Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram- negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti- lipid A mAbs function by neutralizing the toxic effects of LPS.     

Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay.

Myoglobin (Mb) is considered a useful marker for early detection of myocardial infarction and for monitoring cardiac reperfusion after thrombolytic therapy. We developed eight monoclonal antibodies to human cardiac Mb, characterized their epitopic reactivity, and determined which combinations of the antibodies are useful in two-site immunoassays. We configured two of the monoclonal antibodies in a one-step, two-site particle concentration fluorescence immunoassay (PCFIA) for measurement of Mb. The PCFIA has rapid kinetics of reaction, being complete in 15 min, and has a linear analytical range of 20-675 micrograms/L for human Mb. Although the PCFIA has a high-dose "hook" effect, this is of no analytical importance at concentrations of Mb less than or equal to 148,000 micrograms/L. The assay is not subject to interference from icterus (bilirubin less than or equal to 360 mg/L), has no cross-reaction with hemoglobin (less than or equal to 42 g/L), and may be performed with either plasma or serum in approximately 1 h. The intra- and interassay imprecisions (CV) of the method are less than 10% for concentrations of Mb within the normal range and less than 4% at higher concentrations. A comparison of the PCFIA with a commercial radioimmunoassay showed that results of the two assays correlate well (PCFIA = 0.88 x RIA + 18, r = 0.990, n = 171).     

Pancreatic amylase measured in serum by use of a monoclonal antibody immunochemically immobilized to a solid phase

We studied a method for measuring the pancreatic isoenzyme of amylase (EC 3.2.1.1) by use of a mouse monoclonal antibody against human salivary-type amylase (Clin Chem 1985;31:1283) coupled indirectly to particles of polyvinylidene fluoride via polyclonal goat anti-mouse immunoglobulin. These particles, in 200 microL of a suspension, could remove salivary amylase (activity 2200 U/L) from an equal volume of serum in 5 min. Measurement of amylase activity in the supernatant fluids from treated sera thus provided an assay of pancreatic amylase. Precision studies at three activity concentrations yielded within-run CVs of 1.6% to 1.7% (n = 25) and total CVs of 2.2% to 5.1% (20 days). Salivary amylase added to each of 10 sera was completely (99.8%, SD 1.6%) removed. The new method (y) showed the following regression statistics when compared with an electrophoretic method (x): slope = 0.989 (SD 0.019), intercept = -0.220% (SD 1.48%), SEE 4.0%, n = 51. Similar respective regression values were found for urine samples: slope = 0.934 (SD 0.053), intercept = 2.3 U/L (SD 3.2), SEE 8.4 U/L, n = 26. The following respective values were found when the new method (y) was compared with the previously described immunoprecipitation assay (x): slope = 1.02 (SD 0.02), intercept = 2.2% (SD 1.4%), SEE 3.3%, n = 23 sera. Reference intervals for pancreatic amylase activity in serum were established for three different substrates: maltotetraose, maltopentaose, and p-nitrophenylheptaoside.     

Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma

This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2- derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human antibody that detected nuclei, nucleoli, cytoskeletal elements, Golgi complex, or other cytoplasmic components were also isolated in this study. One of these antibodies detected an intracellular antigen that is restricted to cells of neuroectodermal derivation, and a second antibody reacted primarily with cells of epithelial origin. Using these methods to isolate and analyze human monoclonal antibody, it should now be possible to define the repertoire of the humoral immune response to melanoma.     

Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers. STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays. RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies. CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.     

Preparation of monoclonal antibodies to the Fc-fragment of human IgG and the use of their based immunoenzyme conjugates

An original kit containing 17 clones of hybridomas, the producers of monoclonal antibodies (MCAb) against the Fc-fragments of human IgG, has been obtained. The possibility of using horseradish peroxidase conjugates of obtained MCAb as a part of ELISA test systems (Diaproph-Med, Ukraine) was comparatively studied. The application of peroxidase conjugates of two monoclonal antibodies (156C10 and 153H11) as part of five diagnostic kits was shown to provide a high specificity and a high sensitivity.     

Evaluation and comparison of commercially available pregnancy tests based on monoclonal antibodies to human choriogonadotropin

We evaluated eight pregnancy tests: Tandem Icon, beta-hCG Rapid, Pregnastick (immunoenzymometric assays), Neo-Pregnosticon (direct hemagglutination), Pregnospia (sol particle immunoassay), Neo-Planotest (latex agglutination), Gravindex beta-hCG, and beta-hCG Slide Test (both latex-agglutination inhibition), all of which detect human choriogonadotropin (hCG) in urine. We investigated the limits of detection and the responses to the following substances: human lutropin, protein, and blood, and high concentrations of hCG. Using 100 patients' samples, we assessed the diagnostic specificity and sensitivity as well as the accuracy of each kit. The detection limits for Tandem Icon, beta-hCG Rapid, Neo-Pregnosticon, Pregnospia, Neo-Planotest, and Gravindex beta-hCG were as stated by the manufacturers. Only Neo-Planotest gave a false-positive result for lutropin. The three immunoenzymometric assays were not affected by protein or blood, but of these only Tandem Icon did not exhibit prozoning. The five other kits gave false-positive or false-negative results for protein and blood. Tandem Icon performed best, being quick and easy to use and without susceptibility to interfering substances.     

The monoclonal antibody-specific immobilization of erythrocyte antigens assay (MAIEA) in the investigation of human red-cell antigens and their associated membrane proteins

The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) technique is an immunoassay devised primarily for locating blood group antigens on specific red-cell membrane proteins. The assay involves the incubation of intact red cells with two antibodies, one human alloantibody, the other a nonhuman antibody, usually a rodent monoclonal antibody, but polyclonal antibodies of rabbit origin have been utilized. For a positive result, both antibodies must bind to the same membrane protein. The red cells are lysed, the membrane solubilized and the trimolecular complex of two antibodies and membrane protein is captured in a well coated with goat antirodent (or rabbit) immunoglobulin. The immobilized complex is then detected by the use of peroxidase-conjugated goat antihuman (or rodent) immunoglobulin.
Negative results, due to mutual blocking between the human and animal antibodies when their epitopes are close together on the same molecule, have permitted a degree of localization of epitopes on some proteins. This has been most effective in the mapping of Cromer blood group system antigens on the complement control protein domains of decay-accelerating factor (DAF, CD55), but has also proved informative in the clustering of antigens on the Lutheran and Kell glycoproteins.
MAIEA is an effective tool for the identification of antibodies to Knops-system antigens on complement receptor 1 (CR1, CD35) in immunohaematology reference laboratories. These antibodies are clinically unimportant, but must be identified before they can be ignored for transfusion purposes.     

Use of a dual monoclonal solid phase and a polyclonal detector to create an immunoassay for the detection of human cardiac troponin I

OBJECTIVES: We report the development of a fully automated, random access, chemiluminescent immunoassay, for the detection of human cardiac Troponin I (cTnI) in serum and plasma for use on the ACS:180(R) System. DESIGN AND METHODS: This assay format uses a combination of two monoclonal antibodies covalently coupled to paramagnetic (PMP) particles as a solid phase and an affinity purified polyclonal antibody, specific to the N-terminal domain of cTnI (peptide-3 region) labeled with a chemiluminescent compound as the detector antibody. The assay offers excellent low-end sensitivity and precision. RESULTS: No interferences are observed from by blood components such as HAMA and drugs used in cardiac therapy. Patient samples tested on the ACS:180 cTnI assay showed good correlation with the Stratus cTnI assay (ACS: cTnI = 1. 02*Stratus + 0.05 g/L, r = 0.96, n = 1170). CONCLUSION: Paired with the other ACS:180 cardiac assays, myoglobin and CKMBII, the ACS:180 system now offers an excellent panel of cardiac assay for use in rapid and accurate diagnosis of a myocardial event.     

Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus.

ELISA has emerged as a useful alternative to other more costly and complex tests. Polystyrene microhaemagglutination plates have been used as solid phase to absorb Aspergillus fumigatus protein and polysaccharide components for detection of specific antibodies in patients with various forms of pulmonary aspergillosis. IgG and IgE antibodies to the polysaccharide as well as the protein allergens have been found. For the IgE test a double antibody technique has been developed, which is more sensitive than the conventional indirect ELISA.     

Development, characterization, and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells.

BACKGROUND: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5-1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. METHODS: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. RESULTS: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0-419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. CONCLUSION: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.     

Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection

Monoclonal antibodies (IG1, k) directed against a surface component of Plasmodium berghei sporozoites (Pb-44) confer complete protection to mice against a lethal inoculum of parasites. The degree of protection is a function of the number of parasites used in the challenge and of the antibody concentration in serum. Passive transfer of 10 micrograms of antibody per mouse abolished or profoundly diminished the infectivity of 10(3) sporozoites, but much higher amounts of antibody were required for complete protection against challenge with 10(4) parasites. Fab fragments of the monoclonal antibodies were as effective as the intact antibodies in mediating protection as determined by the neutralizing assay. This observation suggests that the antibodies interfere with a parasite function necessary for its infectivity, such as, for example, the ability to penetrate into the target cell or to multiply in the hepatocytes. When sporozoites are incubated with the intact monoclonal antibodies at 37 degrees C, a long filament appears at its posterior end (circumsporzoite precipitation [CSP] reaction). Fab fragments are ineffective at high concentrations. However, if after treatment with Fab, the sporozoites are incubated with rabbit antibodies to mouse k-chains, a strong CSP reaction is observed. We conclude that the CSP reaction can result from the cross-linking of Pb44 and that it has the characteristics of a capping reaction followed by the shedding of the immune complexes.     

Pharmacokinetics and pharmacodynamics of SB-240563, a humanized monoclonal antibody directed to human interleukin-5, in monkeys

The pharmacokinetics (PK) of SB-240563 have been investigated after i.v. and s.c. administration to cynomolgus monkeys. Approximately linear PK was observed following i.v. administration over a 6000-fold dose range (0.05-300 mg/kg). After i.v. dosing, SB-240563 concentration declined in a biexponential manner with a mean terminal half-life of 13 +/- 2 days. The plasma clearance and volume of distribution at steady state were approximately 0.2 ml/h/kg and 70 ml/kg, respectively. Following s.c. administration, SB-240563 was completely absorbed into the systemic circulation. Because interleukin-5 is known to stimulate production, activation, and maturation of eosinophils, eosinophil counts were measured to assess pharmacologic activity of SB-240563. The maximal response (81-96% decrease in eosinophil count relative to baseline) following a single s.c. administration occurred at 3 weeks postdosing. Suppression of eosinophil count also was observed following multiple monthly administrations of SB-240563 to monkeys. The pharmacokinetic/pharmacodynamic relationship was generally well described with an indirect pharmacologic response model with an estimated IC(50) value of 1.43 microg/ml. The combination of a low IC(50) value for reduction of circulating eosinophils and a long terminal half-life suggests the possibility of an infrequent dosing regimen for SB-240563 for treatment of diseases associated with increased eosinophil function such as asthma.     

Platelet-reactive HLA antibodies associated with low posttransfusion platelet increments:a comparison between the monoclonal antibody-specific immobilization of platelet antigens assay and the lymphocytotoxicity test

BACKGROUND: Platelet-reactive HLA antibodies are a major reason for low posttransfusion platelet increments. The clinical importance and value of the test systems for their in vitro determination is still controversial. STUDY DESIGN AND METHODS: A prospective analysis of HLA antibodies was performed in sera obtained once a week for at least 4 consecutive weeks from 55 patients (female/male, 28/27; age: median, 49 years; range, 18-69) undergoing intensive chemotherapy and in need of prophylactic platelet transfusions. All sera (n = 330) were analyzed by the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and by the standard lymphocytotoxicity test (LCT). RESULTS: In the MAIPA, 24.5 percent of sera (81/330) obtained from 22 patients contained HLA antibodies. These were detected significantly more often by the MAIPA assay than by the LCT (24.5% vs. 8.2%). Fifty-five sera (20 patients) were positive in the MAIPA assay only. In 15 patients, HLA antibodies were transient. In 3 patients, HLA antibodies were detected earlier by the MAIPA assay than by the LCT. Significantly more sera obtained at the time of low posttransfusion platelet increments were positive in MAIPA alone, rather than in both MAIPA and the LCT (44% vs. 17%). CONCLUSION: The MAIPA assay is more sensitive than the standard LCT in detecting platelet-reactive HLA antibodies. These MAIPA-positive/LCT-negative HLA antibodies affect the posttransfusion platelet increment.     

Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.     

Development of a sandwich ELISA to detect IgG and IgG sub-class antibodies specific for a major antigen (Ag 7) of Aspergillus fumigatus

A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub-classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin-prick test positive individuals. IgG sub-class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti-IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen-specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.     

A dot-ELISA intended for the specific and simultaneous detection of antibodies directed to antigens derived from Toxoplasma gondii, rubella virus, cytomegalovirus, and type 1 and type 2 herpesviruses

A procedure for the routine and simultaneous laboratory detection of IgG antibodies produced in humans in the course of various infectious diseases is described. The procedure, based on dot-enzyme-linked immunosorbent assay (ELISA), used single nitrocellulose strips onto which several antigens were dotted in close proximity. Optimal conditions were specified that allowed the unequivocal and simultaneous detection of IgG antibodies specifically directed against Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type 1 and type 2 antigens. This technique has proved to be simultaneously specific, sensitive, and reliable, and it has been applied to prenatal screening of sera from pregnant women. It is suggested that this technique should also be used for the screening of large numbers of sera under field trial conditions.     

Use of SAG2A recombinant Toxoplasma gondii surface antigen as a diagnostic marker for human acute toxoplasmosis: analysis of titers and avidity of IgG and IgG1 antibodies

We evaluated the reactivity of IgG and IgG1 antibodies by immunoassays in sera from patients with acute and chronic phases of toxoplasmosis against 2 recombinant antigens, SAG2A (full molecule) and SAG2ADelta (truncated molecule from the epitope recognized by A4D12 monoclonal antibody [mAb]), in comparison with soluble Toxoplasma antigen (STAg). Results demonstrated higher IgG reactivity in acute sera with both STAg and SAG2A than in chronic phase sera, and this difference was more evident for IgG1 antibodies to SAG2A. Low reactivity to SAG2ADelta was found in sera from both phases. ELISA-IgG-SAG2A showed high sensitivity (95%) and specificity (100%). ELISA-IgG1-SAG2A sensitivity was significantly higher (90%) for acute than for chronic (67%) phases. ELISA-IgG avidity using STAg demonstrated high performance for characterizing sera with high avidity (>60%), whereas the ELISA-IgG1 avidity-SAG2A immunoassay was the best to define chronic phase infection. It can be concluded that SAG2A is an antigen that may be used as a diagnostic tool to characterize the acute phase Toxoplasma gondii infection. Also, the epitope recognized by A4D12 mAb may be critical for the recognition of this molecule.     

抗载脂蛋白（a）单克隆抗体的制备及其应用

为建立特异有效的脂蛋白（ａ）检测方法，应用杂交瘤技术，制备出４株抗载脂蛋白（ａ）单克隆抗体，分别命名为Ｍ＿１、Ｍ＿２、Ｍ＿３、Ｍ＿４。各株单克隆抗体与纤溶酶原和载脂蛋白Ｂ等无交叉反应。单抗相加试验及竞争抑制试验结果表明：Ｍ＿１、Ｍ＿４识别同一抗原位点，Ｍ＿２、Ｍ＿３识别另一位点。选用Ｍ＿１、Ｍ＿２、Ｍ＿４３株单克隆抗体建立了测定血清脂蛋白（ａ）浓度的双抗体夹心酶联免疫测定法。样品稀释后测定范围为０．０５～１．６０ｍｇ／Ｌ，批内平均变异系数（ＣＶ）４．２％，批间平均ＣＶ８．７％。测定了１０３２例中老年临床血清标本，脂蛋白（ａ）浓度为１７９，１±１７９．３ｍｇ／Ｌ（ｘ±ｓ）。与多克隆抗体的酶联免疫测定法同时测定了３８份临床标本，结果相似。此法可满足临床脂蛋白（３）浓度检测的需要。