Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Antibody response in laying hens with small amounts of antigen

Chicken antibodies offer many advantages over the traditional mammalian ones. A laying hen produces large amounts of yolk antibodies and the use of yolk antibodies eliminates the painful procedure of collecting blood from the animal. Thus, the use of chicken antibodies will reduce both the number of animals required to produce antibodies and also animal distress. Chicken antibodies also have several biochemical advantages compared to mammalian antibodies: they often increase the signal and reduce interference in many assays. However, the species chosen for antibody production have usually been mammals. This is probably due to tradition, but also to limited knowledge about the production of chicken antibodies. We studied the immune response in the chicken using small amounts of mammalian antigen, and show that a good immune response can be obtained with 0.1–1.0 μg of bovine serum albumin.     

Anti-P30 IgA antibodies as prenatal markers of congenital toxoplasma infection

This study extends a previous study and confirms that the detection of anti-P30 IgA antibodies is very helpful in the diagnosis of acute acquired or congenital toxoplasmosis. Moreover, we demonstrate that an anti-P30 IgA response can be mounted in the fetuses infected by Toxoplasma gondii during their intra-uterine life as early as week 23 of gestation. A double-sandwich ELISA described in our previous work was used to detect anti-P30 IgA antibodies in 1378 human serum samples collected from 551 patients, including 162 fetuses whose mothers had been infected by T. gondii during pregnancy, 46 congenitally infected and 90 uninfected newborns and 253 women suspected of having been infected during pregnancy, including the mothers of fetuses and newborns previously described. Anti-P30 IgA antibodies were detected in all cases of acute toxoplasmosis but in no case of chronic toxoplasmosis: in the majority of cases, the IgA antibody titre fell below cut-off in 3-9 months. Among the 46 congenitally infected newborns, anti-P30 IgA antibodies were detected in sera of 41 infected newborns (38 at birth, two in the first months of life, one in the seventh month of life), while anti-P30 IgM antibodies were detected in only 30 cases at birth and in one case during the first month of life. Among 162 fetuses, anti-P30 IgA response was observed in five infected fetuses, but was not detected in either 152 uninfected fetuses or in five fetuses considered as infected. The absence or presence of anti-P30 IgA antibodies in the fetus is discussed in relation to the date of maternal infection and collection of the fetal blood. It clearly appears from our study that the combined testing of both IgM and IgA in the fetus and the newborn is essential for a more efficient diagnosis of infection.     

PENA方法的建立及与ELISA IgM检测CMV的比较

目的　介绍一种敏感、稳定、快速、简便的实验室检测CMV的方法 ,同时探讨该种新方法与ELISA检测CMV方法的优、缺点。方法　对 5 5 2例病人应用间接荧光免疫法测定细胞核中的特异病毒早期抗体 (PENA)和ELISA法测定IgM抗体。结果　PENA方法 :强阳性 88例 ,阳性率 15 4 9% ,弱阳性 2 73例 ,阳性率 5 9 4 6 % ;ELISA -IgM方法 :阳性 34例 ,阳性率6 16 %。结论　PENA方法操作简便 ,与ELISA方法相比较 ,可对CMV感染进行早期测定及诊断 ,并可区分既往感染和即时感染 ,具有敏感性和稳定性 ,是测定小儿CMV感染的一种较好的方法     

Adsorption of anti-dsDNA antibodies by immobilized polyanionic compounds.

Antibodies to dsDNA are characteristically present in serum from patients with systemic lupus erythematosus (SLE), and have been shown to have the capacity to react with various molecules bearing repeating negative charges. After a number of polymeric or monomeric molecules with differently charged groups and hydrophobic molecules had been coupled covalently as ligands on cellulose gel, the adsorption capacities of the ligands for anti-dsDNA antibodies were evaluated. It was found that gels coupled with polyanionic dextran sulphate (DXS) and polyacrylic acid (PA) and monoanionic sulphanilic acid (SA) absorbed anti-dsDNA antibodies effectively. DXS gel also adsorbed antibodies to ssDNA and heparan sulphate, antigens with repeating negatively charged moieties, while no ligand was able to adsorb anti-nRNP antibodies. The finding that DXS gels adsorbed anti-dsDNA antibody in proportion to their charge density, and that the interaction between anti-dsDNA and DXS gel is broken readily by an increase in ionic strength, indicated that the binding is ionic in nature. Moreover, virtually all F(ab')2 anti-dsDNA became adsorbed onto the DXS gels, suggesting that the binding occurred via specific antigen-binding sites on the antibody molecule. Binding of these polyanion-binding autoantibodies with anionic sites in the glomerular basement membrane may therefore cause the tissue damage observed in SLE.     

Evaluation of Onchocerca volvulus-specific IgG4 subclass serology as an index of onchocerciasis transmission potential of three Gabonese villages

The major objective of this study was to evaluate the usefulness of IgG4 ELISA and Western blot analysis, using a crude extract of Onchocerca volvulus adult worms as antigens, for diagnosing onchocerciasis in a Gabonese paediatric population with mixed filarial infections. The subjects had loaisis, streptocercosis or mansonellosis in addition to onchocerciasis. Control sera from loaisis or mansonellosis subjects residing outside the endemic zone were used to provide the cut-off point for positive results. The IgG4 ELISA had a specificity of 96% but a lower sensitivity of 78·7%. It detected 25 onchocerciasis cases out of 65 individuals who were negative on parasitological examination. Furthermore, the ELISA provided a more accurate picture of onchocerciasis transmission in a village with very low skin microfilartal load. A 27·5-kD antigen was identified on Western blots as a marker of onchocerciasis. The paediatric population provided a reliable window for assessing the parasitologic and serologic parameters in the three villages with disparate levels of onchocerciasis transmission.     

The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases

In 1988 and in 1989 consensus workshops were organized in order to define the interlaboratory concordance in detecting autoantibody specificities in selected sera from patients with rheumatoid disorders and to determine the possible causes of discrepancies. In total 20 sera were tested for the presence of antibodies against nRNP, Sm, Ro (SS-A), La (SS-B), Scl-70, centromeric antigens, ribosomal RNP and Jo-1. The methods used for detection by the 28 European laboratories who participated included immunofluorescence, counter-immunoelectrophoresis, immunodiffusion, immunoblotting and ELISA.

The results showed that only a combination of two or more techniques was able to detect all specificities with an adequate efficiency. Recommendations to improve the efficiency of autoantibody detection and to standardize laboratory protocols are given.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.