Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.     

Murine monoclonal antibodies that identify antigenically distinct subpopulations of human sperm

Murine monoclonal antibodies (MoAbs) were isolated to characterize antigenically distinct subpopulations of human sperm. Spleen cells from Balb/c mice immunized with freshly prepared human sperm, were fused with murine P3-NS1-Ag4-1 myeloma cells by somatic cell hybridization, and supernatants from the IgG-secreting hybridomas were screened by an enzyme immunoassay (EIA) for reactivity against fresh human sperm and a panel of human somatic cells. Two MoAbs, SP1D1 and SP7A7, reacted specifically with human sperm, whereas three others, SP2A9, SP3B3, and SP4F5, cross-reacted with a variety of human somatic cells. The binding of MoAbs were characterized by immunofluorescence, agglutination, and Staphylococcus aureus binding assays. We found that certain MoAbs bound to common antigens of the head and tail, or to tail alone, and had agglutinating activity. However, not all sperm were reactive to antibody, and the binding activity could only be demonstrated in subpopulations of sperm ranging from 5 to 50% of the total number.     

Two-site column enzyme immunoassay for neuron-specific enolase (NSE) in human serum using monoclonal antibodies

A new method for the rapid determination of neuron-specific gamma-enolase (NSE), gamma-subunit of alpha gamma- and gamma gamma-enolase in human serum was developed by employing monoclonal antibodies for the separation method. The assay system consists of 0.1 ml Sepharose 4B column with immobilized rabbit anti-mouse IgG antibodies for the separation of bound label, Fab' fragments of rabbit anti-bovine gamma gamma-enolase IgG labeled with beta-D-galactosidase from Escherichia coli, and F(ab')2 fragments of two mouse monoclonal antibodies to gamma gamma-enolase. Serum samples or standard NSE solutions were incubated at 30 degrees C with the monoclonal antibody fragments. 10 min later, the galactosidase-labeled antibody fragments were added to the mixture, and incubated at 30 degrees C for 30 min. Then the reaction mixture was applied to a micro-column of Sepharose 4B with immobilized anti-mouse IgG antibodies. From the galactosidase activity bound in the column, NSE concentration in the samples could be estimated within 2 h. The minimum detection limit of the assay system was 30 pg/tube, being sufficiently sensitive for the assay of serum NSE with a satisfactory precision. Serum concentrations of NSE determined by the present method correlated well with that by the colorimetric solid-phase immunoassay method.     

An enzyme immunoassay for rapid isotyping of monoclonal antibodies

An enzyme-immunoassay has been developed for rapid isotyping of murine monoclonal antibodies. This assay has several advantages over the commonly used typing methods. It does not require the presence of specific antigen. Typing plates can be prepared in advance and stored frozen. Unequivocal results can be obtained in 2 h, and the urease indicator system produces a vivid colour change that can be read visually.     

Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses.

An enzyme immunoassay for serotyping human rotaviruses in stools and in cell culture was developed. Hyperimmune rabbit antisera to rotaviruses were used as capture antibodies, and rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1, 2, 3, and 4 were used as detection reagents. Partial purification of monoclonal antibodies and inclusion of skim milk powder in antibody diluents contributed to assay specificity. The sensitivity of this assay was greater than that of a direct enzyme immunoassay in which rotaviruses of the appropriate serotype were adsorbed directly to the solid phase. When fecal extracts were concentrated threefold, this serotyping enzyme immunoassay was of equal specificity and approached the sensitivity of electron microscopy for rotavirus detection. This assay is simple and rapid and is suitable for serotyping the large numbers of isolates obtained from epidemiological studies and vaccine trials.     

An ELISA method for quantification of Tamm-Horsfall protein using monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MoAbs) to Tamm-Horsfall protein (THP) were established. The isotype and reaction pattern of the MoAbs with THP from rat, rabbit, guinea pig and man were employed for the selection of clones. At least four epitopes were recognised on human THP. One of these epitopes differed from the others in its dependence on the state of aggregation of the THP. An ELISA procedure was developed for quantification of THP in urine requiring no other sample treatment than dilution in the assay buffer. In this ELISA, THP showed an increased immunoreactivity after freezing.     

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.     

An enzyme immunoassay for the screening of monoclonal antibodies to cyclosporin

An enzyme-linked immunosorbent assay (ELISA) has been developed which allows hybridoma cell cultures to be tested for the presence of monoclonal antibodies specific for cyclosporin A. Immunization of mice with free cyclosporin was found to be preferable to immunization with cyclosporin-carrier conjugates. It is hoped that the availability of monoclonal antibodies to cyclosporin will clarify the contribution of cyclosporin metabolites to immunosuppression and nephrotoxicity.     

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies

Three human and three murine monoclonal antibodies were tested for their reactivity to tetanus toxin and toxoid and used to establish an enzyme immunoassay specific for tetanus toxin. The dissociation constants of the monoclonal antibodies were between 3.91 x 10(-9) and 8.48 x 10(-12). Two human monoclonal antibodies recognized conformation determinants on the toxin, whereas the others reacted to the heavy chain. Only a combination of antibodies of the two species allowed the development of an enzyme immunoassay for the detection of tetanus toxin with a lower detection limit of 1.2 micrograms/l.     

An enzyme immunoassay for cuprozinc superoxide dismutase using monoclonal antibodies. Application for pharmacokinetic study

We developed an enzyme immunoassay for determining human cuprozinc superoxide dismutase (h-SOD) using two kinds of monoclonal antibodies prepared by immunizing h-SOD to BALB/c mice. This method was sensitive and specific enough to determine exogenous h-SOD injected into rats. When intravenously injected into rats, much of the immunoreactive h-SOD accumulated in the kidney and was rapidly excreted in the urine. We observed both a modified and an unmodified form of exogenous h-SOD in rat urine.     

Detection of IgG antibodies to measles virus using monoclonal antibodies for antigen-capture in enzyme immunoassay

An enzyme immunoassay (EIA) for the detection of IgG antibodies to measles viral hemagglutinin (H) has been developed. Monoclonal antibodies were employed as antigen-capture reagents on a polystyrene ball solid phase. The antigen-capture EIA was significantly more sensitive than hemagglutination inhibition (HAI) and somewhat more sensitive than indirect immunofluorescence and indirect EIA for the detection of antibodies to measles virus. The importance of selecting a monoclonal antibody with a high binding affinity, and controlling for non-specific adherence of antigen or antibodies to the solid phase were demonstrated. This method offers not only greater sensitivity than HAI, but also the practicality of reagents capable of standardization and long term storage.     

Development of a sensitive enzyme immunoassay for protein 1/Clara cell 10 kDa protein using monoclonal antibodies]

Protein 1 (P1)/Clara cell 10 kDa protein is a dimer of identical subunits of 70 amino acids. Designed to expand its clinical significance, a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for P1 was developed using two monoclonal antibodies, which were recently generated and characterized in this laboratory. The precision, reproducibility and specificity of the assay were quite satisfactory. Species specificity was also satisfactory. The detection sensitivity was 5 ng/l; high enough to quantify P1 in several body fluids. P1 was distributed in the value of 13 to 25 micrograms/l, 153-4300 micrograms/l and 145-8600 micrograms/l, in serum, bronchoalveolar lavage fluids and seminal fluids, respectively.     

An antibody chimera technique applied to enzyme immunoassay for human alpha-1-fetoprotein with monoclonal and polyclonal antibodies

A 2-step enzyme immunoassay (EIA) for human alpha-1-fetoprotein (AFP) is proposed, which uses covalently coupled anti-AFP IgG and anti-horseradish peroxidase (HRP) IgG (antibody chimera) binding HRP as the marker enzyme immunologically. The use of polyclonal and monoclonal anti-AFP linked to anti-HRP antibodies was compared with a conventional 2-site binding EIA with HRP covalently bound to anti-AFP IgG. The sensitivity of the conventional EIA is increased by the use of an antibody chimera comprising a molar ratio of anti-AFP IgG: anti-HRP IgG of 1:8, especially if monoclonal antibodies are employed. This improved sensitivity may be achieved by a very simple coupling procedure without purification of conjugate and with very crude HRP preparations.     

A monoclonal antibody and an enzyme immunoassay for human Ala-IL-8(77)

We used a relatively small library of 5520 randomly generated single 15-mer peptides prepared by SPOT synthesis as an array of 28.5x19.0 cm to identify epitopes for three distinct monoclonal antibodies, namely anti-p24 (human immunodeficiency virus (HIV)-1) monoclonal anibody (mab) CB4-1, anti-interleukin-10 (IL-10) mab CB/RS/13, and anti-transforming growth factor alpha (TGFalpha) mab Tab2. Initially identified peptide ligands mostly had very low affinities for the antibodies with dissociation constants around 10(-4) M. Subsequent identification of residues critical for the antibody interactions involved complete L-amino acid substitutional analyses. Several substitutions resulted in analogs with dissociation constants in the low micromolar and high nanomolar range. Specifically binding peptides with key residue patterns matching the wild-type epitopes were identified for all three antibodies. In addition, for antibody CB4-1 mimotopes that showed no homology to the known epitope were selected. Our results suggest that a very limited library diversity, although far from covering the entire sequence repertoire, can suffice to rapidly and economically select peptidic antibody epitopes and mimotopes.     

Two-step sandwich enzyme immunoassay using monoclonal antibodies for detection of soluble and membrane-associated human membrane type 1-matrix metalloproteinase

A two-step sandwich enzyme immunoassay (EIA) system for the detection of human membrane Type 1-matrix metalloproteinase (MT1-MMP) was established by using two monoclonal antibodies against recombinant MT1-MMP. MT1-MMP in which samples were reacted with solid-phase antibody and then detected with peroxidase-labeled second antibody. At least 1.25 ng/mL was detected by the EIA system, and linearity was obtained between 1.25 and 160 ng/mL. This EIA system is specific for MT1-MMP and did not show cross-reactivity against several other MMP's examined. Shedding of soluble MT1-MMP into the medium by some cancer cell lines was also detected by this system. However, soluble MT1-MMP in serum from normal and cancer patients was under the detection limit. Membrane-associated MT1-MMP of cancer cell lines was also detected after solubilization of the membranes with extraction buffer containing detergent. Additionally, MT1-MMP in clinical samples was examined. Elevated levels of MT1-MMP were detected in homogenate of cancer tissue compared with the levels for normal tissue and the level of MT1-MMP in tumors correlated with the rate of metastasis to the regional lymph nodes. Thus, we demonstrated that this EIA system is the first to measure MTI-MMP in clinical specimens, thus suggesting its useful for diagnosis of cancer or prediction of malignancy.     

Detection of alphaviruses in a genus-specific antigen capture enzyme immunoassay using monoclonal antibodies.

A genus-specific antigen capture assay using similar combinations of monoclonal antibodies for capture and detection of 24 alphaviruses belonging to the seven serocomplexes was developed. The sensitivity of the test ranged from 10(3.4) 50% tissue culture infective doses/ml for o'nyong-nyong virus to 10(6.1) 50% tissue culture infective doses/ml for Middelburg virus. The antigen capture test uses a combination of cross-reacting monoclonal antibodies directed against the nucleocapsid protein and envelope glycoprotein E1 of Semliki Forest virus.     

Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes

Monoclonal antibodies (MAb) which recognize distinct epitopes on human immunoglobulin E have been used to develop two-site sandwich radio- and enzyme-linked immunoassays for the quantitation of human IgE. In the first step, a purified anti-IgE MAb coated to polyvinyl or polystyrene microtiter plates specifically bound the IgE contained in the samples. In the second step, another anti-IgE MAb (either iodinated or conjugated to beta-galactosidase) directed to a different antigenic determinant was used to estimate the amount of bound IgE. This simple method permitted the determination of IgE concentrations of 10 ng/ml and greater in about 3 h. Coefficients of variation on a single day did not exceed 7.5% for IgE levels, covering a wide range of the standard curve. The values obtained on serum samples showed a good correlation with those obtained using the paper radioimmunosorbent test (PRIST).     

Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies

Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.     

Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies

A two-site sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Monoclonal anti-beta-hCG antibody was used for coating microtitration plates and monoclonal anti-alpha-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or beta-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r = 0.968) with the values obtained by radioimmunoassay.     

Development of an enzyme immunoassay for human placental lactogen using labelled antibodies.

An immunoassay for the determination of human placental lactogen (HPL) has been developed using specific antibodies labelled with horseradish peroxidase. A method is presented in which the enzyme : antibody conjugate is purified with an antigenic solid phase to ensure the production of conjugates with intact immunological activity. Serum components interfere with the assay but this problem can only be avoided at the expense of a precise and practical assay for the determination of HPL.