl-bovine serum albumin and streptavidin. The serum volume used was limited to 10 microL due to serious serum interference, and the detection limit of HIV-1 p24 antigen was 240 fg/mL serum. In the present study, HIV-1 p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-affinity-purified rabbit anti-HIV-1 p24 IgG and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate in the presence of excess nonspecific rabbit IgG. The immune complex of the three components formed was trapped on polystyrene beads coated successively with biotinyl-bovine serum albumin, streptavidin and biotinyl-affinity-purified (anti-2,4-dinitrophenyl group) Fab'. After washing, the immune complex was eluted from the polystyrene beads with excess epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads coated with affinity-purified goat (antirabbit IgG) IgG.The serum volume used was increased to 90 microL with only slight serum interference, and the detection limit of HIV-1 p24 antigen was lowered 9-fold to 26 fg/mL serum.     

A new enzyme immunoassay for prolactin in serum or plasma

This enzyme immunoassay (EIA) of human prolactin (hPRL) involves incubation of sample and anti-hPRL antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) in tubes coated with a second antibody to hPRL. The test can be performed within 60 min. No reaction of the antibodies with human placental lactogen and human somatotropin is detectable. The presence of detergent allows assay of both serum and plasma. Precision was improved by including polyethylene glycol in the reaction mixture. To optimize analytical recovery, we added protease inhibitor. Assay of the EIA standards shows good correlation with results for World Health Organization reference preparations. The measurable range is 1 to 400 micrograms/L. Intra- and interassay CVs are about 5%. Comparisons with two RIAs and two other EIAs show reasonably good correlations. The components of our EIA are stable for 18 months.     

Molecular and electrical heterogeneity of circulating human erythropoietin measured by sensitive enzyme immunoassay

BACKGROUND: We set up a sensitive sandwich enzyme immunoassay (EIA) of human erythropoietin (EPO) and further investigated molecular weight and structural heterogeneity, and electrical heterogeneity of the circulating EPO in normal human plasma without any concentrating procedure. MATERIALS AND METHODS: We set up a sensitive sandwich EIA of EPO using anti-EPO rabbit Fab'-peroxidase conjugate and anti-EPO IgG coated polystyrene balls. Standard EPO and plasma samples obtained from three normal subjects were applied to gel chromatographic analysis. Plasma samples obtained from three normal subjects were subjected to preparative isoelectric focusing with a column (pH 3.5-10). RESULTS: The minimal detectable quantity was 0. 15 mIU mL-1 using 100 microL sample. There was a good parallelism between diluted human plasma samples and the standard EPO in the assay. Rat plasma EPO was cross-reacted in the assay. There was a good correlation between EPO levels determined by the EIA and those of radioimmunoassay (y = 0.897x + 0.564, r = 0.957). Gel chromatographic analysis of standard EPO revealed a single peak. On the other hand, gel chromatographic analysis of unconcentrated plasma samples obtained from three normal subjects revealed at least four components of immunoreactive EPO. Preparative isoelectrical focusing revealed at least four major components and some other small peaks in normal human plasma samples. CONCLUSION: These findings indicate the first evidence for molecular and electrical heterogeneity of circulating EPO in normal human plasma without any concentrating procedure.     

Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.     

Immunoradiometric assay of atrial natriuretic peptide in unextracted plasma

We have developed and validated a two-site liquid-phase immunoradiometric assay (IRMA) of atrial natriuretic peptide (ANP) in unextracted human plasma. Both radiolabeled rabbit anti-ANP IgG and polyclonal mouse anti-ANP must bind to ANP for detection, and the assay is specific for peptides with both an intact C-terminus and a disulfide bridge. The assay sensitivity (detection limit) is 0.96 pmol/L, and the working range is 2.3-300 pmol/L, with the hook effect occurring above 500 pmol/L. Results for diluted plasma from normal subjects and from patients with renal failure paralleled the standard curve; analytical recovery of ANP added to such samples averaged 94%. The between- and within-assay CVs at 8 pmol/L were 10% and 5%, respectively. The assay is sufficiently sensitive and precise to detect the postural change in ANP concentrations in normal subjects.