Quantitation of mouse monoclonal antibody and human anti-mouse antibody response in serum of patients

The assays required to study pharmacokinetics of monoclonal antibodies and human anti-mouse antibody response in man, utilizing enzyme linked immunosorbent assay, have been at best semi-quantitative. We have developed and well characterized a quantitative assay for measurement of murine monoclonal antibodies and human anti-mouse antibody in circulation of patients injected with murine monoclonal antibodies. The sensitivity of mouse IgG assay is approximately 1 ng/ml and cross reactivity of IgG's from other species were less than 0.01%. The pre-therapy samples from 30 patients treated with monoclonal antibody CO17-1A as well as serum from 54 individuals had no detectable human anti-mouse antibody indicating specificity. The sensitivity of this assay was approximately 10 mg/ml. The utility of these assays was demonstrated in a group of patients receiving 400 mg of monoclonal antibody CO17-1A. The half-life and the degree of immune response can be measured using these assays.     

A monoclonal antibody applicable for determination of C-peptide of human proinsulin by RIA

BALB/c, (BALB/c x B10.A)F1 and (BALB/c x B10)F1 hybrid mice were immunized with C-peptide of human proinsulin. The (BALB/c x B10.A)F1 hybrids were the best responders and yielded 3 hybridomas secreting specific monoclonal antibodies. One of them, C-PEP-01, bound the C-peptide with high affinity (Kas = 1.1 x 10(9) l/mol), cross-reacted fully with human proinsulin but not with insulin, glucagon or somatostatin and apparently recognized the regions of C-peptide comprising amino acid residues 8-13 and 25-31. A RIA system could be set up employing this monoclonal antibody suitable for estimation of C-peptide concentrations in a diagnostically useful range (1-50 ng/ml).     

A monoclonal antibody inhibiting human placental Fc gamma-receptor activity

Fc gamma receptor (FcR) from human placenta was solubilized using EDTA and 2-mercaptoethanol and purified by affinity chromatography on human IgG-coated Sepharose 4B. BALB/C mice were immunized with FcR and monoclonal antibodies were obtained by growing hybridoma cells following fusion of spleen cells with P3 X 63Ag8 myeloma cells. Using an immunofluorescence technique, the IgG1 monoclonal antibody secreted by clone B1D6 stained the FcR-positive areas in sections of placental tissue. The endothelium of the foetal stem vessels stained more strongly than did the trophoblasts. The antibody also inhibited the haemadsorption to placental tissue of erythrocytes (E) sensitized with IgG antibodies (A), (EA), and inhibited the agglutination of EA by FcR. The data indicate that the monoclonal antibody reacts with the placental FcR at the binding site for IgG, or with an epitope close to the binding site. Apparently, the FcR in different anatomical areas in the placenta have a common antigenic determinant.     

Immunoinhibitory determination of CK-MM subbands by monoclonal antibody]

The measurement of CK-MM isoform by monoclonal antibody and its clinical application were summarized. The tissue type of CK-MM3 isoform was completely inhibited, MM2 isoform was about 57% and the serum type of CK-MM1 isoform was not inhibited by use of monoclonal antibody. This method enabled selective immunoinhibitory measurement of tissue type of CK-MM isoform. The heat lability and inhibition by EDTA suggested that it is a metal-depending enzyme. The analysis of MM isoform offers a promising alternate, non-invasive method to detect and follow up AMI and successful reperfusion. The MM3/MM1 ratio has been found to peak at about 2 to 6 hours, making it a quicker responding parameter to AMI than MM3 alone and significantly more responsive than the total CK or CK-MB. An MM3/MM1 ratio greater than 1.0 appears to be a practical cutoff point for detection of the pathological release of MM3 from tissue. Reperfusion therapy should be instituted within about 2 to 4 hours of AMI onset.     

The detection of antithyroglobulin activity in human serum monoclonal immunoglobulins (monoclonal gammopathies)

The sera of 159 patients with monoclonal gammopathies were examined for the presence of anti-thyroglobulin (Tg) activity. An enzyme-linked immunosorbent assay was employed. Thirty-one (19.5%) sera were found to bind Tg. The activity against Tg was further confirmed by using purified immunoglobulins and employing competition assays. The anti-Tg antibodies were found in the sera of patients with IgG, IgM and IgA gammopathies. Anti-Tg antibodies were more frequent among patients with IgG gammopathy. Autoantibodies to Tg are found in patients with Hashimoto's thyroiditis, Graves' disease and occasionally in patients with thyroid carcinoma. Natural autoantibodies directed against human Tg have been detected, as well, in healthy subjects. None of the patients in the present study whose serum was found to contain high titers of anti-Tg human monoclonal antibodies had any clinical or biochemical evidence of thyroid disease. Our results of a high incidence of anti-Tg activity in the sera of patients with monoclonal gammopathies support previous reports of autoantibody properties characteristic of these immunoglobulins.     

具胆碱酯酶活性的抗人血清丁酰胆碱酯酶单克隆抗体3A5

目的研究具抗原酶活性的抗人血清丁酰胆碱酯酶单抗3A5的催化特性及活性位点。方法利用酶促反应动力学方法测定3A5的Km及Kcat/Km;根据胆碱酯酶抑制剂对其催化活性的影响情况分析其活性位点。结果3A5催化丁酰胆碱酯酶特异性底物的水解符合米.曼氏方程规律,但Kcat/Km明显小于抗原酶;丝氨酸蛋白酶抑制剂PMSF明显抑制其胆碱酯酶活性,胆碱酯酶活性中心催化位点抑制剂吡啶斯的明及活性中心阴离子位点抑制剂四甲基铵则分别对其产生竞争性及非竞争性可逆抑制。结论3A5具有与抗原酶相似但较弱的催化活性,具有与天然胆碱酯酶相似的活性中心催化位点及阴离子位点。     

Quantitative assay of a human monoclonal IgM antibody (HA-1A) in human serum

A rapid, specific and sensitive radiometric assay was developed capable of quantitating serum levels of HA-1A, a human IgM monoclonal antibody to endotoxin. 'Private' anti-idiotypic murine monoclonal antibodies were produced and utilized in the assay to avoid cross-reactivity with normal human IgG, IgM, IgA, IgE or IgD. The presence of E. coli or gram-negative lipopolysaccharide in the sera did not affect the ability of the assay to detect HA-1A. The sensitivity of the assay was calculated to be 25 ng/ml with an interassay coefficient of variation of less than 10%. In one patient given 100 mg of HA-1A, peak serum concentration was 101.5% of the predicted value with a mean plasma half life of 24.5 h. This assay will be useful in establishing the pharmacokinetics of HA-1A and in monitoring serum levels during phase II and phase III clinical trials.     

Preparation and characterization of a monoclonal antibody that inhibits human neutrophil elastase activity

Neutrophil proteases are believed to play a role in the pathogenesis of a variety of human diseases. While many studies of proteases in models of disease have focused on elastase, neutrophils contain several proteases some of which share a high degree of homology. This report describes the production and characterization of an IgG1 murine monoclonal antibody (AHN-10) that reacts with human neutrophil elastase but not with the other major neutrophil neutral proteases: cathepsin G, proteinase 3, collagenase, or the newly purified neutral protease, esterase N. AHN-10 inhibited the elastinolytic activity of purified human neutrophil elastase and could detect elastase in alcohol-fixed cytospin preparations. The epitope recognized by AHN-10 was resistant to treatment with NaIO4, suggesting that the epitope is not a carbohydrate. AHN-10 should be useful for the immunolocalization of neutrophil elastase in tissue specimens and as a stable source of characterized antibody for quantitative identification of neutrophil elastase.     

Binding of one monoclonal antibody to human Ia molecules can be enhanced by a second monoclonal antibody

A monoclonal anti-human Ia antibody, PTF 29, was tested for its ability to bind purified, 125I-labeled human Ia preparations. It was found that the binding level increases considerably in the presence of a second monoclonal antibody. Experimental conditions were selected under which only the binding of PTF 29 and not the binding of the second antibody could be determined. Under these conditions, it was found that "helped" PTF 29 binding has higher affinity and is exerted on a molecular subset different from that bound by PTF 29 alone. This phenomenon, while not easily accommodated in the present conceptual framework of the human Ia system, appears in itself interesting and may have more general implications.     

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15–25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg–sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.     

A human X-linked antigen defined by a monoclonal antibody

We have constructed hybrids between human thymocytes and the mouse thymoma BW5147. These hybrids, and others, have been used to show that the expression of a thymocyte antigen is controlled by an X-linked gene.     

A human leukocyte antigen identified by a monoclonal antibody

Leukocyte antigen of approximate molecular weight 200,000 daltons have beendescribed in mouse, rat, and man. We described here the reactivities of a monoclonal antibody. GAP 8.3, which identified such an antigen on human leukocytes. We found the leukocyte antigen H-T200 on T and B lymphocytes, granulocytes, monocytes, and platelets, but not on erythrocytes or nonhematopoetically derived cells. Resting and activated T cells had more antigen or their surfaces than did resting B lymphocytes and EBV-transformed B cells, respectively. The leukocyte antigen was detected on approximately 75% of bone marrow cells; cells of the erythroid series comprised the negative population. The GAP 8.3 antibody and its F(ab′)₂ fragments had no effect on in vitro stimulation of peripheral blood cells by mitogens or allogeneic cells. Antigen isolated from T cell lines had a higher electrophoretic mobility than did antigen from B cell lines; antigen from the mycloid line U937 comigrated with that from B cell lines. In addition, we detected very small but reproducible differences in the electrophoretic mobility of the antigen on two T cell lines.     

Characterization of human anti-hrB-like monoclonal antibody

To develop a blood typing reagent with Rh specificity, peripheral blood lymphocytes were isolated from a multiparous woman with apparent alloanti-Ce and fused with FIS heteromyeloma cells. One hybridoma cell line, FOR-2E3, secreted human IgG, which agglutinated all human red blood cells except R2R2, RZRZ, Dc-, D--, Rhnull, and e+ hrB-. The supernatant is useful as a reagent to screen for hrB- blood donors and to differentiate haplotypes with C and e in cis from haplotypes with C and e in trans.     

Detection of rotavirus in human stools by using monoclonal antibody.

A monoclonal antibody, 3F7, that reacts with the common rotavirus antigen on the sixth viral gene product was prepared. It was used in a direct monoclonal antibody radioimmunoassay (RIA) as a diagnostic reagent for detection, in 3.5 h, of rotavirus in human pediatric stool specimens. In the 177 samples tested, a concordance of 96% was seen between the monoclonal RIA and the well-established and commonly used commercially available Rotazyme test. Six discrepant specimens that were positive by monoclonal RIA but negative by Rotazyme were shown to be positive by either electron microscopy or confirmatory blocking immunoassay. A seventh discrepant specimen was positive by Rotazyme and negative by monoclonal RIA as well as by both direct and immune electron microscopy. The monoclonal RIA test appears to be highly sensitive and specific, and merits additional evaluation as a rapid, convenient diagnostic assay that can reduce currently encountered problems associated with diagnosing rotavirus infection by immunoassay.     

单克隆抗体ELISA双抗体夹心法测定人白细胞介素2

本文叙述了测定人白细胞介素2(IL-2)含量的ELISA双抗体夹心法。用辣根过氧化物酶标记IL-2单克隆抗体。聚苯乙烯反应板预先用0.1％戊二醛处理,然后以单克隆抗体包被。本法批内变异系数CV=3.99％,批间变异系数CV=5.96％,标准曲线的测定范围为3.1～100u/ml,检测下限为1.55u/ml。应用本法已测定了2S例正常人和11例恶性肿瘤患者的IL-2含量。     

A novel monoclonal antibody with catalytic activity against beta human chorionic gonadotropin

In this study, we report for the first time, production of monoclonal antibody (MAb) against beta human chorionic gonadotropin (hCG) with proteolytic activity. MAb "7D9" was raised in Balb/C mice using purified human chorionic gonadotropin. Immunoblot analysis and enzyme-linked immunoabsorbent assay (ELISA) showed that this MAb reacts with beta hCG. The epitope for this antibody appears to be located in the C-terminal of beta chain as suggested by the absence of cross-reaction with other glycoprotein hormones such as FSH, TSH and LH. Our data reveal that this MAb is very unstable and has autodegradation characteristics. Zymogram analyses also show that 7D9 MAb has a high level of hydrolytic activity against different substrates such as casein and gelatin. This proteolytic activity can be inhibited by EDTA. These findings demonstrate the proteolytic character of 7D9 MAb and consequently explain its instability.     

Characterization of a monoclonal antibody inhibiting the biological activity of human chorionic gonadotrophin

A mouse monoclonal antibody (MCA), raised against human chorionicgonadotrophin (HCG) and coded 130A, was characterized with severaltypes of immunoassay and with in-vitro and in-vivo bioassays.MCA 130A binds strongly to the intact HCG molecule but lessso to either -or -subunit; The dissociation constant of theMCA-HCG complex was found to be in the order of 70 pmol/l. MCA130A inhibits the biological activity of HCG very effectively,both in vitro and in vivo. In a competitive immunoassay MCA130A binds 30 x better to HCG than to human luteinizing hormone(HLH). The selectivity in the bioassays was much higher, e.g.in vitro, 50% inhibition of HLH-stimulated testosterone productionrequires 2600 x as much MCA as is needed for inhibiting HCG-stimulatedproduction. Possible reasons for this difference in selectivityare discussed     

Evaluation of a solid-phase monoclonal antibody-based enzyme immunoassay for insulin in human serum

A 'sandwich-type' enzyme immunoassay for the measurement of serum insulin is described in which a monoclonal antibody-alkaline phosphatase conjugate and an antibody-immobilized polystyrene solid phase are used. Serum samples of 50 microliters can be analyzed and the enzyme immunoassay is as sensitive as the conventional radioimmunoassay for insulin. The results obtained with ELISA correlate well with those of the radioimmunoassay (r = 0.9844) and the between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (2-200 microIU/ml). The sensitivity is 2 microIU/ml and this can be increased by longer incubation times. The crossreaction with porcine insulin is 45%, with bovine insulin 30% and with human proinsulin 20%.