Characterization of monoclonal antibodies to human interferon-gamma and their application to enzyme-linked immunosorbent assay (ELISA)

The authors obtained two mouse monoclonal antibodies, G-208 and G-166, to recombinant human interferon-gamma (rH-IFN-gamma). Immunologically, they were classified as IgG1-K subclass. G-208 neutralized the antiviral activity of natural and recombinant human IFN-gamma, but did not bind to heat-denatured rH-IFN-gamma. G-166 was able to bind to rH-IFN-gamma as well as to heat-denatured rH-IFN-gamma, but it did not bind to natural human IFN-gamma (nH-IFN-gamma). A sandwich enzyme immunoassay specific to H-IFN-gamma molecule was developed using polyclonal rabbit anti-nH-IFN-gamma antibody and G-208. This assay monitors only biologically active H-IFN-gamma molecule. Thus, this method may be used for the direct determination of H-IFN-gamma instead of determination of antiviral activity of H-IFN-gamma.     

Dot enzyme-linked immunosorbent assay (dot ELISA) for antibodies to Encephalitozoon cuniculi

A dot-ELISA procedure was developed to detect antibodies against Encephalitozoon cuniculi. Sera from 84 rabbits, 22 dogs, 18 squirrel monkeys and 200 mice were tested by dot-ELISA and most also were tested by immunofluorescence. Comparison of the two tests showed that there was excellent agreement of the results (Kappa values greater than or equal to 0.74) in all four species. Dot-ELISA is a simple, quantitative, rapid alternative to immunofluorescence when large numbers of serum samples must be evaluated.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Pasteurella pneumotropica in murine colonies

An ELISA for the detection of class specific IgG antibodies to Pasteurella pneumotropica was developed for the serological diagnosis of infections in mouse colonies. Heat inactivated whole cell preparations of an isolate of P. pneumotropica biotype Heyl (strain P 166) served as antigen for the ELISA procedure and for immune serum production in germ-free Han:NMRI mice. Cross reactions with the autochthonous flora of Han:NMRI SPF-mice were not observed, but were evident when a P. pneumotropica antiserum was tested against other antigens of the Pasteurella-Actinobacillus group. According to the reclassification of this bacterial group proposed by Mutters et al. (1), strains of the following species were tested: P. anatis, P. canis, P. dagmatis, P. langaa, Pl multocida sub. multocida, P. pneumotropica biotype Jawetz, P. stomatis, Actinobacillus equuli and A. lignieresii. Clear cross reactions could be shown with P. pneumotropica biotype Jawetz and A. equuli and to a lesser extent with P. anatis. Antibody formation profiles after nasal infection of Han:NMRI mice exhibited a primary rise of IgG-type antibody titer between 17 to 21 days post infection. Investigations of different mouse colonies free and infected with P. pneumotropica revealed good correlations between serological and bacteriological findings.     

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome—red-absorbing form and phytochrome—far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

Rapid enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to Candida albicans

A rapid enzyme-linked immunosorbent assay (ELISA) where the performance time was shortened to 4h was compared with counter-immunoelectrophoresis (CIE) and a standard ELISA procedure for the detection of IgG antibodies to Candida albicans in 61 patients with suspected invasive candidosis. Using a C. albicans cytoplasmic antigen the rapid ELISA compared well with CIE and the standard ELISA. Seventeen sera that reacted with two concentrations of C. albicans antigen in CIE were also positive in both forms of ELISA. Four sera that were CIE-negative were positive in the standard ELISA and three were also positive in the rapid ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of different forms of candidosis.     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.     

Comparison of liposome immune lysis assay (LILA) and enzyme-linked immunosorbent assay (ELISA) for detection of antibodies

Anti-α-chymotrypsinogen A antibody was assayed by both enzyme-linked immunosorbent assay (ELISA) and liposome immune lysis assay (LILA). The detection limit was slightly affected by the measurement conditions in ELISA; however, it was possible to control the detection limit and to achieve a lower level by adapting the measurement conditions in LILA. LILA is believed to offer a simple and highly sensitive method for measuring the concentration of antibody in serum.     

A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.     

Detection of antibodies to Sendai virus in mouse sera by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of Sendai virus infection in mice was evaluated. A large-scale survey of infected mice showed that ELISA is approximately 100 times more sensitive than the hemagglutination-inhibition or complement-fixation test. Although a few SPF mice showed false-positive reactions at a serum dilution of 1:40, further dilution to 1:80 eliminated the non-specific reaction. It was shown that ELISA is a highly satisfactory method for examination of Sendai virus infection in mice.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

Use of a monoclonal antibody to estrone-3-glucuronide in an enzyme-linked immunosorbent assay (ELISA)

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.     

Development and optimization of an enzyme-linked immunosorbent assay employing two murine monoclonal antibodies for absolute quantitation of human beta-glucuronidase.

We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) for absolute quantitation of human beta-glucuronidase. This is a double antibody sandwich system employing two murine monoclonal antibodies specific for human beta-glucuronidase developed in our laboratories. The method involves (a) coating of the high binding polystyrene microtitration plate with the first antibody (7B6 IgG), (b) blocking of remaining active sites with 3% bovine serum albumin in phosphate-buffered saline, (c) application of samples, (d) addition of the biotinylated second antibody (6D2 IgG), (e) addition of streptavidin-horseradish peroxidase, and (f) development of color with o-phenylenediamine dihydrochloride-H2O2 and reading in a microplate reader at a wavelength of 490 nm. The method is highly sensitive with an optimal range of 10 to 100 ng/ml of the enzyme and is reproducible with intraday and interday precisions of 3.2 and 4.1%, respectively. The enzyme contents of 20 urine and 20 bile samples quantitated by this ELISA method were, respectively, 148 +/- 101 and 6380 +/- 3780 ng/ml (means +/- SD) which correlated well with their enzyme activities. Such a method for absolute quantitation of human beta-glucuronidase is essential for studying its pathophysiologic roles in cholelithiasis and carcinogenesis and can also be used clinically as an indicator for tissue damage or malignancy.     

Characterization of mouse monoclonal antibodies to human interferon-gamma

Mouse monoclonal antibodies (mAb) to human interferon-gamma (HuIFN-gamma) were characterized. The mAbs studied--E4-18, G4-15, and SAT-1--which are all IgGl-type, reacted to all HuIFN-gamma molecular species, both glycosylated and non-glycosylated. Affinity constants calculated of E4-18 and G4-15 didn't have considerable differences for both kinds of HuIFN-gamma (1-3 x 10(8) liter/mol), but SAT-1 had a difference--a higher value (10(10) liter/mol) for the former than for the latter (8 x 10(8) liter/mol). In epitope specificity, the results suggested that E4-18 and G4-15 recognized an overlapped region remote from the region of SAT-1. Competition experiment using synthetic peptides suggested that epitope of G4-15 is around N9-26 of the HuIFN-gamma sequence. Those mAbs could be used for sandwich radioimmunoassay of HuIFN-gamma using double mAbs in two combinations, one (G4-15/E4-18) based on dimer forms of HuIFN-gamma and the other (SAT-1/E4-18) based on epitope difference. The mAbs are all neutralizing antibodies in which SAT-1 neutralized at a lower concentration than did G4-15, and at a much lower one than did E4-18. The receptor binding of HuIFN-gamma was inhibited by mAbs G4-15 and SAT-1. Efficacy of G4-15 and SAT-1 for the inhibition correspond with that for neutralization.