Preparation and specificity of avian anti-GM2(NeuGc) ganglioside antiserum

Antibodies to N-glycolyl neuraminic acid-containing GM2 ganglioside, GM2(NeuGc), were prepared by immunizing chickens. The specificity of the antibodies was examined by the double immunodiffusion test and solid-phase radioimmunoassay (RIA). One(C-4) of two antisera produced did not cross-react with GM3(NeuGc) but the other(C-3) did as assessed by the double immunodiffusion test. In RIA, the antibody activity of C-4 antiserum was detected only in the IgG fraction. Specificity of the serum was examined using authentic glycolipids which were structurally related to GM2(NeuGc). The antiserum showed a high specificity for the homologous ganglioside by either an RIA or an inhibition assay. This antiserum is a useful tool for the detection of GM2(NeuGc) in human and animal tissues under normal and/or disease condition.     

Validation of a specific radioimmunoassay to measure plasma cortisol in the fetal sheep.

A procedure utilizing co-chromatography and complementary antiserum comparisons was employed to assess the specificity of a cortisol radioimmunoassay for use in the chronically catheterized fetal sheep preparation. Complementary antiserum comparisons is a technique by which two different cortisol antisera, prepared from conjugates attached at opposite ends of the cortisol molecule, were used to determine cortisol concentrations in the same ovine fetal plasma specimens. Results were not significantly different between the two groups, each measured by a different antiserum. This procedure may be used to assess assay specificity in any species in which steroid radioimmunoassays are being adapted.     

A radioimmunoassay for chironomid hemoglobin

A double antibody, competitive radioimmunoassay was developed for the quantitation of stage-specific hemoglobin from the insect Chironomus thummi. The radioimmunoassay will detect as little as 150 picograms of Hb 3, a hemoglobin synthesized and secreted into the hemolymph of larvae during the 4th, but not the 3rd instar. The assay also detects cross-reacting hemoglobins purified from 4th instar larvae and in freshly laid eggs. Application of this radioimmunoassay is discussed in light of the cross-reacting Hbs in the insect.     

Dirofilaria immitis: performance and standardization of specific antibody immunoassays for filariasis

The factors associated with the development, optimization, and validation of immunoassays for the detection of parasite-specific antibody in filariasis infections were investigated using the dog heartworm, Dirofilaria immitis as a model. We examined two assays, the Protein A solid-phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent assay (ELISA), for quantitation of specific antibody to the parasite in canine serum. Precision, reproducibility, and parallelism were examined using response-error relationships and precision profile analyses. A staphylococcal Protein A saturation analysis was applied to the standardization of IgG anti-parasite antibody reference sera in weight per volume units (microgram/ml). Using the mean minimal detectable dose + 3 SD and an intraassay precision profile less than 10% coefficient of variation (CV) as criteria for assay sensitivity, the SPRIA and ELISA displayed comparable positive thresholds of 1 microgram/ml IgG anti-parasite antibody/ml of serum. Both assays demonstrated good reproducibility (less than 15% interassay CV) and parallelism (less than 20% interdilutional CV) over their working ranges (SPRIA: 1-40 micrograms/ml; ELISA: 1-5 micrograms/ml). Specificity of each assay was enhanced by preadsorption of cross-reacting antibodies in canine serum (i.e., specific for Toxocara canis antigens) with solid-phase antigen prior to assay. Methods for comparing different immunoassay designs are considered in relation to the variables that influence the assays' performance characteristics.     

Identification and distribution of immunoreactive peptide YY in the human, canine, and murine gastrointestinal tracts: species-related antibody recognition differences

A radioimmunoassay using two antisera (antibody 80 and antibody 213) from rabbits immunized with porcine peptide YY has been characterized for both sensitivity and specificity. To determine the distribution of peptide YY in the gut, fresh tissue specimens from the human and canine gut were separated into mucosal-submucosal and muscularis externa layers by microdissection. These tissues and transmural specimens from murine gut were acid-extracted and neutralized, followed by radioimmunoassay using each antiserum. Immunoreactive peptide YY in canine and murine gut was present in similar concentration and distribution using each antiserum, with highest concentrations in the mucosal-submucosal layer of the descending colon. Using antibody 213, immunoreactive peptide YY throughout the human gut was measured only at the lower detection limit of the radioimmunoassay. By contrast, using antibody 80, peptide YY in human gut was present in a distribution similar to canine and murine gut. Using antibody 80, one major immunoreactive species was identified with C18 reverse-phase high-performance liquid chromatography in extracts of human, canine, and murine colon. These results suggest species-related antibody recognition differences. The similar concentrations of peptide YY in canine and murine gut determined with the two antisera are consistent with the hypothesis that the amino acid sequences of canine and murine peptide YY are similar to porcine peptide YY. Using antibody 213, the low concentrations of immunoreactive peptide YY found in human gut are consistent with the hypothesis that human and porcine peptide YY have different amino acid sequences. Antisera prepared by immunization with porcine PYY must therefore be carefully characterized prior to studies using human sera or human tissue extracts.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

Immunological studies on ovine fetuin: radioimmunoassay and comparison with related antigens

A radioimmunoassay for fetuin is described. It uses [125I]fetuin and rabbit antisera. The bound antigens are precipitated by anti-rabbit globulins coupled to Sepharose. This assay has been used to compare the immunological properties of ovine and bovine fetuin. It allows to detect a serum antigen cross-reacting with fetuin in pig species. A very faint reaction is observed with human fetal serum, that does not support the existence of a fetuin like antigen in human.     

Functional cholesteryl binding agents: evaluation of viability and specificity of antibodies bound to modified phospholipid vesicles

A cholesterol derivative was incorporated into small unilamellar phospholipid vesicles, and antibodies were bound covalently to the vesicles. More than one antibody was bound to each vesicle. The antigen binding viability and specificity were determined using a modified radioimmunoassay and an in vitro cell assay. Both of these tests showed good antibody activity and specificity. The antigen affinity of the bound antibodies was higher than for the unbound antibody due to more than one viable antibody being bound to each vesicle. The modified vesicles can be used as immunodirected drug delivery systems for both diagnosis and therapy.     

Detection and characterization of monoclonal antibodies to platelet membrane proteins

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.     

Radioimmunoassay of 17-hydroxyprogesterone in serum with or without thin-layer chromatographic purification.

A radioimmunoassay for the measurement of 17-hydroxyprogesterone (17-OHP) is described. The antigen 11-desoxycortisol-21-hemisuccinate-bovine serum albumin has been used to produce two antisera of different antibody populations in the same animal. The thin-layer chromatographic system described can be used to separate all the cross-reacting steroids investigated from 17-OHP. A simplified method is also presented using preliminary solvent extraction only. The mean 17-OHP levels measured, using this method, were, for normal men, 0.86 +/- 0.19 ng/ml (SD), for normal females 0.30 +/- 0.19 ng/ml in the follicular phase and 1.72 +/- 0.18 ng/ml in the luteal phase of the menstrual cycle, and 0.45 +/- 0.17 ng/ml in normal postmenopausal women.     

Comparison between different rabbit antisera against the glucocorticoid receptor

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.     

Turbidimetric Method for the Assay of Antiviral Antibodies

A rapid, simple assay method has been developed for antiviral antibodies. The technique has been applied to antisera, immune γ-globulins, and immunospecifically purified antibody for two strains of influenza virus, Asian 305 and PR8, and to antisera to tobacco mosaic virus. Turbidity changes due to the specific interaction of a virus with its antibody were measured by the increase in optical density in a sensitive wavelength region, e.g., 436 nm. Successful application of the method required that nonspecific effects which give rise to turbidity changes be eliminated. This was accomplished by proper choice of ionic strength (0.3 m) and pH (5.5), and by the addition of normal serum or serum albumin to the virus before contact with the antibody. Sensitivity of the method allowed quantitation of antibody down to the level of 10 μg of antibody protein per ml. The specificity of the reaction causing the turbidity change was established by experiments which showed that precipitation of virus-antibody complexes removed the reactive component in the serum, and by the absence of turbidity changes for nonspecific pairs (virus plus unrelated antisera).     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Assessment of the specificity of norethisterone radioimmunoassays

Specifities of 4 different norethisterone (Nor) antisera (coded A,B,C, and D) were evaluated and compared by cross-reaction studies to relate the antiserum specificity to the overall specificity of the radioimmunoassay (RIA), as established by plasma levels measured in women regularly taking the microdose of Nor (300 mcg/day). Using any of the 4 antisera, no significant deviation from parallelism were found among graded doses of authentic Nor and increasing volumes of plasma from women taking Nor for contraception. Cross-reaction studies preceded by chromatography to decrease plasma blanks are described, with each antiserum compared to the others for its efficacy in estimating plasma Nor values. It was concluded that 1) the significance of cross-reaction studies as well as that of a parallelism test for assessing overall specifity of the RIA is limited; 2) a single chromatography before RIA improves assay specificity but may not be sufficient to remove all interfering compounds; and 3) a comparison of direct and chromatographic procedures using several different antisera is useful for selection of the relatively most specific RIA procedure. These study results indicated that either antiserum C or D (preceded by chromatography) will yield better results than A or B.     

Affinity enhancement of bispecific antibody against two different epitopes in the same antigen.

For the enhancement of antibody binding affinity, a bispecific antibody against two different epitopes in human chorionic gonadotropin hormone, one is in alpha-subunit and the other is in beta-subunit, was prepared by chemical recombination using 5,5'-dithiobis(2-nitrobenzoic acid). The epitopes recognized by antibodies were investigated by competitive radioimmunoassay, two-site sandwich radioimmunoassay and additivity assay and a proper epitope pair was chosen for preparation of the bispecific antibody. This bispecific antibody has dual specificity and as much as 17.2-fold higher affinity than that of monoclonal antibody with higher affinity by dual antigen binding radioimmunoassay and Scatchard plot analysis.     

Further characterisation of a polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity

Synthetic polynucleotides irradiated with far (254 nm) or near (320 nm) UV-light were used to characterise 3 different radioimmunoassay systems. Antiserum raised against DNA irradiated with a high dose of far-UV-light was found to have at least 2 antibody populations. A competitive assay in which the labelled antigen was irradiated at 254 nm was found to be specific for Pyr(6-4)Pyo adducts, the antibody-binding sites being sensitive to a secondary photolytic dose of 320-nm light. When the labelled antigen was irradiated with 320-nm light the assay was specific for cyclobutane dimers. This assay had the same specificity as one consisting of labelled DNA irradiated with 254-nm light and an antiserum raised against DNA irradiated at 320 nm in the presence of acetophenone. These assay systems were used to demonstrate the dose-dependence of the induction and photolytic degradation of Pyr(6-4)Pyo adducts by a near-UV-light source.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.     

Immunochemical and biochemical investigation of hexosaminidase S.

Hexosaminidase S (HEX S), the residual isozyme found in tissues and body fluids of children with the O variant of GM2 gangliosidosis, was purified from tissues of variant individuals and biochemically and immunochemically characterized. This enzyme has an apparent molecular weight of 103,000 with an isoelectric point of 4.2, is heat labile to the same extent as HEX A, and loses most of its activity following heating for 30 min at 50 degrees C. HEX S reacts immunologically with the antisera against either HEX A or B, but the reaction is considerably stronger with the anti-A serum or with antibody preparations which react exclusively with the A isozyme. Results obtained by a radioimmunoassay using the various antisera indicated that there is no antigenically cross reacting material which lacks enzymatic activity in the variant tissues. These findings are in accord with a suggested molecular structure of two subunits, each composed of two alpha chains (alpha2 alpha2) for HEX S; it also implies that alpha and beta chains have some structural similarity which is manifested in antigenic cross-reactivity.