Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies

A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species is discussed.     

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin

OBJECTIVE: To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. ANIMALS: 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. PROCEDURE: Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. RESULTS: MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes. CONCLUSION: MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells.     

Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay

Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium. Veillonella, Enterococcus avium and S. typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture. The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids. The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures. The ELISA was capable of detecting 10(4) cells per ml of the bacteria. The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E. avium, S. typhimurium and Veillonella, respectively. The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation.     

Enzyme-linked immunosorbent assay for measurement of JNK, ERK, and p38 kinase activities

A rapid enzyme-linked immunosorbent assay for the enzyme activity measurement of three well-known mitogen-activated protein (MAP) kinases, JNK2, ERK2, and p38 is described. The assay involves immobilization of the respective kinase substrates c-Jun, Elk1, or ATF2 on microtiter plates, addition of the kinase reaction mixture, and measurement of substrate phosphorylation using phospho-epitope-specific antibodies. This novel procedure represents a marked improvement to conventional radioactive MAP kinase assays in terms of quantification, precision, performance at physiological ATP concentration, high throughput, time consumption and amenability to automation. In addition to the standard solid phase assay using plastic-bound protein substrates, we developed an alternative solution phase protocol using soluble protein substrates. By comparing the results of the two assays, we found that MAP kinases retained much of their substrate specificity in the phosphorylation of immobilized protein substrates. Interestingly, we observed a strong preference of JNK2 and p38 for the phosphorylation of dimeric over monomeric substrates. We further characterized the kinase inhibitory activity of olomoucine, staurosporine, and SB 203580 for JNK2, ERK2, and p38. Taken together, this assay could assist in the biochemical characterization of MAP kinases and in identifying potent and specific inhibitors of these enzymes.     

Production and characterization of genus and species specific monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi

Fourty-four primary brain tumors were studied by immunohistochemistry. 29 (67.2%) expressed HLA-Dr. The incidence and number of tumor cells positive for HLA-Dr correlated with the histological type of brain tumor and increased with the degree of malignancy. The mononuclear cells infiltrating in these tumors were mostly CD45ROT cells and macrophages. The former consisted mainly of CD4 and CD8 subsets. The numbers of CD45RO+ and CD8+ T cells in HLA-Dr-group were more than any of the HLA-Dr+, +2, +3 groups. The numbers of CD4 subset and macrophages were not affected by the level of HLA-Dr expression. These results suggest that the HLA-Dr expressed by brain tumor cells selectively inhibit CD8 subset which participates in immunoreaction against brain tumors in situ.     

The characteristics of monoclonal antibodies and their antigens associated with human sperm acrosome reaction. I. The induction of acrosome reaction and monoclonal antibody production

A low blood glucose level is associated with impairment of higher cerebral function and an increase in cerebral blood flow. This study examined whether there are differences in the physiological responses to hypoglycaemia between the cerebral hemispheres. Eight healthy men participated in two hyperinsulinaemic glucose clamp studies: after 60 min at 4.5 mmol/l, blood glucose was either lowered to 2.0 mmol/l and "clamped" there for 60 min (hypoglycaemia) or continuously maintained at 4.5 mmol/l (euglycaemia). Cardiac output, middle cerebral artery velocity (transcranial Doppler) and cerebral blood flow (133-xenon inhalation) were measured during the studies. Neuropsychological tests were used to determine whether hypoglycaemia caused differential impairment of hemispheric cognitive function. Hypoglycaemia was associated with symmetrical impairment of cognitive function in both cerebral hemispheres and a rise in cardiac output (from 5.5 [0.2] to 8.7 [0.2] l.min-1, p < 0.0001, mean [standard error]), middle cerebral artery velocity (from 55 [2.6] to 64 [2.8] cm.s-1, p < 0.002), and global cerebral blood flow (from 56 [2.6] to 69 [2.9] ml.100 g-1.min-1, p < 0.005 compared to pre-insulin values). There were no differences in the blood flow response during hypoglycaemia between hemispheres and the increase in blood flow did not correlate with either the change in cardiac output or rise in plasma catecholamine levels. After 120 min of hyperinsulinaemic, euglycaemia, global cerebral blood flow rose significantly above baseline (from 58 [2.4] to 63 [2.2] ml.100 g-1.min-1, p < 0.05). In conclusion, using the techniques described, the physiological and cognitive responses of each cerebral hemisphere to hypoglycaemia were symmetrical.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzyme linked immunosorbent assay (ELISA) for detection of seminal fluid using a monoclonal antibody to prostatic acid phosphatase

A microtitre plate format enzyme linked immunosorbent assay (ELISA), employing commercially available PASE/4LJ mouse monoclonal hybridoma antibody is described. The technique is a solid phase indirect ELISA for prostatic acid phosphatase, applicable to specific detection of semen. Maximal detectability was found to be one hundred thousand fold dilution of pooled seminal plasma. No cross reactivities with human vaginal fluid, blood, saliva, female urine, nasal discharge, earwax, sweat or faeces have been found.     

Development of a monoclonal antibody to 6 beta-hydroxycortisol and its application in an enzyme-linked immunosorbent assay (ELISA) for 6 beta-hydroxycortisol in urine

A novel monoclonal antibody to 6 beta-hydroxycortisol (6 beta-OHC) was generated and incorporated into an antigen-coated indirect enzyme-linked immunosorbent assay (ELISA) using 6 beta-OHC-protein conjugate as the steroid-coating antigen. The monoclonal antibody is specific to 6 beta-OHC and 6 beta-OHC-3-carboxymethyloxime. Cross-reactivity with other structurally related steroids such as cortisol, cortisone, and 6 beta-hydroxycortisone was less than 10%. Two different clones (clone 5C1 and 19F) of the monoclonal anti-6 beta-OHC antibody have been developed, each with slightly different sensitivity and specificity. The sensitivity of the MAb clones was not significantly improved when compared to the rabbit polyclonal antibodies in this study, but still within the accepted detection limit for 6 beta-OHC in both human and laboratory animals. The assay had a detection limit of 200 ng/ml, an intraassay variation of 6.4% and an interassay variation of 7.3%. The application of the anti-6 beta-OHC-MAb-based-ELISA was tested by measuring the urinary output of 6 beta-OHC in human before and after enzyme induction by rifampicin treatment. The mean 24-h urine output of 6 beta-OHC in human subjects was 485 +/- 100 micrograms and 1478 +/- 281 micrograms before and after rifampicin administration, respectively. In conclusion, the monoclonal anti-6 beta-OHC antibody developed in this study has the required specificity and sensitivity as an alternative method for measuring urinary 6 beta-OHC in the detection of enzyme induction or enzyme inhibition of CYP3A in humans and laboratory animals.     

Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes

Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.     

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.     

Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane

A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.     

Immobilization of small molecules on solid matrices: a novel approach to enzyme-linked immunosorbent assay screening for saxitoxin and evaluation of anti-saxitoxin antibodies

A novel enzyme-linked immunosorbent assay (ELISA) technology was developed for detecting saxitoxin or evaluation of anti-saxitoxin antibodies, which is based on non-covalent immobilization "free' saxitoxin to Maxisorp microtitre plates. The effect of pH on immobilization was studied in media with wide-range buffering capacities (piperazine-glycylglycine and barbiturate buffers). Increasing pH resulted in better responses, although this was mainly due to non-specific interactions. At pH 10.0, however, saxitoxin immobilization was quite effective and specific. The same pattern was found under four different conditions; absence vs presence of bovine serum albumin precoating and absence vs presence of 150 mM NaCl. The best results (high specific response) were achieved with bovine serum albumin precoating in the presence of 150 mM NaCl. The method of choice involved precoating Maxisorp with 5 micrograms/ml albumin followed by addition of 5 microM saxitoxin in 0.01 M piperazine-glycylglycine buffer, pH 10.0. The efficacy of this technology was demonstrated on a polyclonal rabbit anti-saxitoxin antibody and compared with a conventional ELISA of saxitoxin using saxitoxin-bovine serum albumin conjugate as the coating antigen. In the experiments investigating cross-reactivities of various saxitoxin derivatives based on a competitive assay, significantly greater sensitivity was achieved with the novel approach, e.g. 35 pM saxitoxin could be detected (3 x 10(4) times lower concentrations than using the conjugate). The assay works well with mussel tissue homogenates, and because it does not require the use of the covalent saxitoxin-carrier conjugates it offers a simpler alternative to the traditional ELISA for saxitoxin.     

Immunoreactivity, stability, pharmacokinetics and biodistribution of a monoclonal antibody to human leukemic B cells after three different methods of radioiodination

Dal B02, a murine monoclonal antibody against human chronic lymphocytic leukemia (CLL) was radioiodinated using chloramine T (Chl.T), Bolton-Hunter (B-H) or N-succinimidyl-p-iodobenzoate (PIB). The preparations had comparable radiochemical purity (> 97%) and immunoreactive fraction (65-80%) but the Chl.T-based product was most susceptible to deiodination and loss of immunoreactivity. After i.v. injection into CLL-xenografted nude mice, the preparations had identical patterns of clearance from the blood but the PIB-based product led to more radioactivity in liver and spleen and less in the thyroid compared to the other preparations. The Chl.T-based product showed loss of immunoreactivity in circulation and less tumor-localized radioactivity 168 h after administration. The differences between the B-H-based and PIB-based products were less impressive than between PIB-based and Chl.T-based products.     

Anti-seminal plasma antibodies associated with infertility: II. Comparative investigation of human antibody binding to normo-, astheno-, and azoospermic seminal plasma antigens

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1:1 and the hybridization efficiencies were approximately 99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells.     

Cancer imaging with radiolabeled antibodies: new advances with technetium-99m-labeled monoclonal antibody Fab' fragments, especially CEA-Scan and prospects for therapy

The use of radiolabeled anticancer antibodies to detect cancer sites by external scintigraphy has had a relatively long history. With the advent of monoclonal antibodies (MAbs), which precluded the need for purifying the antibodies by laborious purification steps, there was a surge of interest and efforts to develop these reagents for both imaging and therapy applications (1). Today, many thousands of patients have received different forms and doses of MAbs for various purposes, and four MAb-based products have been licensed for manufacture and sale in the U.S. (2,3). This article describes the most recent MAb product to be approved in the U.S. for colorectal cancer imaging, including discussions of using this agent and its therapeutic counterpart in several cancer types.     

Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics

The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories (< 4-, 4-8, and > 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.     

A new Combi test for simultaneous detection of antibodies to viral capsid, early and EBNA antigens of Epstein-Barr virus

In order to facilitate the differentiation between a recent (acute) and a past Epstein-Barr virus (EBV) infection, the Combi test was developed. This test is an anticomplement immunofluorescence test (ACIF) requiring only a single serum dilution to be tested on a single cellular spot. The cell line used expresses viral capsid antigen (VCA) and early antigen (EA) in about 5 to 10 percent of the cells as well as EBV nuclear antigens (EBNA) in more than 90 percent of cells. A satisfactory agreement between the Combi test and other tests for antibodies to EBV was obtained (IgG and IgM antibodies to VCA by IFA and EIA and antibodies to EBNA by ACIF including tests for heterophile and complement-fixing antibodies). When the standard serological tests gave negative results, the Combi test was also negative (absence of any fluorescence in the cells). Serologically confirmed recent (acute) infections lead to specific fluorescence in only 5 to 10 percent of the cells, while past infections result in fluorescence in 90 percent or more of the cells. For the diagnosis of a reactivated EBV infection or of EBV-associated malignancies, other tests should be employed. The test is based on the measurement of the activation and specific distribution of the C3 component of complement; the antibody class differentiation is therefore not necessary. The presence of rheumatoid factor (RF) and the IgG competition phenomenon do not influence the results of the Combi test. An introduction of the Combi test will enable a simplified, less expensive and more reliable serodiagnosis of EBV infections.     

Radioimmunoassay of oestrone in plasma. A comparison of different methods with respect to the relation between assay specificity, sample purification and antibody specificity

A radioimmunoassay (RIA) for oestrone (Oe1) in plasma was developed using an ether extraction, a partition between NaOH and light petroleum, and a TLC as sample purification and an antiserum cross-reacting with Oe2 for the final assay (Method A1). The reliability criteria are given in detail. In order to simplify this method a highly specific antiserum was developed by using Oe1-3-hemisuccinate-BSA as an antigen. Using this antiserum for the final assay but omitting the TLC (Method B) the Oe1 concentration in male plasma was 76% overestimated (Method B compared with Method A1). In pregnancy plasma Oe1 could specifically be determined after a simple ether extraction (Method C). It was concluded that the use of a highly specific antiserum (as determined by cross-reaction studies) for the final assay does not necessarily imply that a chromatographic sample purification can be omitted without loss in assay specificity. This appears to be true mainly in cases where the steroid concentration of the sample is low. Normal values of Oe1 from 80 healthy adult males were ascertained by Method A1. Age group I (22-61 years, n = 48) ranged from 1.22-5.60 ng/100 ml, median 2.81; age group II (67-90 years, n = 32) from 1.55-6.40, 100 median 3.41. The small increase of Oe1 with age was highly significant.     

Monoclonal antibody for the demonstration by ELISA of antibodies to protein p24 of enzootic bovine leukosis virus in individual and pooled blood serum and milk samples

A sensitive and specific ELISA for the demonstration of antibodies to the protein p24 of enzootic bovine leukosis virus (EBVL) using a 'capture' monoclonal antibody to this protein (MAb p24) was developed. The method is sensitive enough to detect the international reference serum E4/10 in pooled blood serum samples collected from up to 50 cows, or, if a 10-fold concentrate of milk whey is tested, in samples of bulk milk collected from up to 400 cows. The application of MAb p24 has considerably increased not only the sensitivity, but also the specificity of ELISA. Moreover it is possible to differentiate reliably between positive and 'false positive' reagents by testing a suspicious sample in a pair of wells of which one is coated with MAb p24 alone and the other with the complex MAb p24 + EBLV antigen and the subsequent calculation of 'specific absorbance'. This method, showing the highest sensitivity of detection of antibodies to EBLV p24 described so far, can become an effective tool on the sanitation of infected herds as well as in checks of the EBL-free status. A diagnostic kit suitable for commercial manufacture has been devised.     

Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes

The surface plasmon resonance (SPR) wavelength of colloidal gold particles coated with a monoclonal antibody is red-shifted when the antibody interacts with its specific ligand. This shift results from the change in the refractive index of the particles as induced by ligand binding. This property is used to monitor in real-time the association and dissociation kinetics of the interaction in solution. The monitoring is performed in a clinical chemistry automated analyzer during a few minutes of incubation at 37 degrees C. Data treatment allows calculation of the affinity constant of the interaction. The SPR wavelength shift does not necessarily require agglutination or aggregation of the particles to occur since particles coated with one monoclonal antibody specific for a single epitope on the ligand can be used in the procedure. The affinity constants measured by this procedure correlate with those calculated from Scatchard plots or BIAcore data.