An enzyme-linked double antibody immunoassay to measure murine immunoglobulins--its application to determine the specific activity of radiolabeled monoclonal antibodies

A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with radioactive isotopes. A combination of radioactive counting and measurement of the absorbance of a peroxidase catalyzed reaction permits accurate determination of the specific radioactivity of labeled monoclonal antibodies.     

Production and characterization of a monoclonal antibody to chloramphenicol

A monoclonal antibody to chloramphenicol (CAP) was produced. After immunization of BALB/c mice with CAP base coupled to human serum albumin and incubation of the stimulated splenocytes in vitro in the presence of antigen for three days, these splenocytes were hybridized with X63‐Ag8·653 myeloma cells. The antibody, designated 6A10, proved to be IgG2b, and it had a detection limit for CAP of 10 ng/ml (0·5 ng/assay) in the direct enzyme immunoassay using horseradish peroxidase‐labelled CAP. The cross‐reactivities with CAP base, p‐nitrobenzyl alcohol, and p‐nitrophenol were 5·0, 0·94, and 0·007%, respectively. No cross‐reactivities were observed with penicillin, tetracycline and thiamphenicol, respectively.     

Production of a specific monoclonal antibody against mercury-chelate complexes and its application in antibody-based assays

In this study, monoclonal antibodies (mAbs) against the chelated Hg^{2 +} were prepared. The immunoconjugate (chelated Hg^{2 +}, or KLH-ITCBE-Hg^{2 +}), was generated by coupling of Hg^{2 +} to keyhole limpet hemocyanin (KLH) with a bifunctional chelator 1-(4-isothiocyanobenzyl)- ethylenediamine N, N, N’, N’-tetraacetic acid (ITCBE). Five hybridoma cell lines secreting antibodies that specifically reacted with bovine serum albumin (BSA)-glutathione-Hg2+, but not with BSA-glutathione, were isolated from the fusion between the spleen cells of a mouse immunised with KLH-ITCBE-Hg2+ and SP2/0 myeloma cells. A clone, B/B11, was chosen for further development of a specific and rapid immunoassay for Hg^{2 +}. In this context, we established an effective competitive indirect enzyme-linked immunosorbent assay (ELISA) based on the mAb for Hg^{2 +}. The assay is specific to mercury with an IC₅₀ value of 1.589 µM and the lowest detection limit of 0.009 µM. The immunoassay was applied for detection and measurement of mercury in various water samples and in greengrocery samples. The recoveries from ultrapure water, tap water, and pool water were in the range of 94.38–109.67%, and in greengrocery samples were from 85.12 to 92.68%. Overall, the optimised ELISA based on mAbs is a convenient and efficient analytical tool for monitoring mercury residues in water and in the greengrocery samples.     

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA)

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

A competitive solid-phase radioimmunoassay for translational factors employing monoclonal antibodies

Monoclonal antibodies produced by the hybridoma techniques were purified by chromatography on DEAE Affi-Gel blue, and covalently coupled to Affi-Gel 10 to purify their antigens. The purified components were used to develop a sensitive competitive radioimmune assay for the quantitative determination of translational factors, as described here with a monoclonal antibody directed against yeast elongation factor 3. Antigen was adsorbed to polyvinyl chloride plastic surfaces and a limiting concentration of monoclonal antibody necessary to bind to the adsorbed antigen was determined. Varying concentrations of purified antigen and of samples containing unknown amounts of antigen were then mixed with the limiting concentration of monoclonal antibody, prior to or at the same time as the reaction of the antibody with the surface-adsorbed antigen. The amount of monoclonal antibody that bound to the surface-adsorbed antigen was determined with a second antibody, radioactive goat anti-mouse antibody. The addition of the free antigen preparations to the monoclonal antibody served to compete for the antibody with the antigen adsorbed to the plastic surfaces. The concentration of antigen in the unknown samples was estimated from the titration curves obtained with varying concentrations of pure antigen. This technique did not require isotopic labeling, modification or derivatization of the monoclonal antibody or its antigen.     

骨桥蛋白单克隆抗体的制备及其特异性分析

为了研制骨桥蛋白(OPN)特异性单克隆抗体(mAb),并鉴定其特异性。本研究在毕赤酵母中表达具有良好免疫原性的重组人OPN蛋白的基础上,用重组人骨桥蛋白(rhOPN)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选杂交瘤细胞,并结合免疫印迹对抗体的特异性进行鉴定,通过竞争抑制试验对单克隆抗体识别的抗原位点进行分析。结果共获得4株能够识别OPN不同抗原位点的mAb,亚类测定显示,3株为IgG1,1株为IgG2a。这些mAb能与重组人OPN特异性结合。本研究的四株抗体中,只有4G2B5能够检出与肿瘤转移密切相关的OPN-c的条带,预示该抗体可用于判断肿瘤预后和高转移肿瘤的临床检测。本研究成功获得了针对骨桥蛋白的特异性mAb,同时为进一步研究OPN蛋白的结构和功能提供了重要工具。     

Strain‐specific monoclonal antibodies to Penicillium bilaii

Monoclonal antibodies (IgM class) to Penicillium bilaii (isolate PB‐50) were developed and their specificity was determined against various fungi using enzyme‐linked immunosorbent assays. Cross‐reactivity in the range 0–6.5% was obtained for Fusarium, Aspergillus, Paecilomyces and Trichoderma species while 1.5–9.5% was obtained for P. lapidosum, P. crustosum, P. hiramayae, P. spinolosum and P. implicatum. Strains of P. bilaii (ATCC 22348 and ATCC 18309) yielded a relative cross‐reactivity of 15.9 and 10.8%, respectively. An immunochemical test for the detection of PB‐50 mycelia in plant and soil samples was devised using monoclonal antibodies. Several procedures have been proposed to show the feasibility of the application of developed monoclonal antibodies to P. bilaii PB‐50 detection in plant and soil samples. The immunodiffusion blotting method was superior to other tests for the detection of PB‐50 in infected plant samples.     

[125I]protein a solid-phase assay for detection of monoclonal antibody

Summary The production of monoclonal antibodies involves testing hundreds of hybridoma supernatant samples for antibody content. This necessitates a rapid detection assay which can separate antibody of interest from antibodies that react nonspecifically. Solid-phase radioimmunoassay is one such method.     

Production and characterization of monoclonal antibodies cross‐reactive with major aflatoxins

Monoclonal antibodies cross‐reactive with four major aflatoxins (AFs) were produced by fusion of P3/NS‐1/1‐AG4–1 murine myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with aflatoxin B3‐hemisuccinate conjugated to bovine serum albumin. Six stable clones were obtained. Isotyping by enzyme‐linked immunosorbent assay (ELISA) revealed that the antibodies produced by all but two of the clones were of the IgG₁ type. Of the remaining clones, one produced IgG₂₁, and the other IgA. Competitive radioimmunoassay using tritiated AFB₁ as the marker ligand revealed that two clones produced antibody that cross‐reacted well with both AFB₁ and AFG₁; one clone produced an antibody that had good specificity toward AFB₁. The relative cross‐reactivities (RCR) of antibody produced by clone 575B8F12 for AFB₁, AFB₂, AFG₁, and AFG₂ were 100, 5, 153, and 6, respectively. The RCR of antibody produced by clone 575G4H7 for the above AFs were 100, 40, 153, and 40, respectively. The RCR of antibody produced by clone 585D4D6 for the above AFs were 100, 40, 152, and 23, respectively. Antibodies produced by the other three clones were inadequate for immunoassay because their affinities for the AFs were 100 times less than the three clones described above.     

The use of monoclonal antibodies in (reverse) passive haemagglutination tests for herpes simplex virus antigens and antibodies

Three monoclonal antibodies against herpes simplex virus type 2 have been tested for their suitability as reagents in reverse passive haemagglutination. Two of these antibodies with specificity for virus glycoprotein D, when linked to red blood cells, were able to capture antigens without being agglutinated, but addition of immune serum subsequently led to agglutination. Haemagglutination using these monoclonal antibody-linked, antigen-captured red cells was readily applicable to testing human sera for antibodies to herpes simplex virus and the titres obtained correlated with those from virus plaque neutralisation tests. The procedure has been termed "Specific Antigen Capture Passive Haemagglutination." A further monoclonal antibody with specificity for the major DNA-binding protein of type 2 herpes virus-infected cells (a nonstructural protein) showed conventional reverse passive haemagglutination when linked to red blood cells and was specific for type 2 herpes simplex virus. The nature and potential uses of these simple reverse passive haemagglutination procedures using monoclonal antibody reagents are discussed.     

Production and characterization of antibodies against neosaxitoxin utilizing a novel immunogen Synthesis procedure

Polyclonal antibodies against neosaxitoxin (neoSTX) were produced in rabbits after immunization with a neoSTX—glucose oxidase conjugate. This method used a novel approach for immunogen synthesis, which required only trace amounts (28 ug) of neoSTX for the coupling reaction. A competitive indirect enzyme immunoassay (EIA), with saxitoxin conjugated to bovine serum albumin as the solid phase antigen, was established using these antibodies. The detection limit for neoSTX was 17.8 pg ml^‐1. Relative cross‐reactivities of the antibodies with gonyautoxin 1/4 (GTX1/4), saxitoxin (STX), gonyautoxin 2/3 (GTX2/3), decarbamoylsaxitoxin (dcSTX) and N‐sulphocarbamoylsaxitoxin (C1/2) were 104, 3.36, 3.35, 0.11 and 0.04% respectively. Thus, the EIA described here was most specific for neoSTX and GTX1/4.     

Generation of hybridoma antibodies to double-stranded DNA from non-autoimmune BALB/c strain: studies on anti-idiotype

A hybridoma obtained between normal spleen cells from BALB/c mice (a non-autoimmune strain) and SP2-O-Ag 14 myeloma cell line was designated as HB2. These hybrid cells produced an IgM kappa-anti-ds-DNA antibody, but their specificity was limited to some polydeoxyribonucleotides such as natural ds-DNA from calf thymus, poly dG-poly dC, poly d(GC) and poly d(GC)-poly d(GC). In contrast, poly dA-poly dT, poly d(AT) were not recognized. The configuration of the nucleic acid helix plays a small role if any, in the building of the epitopes recognized by the hybridoma HB2 antibodies, while the presence of G and C appeared to be essential. These epitopes could not be found on ss- and ds-polyribonucleotides. B cells able to produce anti-ds-DNA antibodies are therefore present in non-autoimmune BALB/c mice, but not enough to produce the corresponding antibodies at a detectable level in the serum. Following immunization of BALB/c mice with hybridoma HB2 monoclonal antibodies, anti-idiotype antibodies were obtained which also recognized idiotopes present in the serum from both murine MRL/1 and human systemic lupus erythematosus (SLE).     

Production of monoclonal antibodies for the determination of s‐triazines with enzyme immunoassays

Two triazine derivatives, ametryn sulphoxide (2‐ethylamino‐4‐isopropylamino‐6‐methyl‐sulphoxide‐1,3,5‐triazine) and dichloroatrazine (2,6‐dichloro‐4‐isopropylamino‐1,3,5‐triazine) were conjugated to bovine serum albumin (BSA) and used for the immunization of BALB/c mice. Hybridomas were produced by cell fusion of immune spleen and mouse myeloma cells (PAI‐B₃AG8.I). After screening with a competitive enzyme‐linked immunosorbent assay (ELISA), four anti‐triazine monoclonal antibodies from permanent hybridoma cell lines were selected for further characterization. Cross‐reaction studies with the antibodies developed against the ametryn sulphoxide conjugate showed strong affinities to terbutryn and prometryn. ELISAs with antibodies from clone AS‐K1F4 reached a detection limit of approximately 0.1 μg/l for terbutryn, and with AS‐K1A11 antibodies a limit of 0.3 μg/l for prometryn was attained. Cell lines from mice immunized with the dichloroatrazine‐conjugate produced antibodies with the highest affinities to aziprotryn. The detection limit of the corresponding ELISA was 1 μg/l (clone DC‐C3K5). The scope of the monoclonal antibodies for use in pesticide residue analysis is discussed with respect to polyclonal antibodies.     

A human monoclonal antibody against HLA-Cw1 and a human monoclonal antibody against an HLA-A locus determinant derived from a single uniparous female

Abstract: Two human monoclonal antibodies (HuMAbs) with widely different HLA specificities were raised from a uniparous HLA-seropositive female. Screening against a large panel of serologically HLA-typed lymphocytes in the complement-dependent cytotoxicity test showed that one of these HuMAbs, VP6G3, was specific for HLA-Cwl, thereby constituting the first HuMAb against an HLA-C locus product. The second HuMAb, VP5G3, was directed against an HLA-A-encoded determinant shared by HLA-A11, -A25, -A26 and -A66. The epitopes responsible for binding were determined by comparing the aminoacid sequences and were pinpointed to the 6K/9F combination for HuMAb VP6G3, and 163R with a critical contribution of aminoacids present at positions 166/167 for HuMAb VP5G3.     

Validation of a monoclonal antibody‐based indirect enzyme immunoassay method for picloram detection in soil and plants

Picloram (4‐amino‐3,5,6‐trichloro‐2‐pyridinecarboxylic acid) residues in authentic soil and plant samples were determined by indirect enzyme immunoassay (EIA) and by gas chromatography (GC). Results obtained by EIA methods correlated well with the GC results with coefficients of determination ranging from 0.778 to 0.891. Picloram levels determined by EIA in soil extracts obtained by a potassium hydroxide extraction method (EIA‐1) underestimated picloram levels determined by GC. Conversely, EIA determinations of soil extracts obtained by a more rigorous acetonitrile extraction method (EIA‐2) yielded picloram levels that overestimated levels determined by GC. First order linear regression models using EIA determinations as the independent variable resulted in dependent variable coefficients of 0.325 and 1.42 for EIA‐1 and EIA‐2 respectively. Picloram levels determined in plant extracts were approximately equivalent by EIA and GC methods. Using a point on the standard curve representing 5 ng ml^?1 picloram as a threshold value, the EIA method effectively identified samples as either positive or negative for picloram residues based on the results obtained by GC analysis.     

Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6.

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of C-reactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.     

Production of monoclonal antibodies for detection of antigens in both their native and denatured states

Different immunization procedures were tested to find methods to enhance the proportion of monoclonal antibodies reacting equally well with native and denatured proteins for use in food analysis. Antibodies to soybean trypsin inhibitor with the desired characteristics were obtained by immunization with the denatured protein. Antibodies to ovomucoid were obtained by long‐term immunization. The monoclonal antibodies to ovomucoid were of the IgM class.     

Measurement of squirrel monkey serum IgG levels by a two-site sandwich radioimmunoassay with monoclonal antibodies

Monoclonal antibodies against the squirrel monkey Saimiri sciureus IgG have been produced for a more specific analysis of the antibody-related immunological aspects in experimental human or monkey malaria. Two monoclonal antibodies, 3D8/D5 and 3F11/G10, out of 64 reacted with distinct epitopes on the IgG present throughout the complete population without interfering with each other. The 2 monoclonal antibodies were used to develop a highly specific, reliable and sensitive two-site sandwich radioimmunoassay for the measurement of the serum IgG levels in 83 animals. The antibodies also allowed us to produce by a simple immunoabsorbent technique a highly purified IgG standard easy to calibrate and store. The assay permits the detection of IgG levels as low as 0.48 ng/ml. The standard curve is linear between 3.9 and 125 ng protein/ml and allows by a simple mathematical equation an accurate measurement of the serum IgG levels.     

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus is described. It requires only a single tube culture showing viral cytopathology and can accommodate multiple specimens in a single assay. The monoclonal antibodies confer absolute specificity and the use of horseradish peroxidase-conjugated staphylococcal protein A or antiglobulin permits easy visual interpretation of the results following the 2-3 hour assay. Although it is possible that an occasional wild strain of HSV might escape recognition by monoclonal antibody, this potential problem has not been observed among the more than 500 clinical isolates tested to date, all of which have yielded an unequivocal result.