Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.     

Immunoperoxidase slide assay (IPSA)--a new screening method for hybridoma supernatants directed against cell surface antigens compared to other binding assays

A modified immunoperoxidase assay on microscope slides (IPSA) was adopted as a screening procedure for hybridoma supernatants with specificity for human lymphocyte subpopulations. The method proved to be suitable for testing a large number of hybridomas with a minimum of cells and reagents. In addition, the slide-technique yields considerable information about the type and morphology of target cells. This allows a first differentiation between cell types in the assayed preparation, e.g. between lymphocytes and monocytes. Compared to indirect immunofluorescence, solid-phase radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) with intact cells as antigen, IPSA turned-out to be as sensitive as RIA and superior to indirect immunofluorescence and ELISA. Nevertheless, none of the assays used detected all supernatants binding to cells in the right manner and could, thus, be considered ideal. In our hands, the combination of RIA and IPSA proved to be an excellent system for screening procedures on lymphocytes.     

Release of proenkephalin-derived opioid peptides from rat striatum in vitro and their rapid degradation

In a previous paper we demonstrated that the heptapeptide [Met]enkephalyl-Arg6-Phe7 was released from rat striatal slices by high K+ concentration and rapidly degraded by peptidases, even in the presence of the neutral endopeptidase 24.11 ("enkephalinase")-inhibitor, thiorphan (0.1 microM), the angiotensin-converting enzyme inhibitor, captopril (1 microM), and the aminopeptidase inhibitor, bestatin (20 microM). In this study the pattern of degradation of exogenous [3H]heptapeptide by rat striatal slices has been studied. The angiotensin-converting enzyme and aminopeptidase(s) were partly responsible for this degradation. In addition an enzymatic activity that cleaved the Phe4-Met5 bond was involved in the degradation of the heptapeptide by striatal slices. This activity was inhibited by the dipeptide Leu-Arg (1 mM) and the tripeptide Leu-Arg-Leu (1 mM). The simultaneous presence of thiorphan (0.1 microM), captopril (1 microM), bestatin (20 microM) and Leu-Arg (1 mM) almost completely inhibited the degradation of [3H]heptapeptide by striatal slices. In the presence of these peptidase inhibitors a concomitant release of [Met]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8 was evoked by KCl or veratridine. The K+-evoked release was by a Ca2+-dependent mechanism and the release evoked by veratridine was blocked by tetrodotoxin. In both cases the ratio of [Met]enkephalin to heptapeptide amounts released was close to that found in their common precursor, proenkephalin. Thus the enkephalinergic neuron appears to be capable of synthesizing, from a unique precursor, four different putative opioid neurotransmitters, namely [Met]enkephalin, [Leu]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8, to store these peptides and to release them upon depolarization.     

Carbon-protein covalent conjugates in non-instrumental immunodiagnostic systems

An original technique for obtaining stable conjugates using colloid carbon particles as indicator labels has been developed. The reliability and stability of the diagnostic reagents obtained is provided by covalent binding of various affine compounds on the surface of carbon particles. The stability of the reagents has been studied under various storage conditions for 3-10 years. It was shown that even when storage conditions were outside the optimal range, some conjugates preserved their analytical characteristics for 10 years. A number of systems for analytical detection of various ligands have been designed based on the carbon-protein conjugates synthesized. The major characteristics of these systems are their high sensitivity and specificity, reliability, and reproducibility, simplicity in operation, and quick results.     

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.     

Development of a sensitive solid-phase radioimmunoassay technique for quantification of class-specific Escherichia coli antibodies

A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions. Preparation of affinity-purified anti-E. coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 microliters of sample. This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection. RIA also compares favourably with ELISA in sensitivity and sample size required. Affinity-purified standards may also be used to quantify the ELISA test.     

An enzyme immunoassay for the detection of staphylococcal protein A in affinity-purified products

Rabbit antiserum, specific for protein A from Staphylococcus aureus, was conjugated to alkaline phosphatase and used in a double antibody solid-phase enzyme immunoassay. The assay was developed to monitor eluate from a large-scale protein A-Sepharose affinity column used to purify monoclonal antibodies for human clinical trials. The assay detected soluble protein A in the presence of immunoglobulin at concentrations as low as 4 ng/ml. Analysis of the product purified by affinity chromatography revealed the presence of protein A at ng/ml concentrations. The assay developed here can provide a reliable and convenient method for detecting soluble protein A.     

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

Antibody production by human X human hybridomas in serum-free medium

Four human X human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human X human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies.     

Simultaneous screening for two different antibodies in ELISA by combining two solid phases: microtiter plate and Falcon assay screening test system lid

An enzyme-linked immunosorbent assay (ELISA) is described that used a combination of 2 solid phases: a microtiter plate covered with a Falcon assay screening test (F.A.S.T.) system lid. By coating each solid phase with a different antigen, it was possible to simultaneously detect 2 different antibodies. The results of the combined test were compared and found similar to those obtained in separate assays on the same solid phases by the usual ELISA method. Further, the combined test was more economical in time, work and materials than 2 separate tests for the 2 antibodies. This method was used in a serological survey for 2 turkey viral infections: hemorrhagic enteritis and paramyxovirus type 3.     

A potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. The antigen-antibody complex was precipitated with normal human serum as the carrier protein, followed by the addition of rabbit anti-human IgG F(ab')2 serum. With this method, different H-D antigen-active molecules were compared for heterophile H-D antigen potency with reasonable sensitivity detecting about 0.3 ng of cold glycoprotein. 8 different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attempt to correlate expression of H-D antigen on tissues with elevation of H-D antibodies. The results showed that all patients' tissues expressed the antigen(s) but only 3 of them had abnormal levels of H-D antibodies. This could have been due to excess antigens in circulation or immune complexes.     

Cross-reactivity between solid-phase immunoassay plates and intermediate filaments demonstrated by human monoclonal antibodies

While screening supernatants of human-human hybridomas for rheumatoid factor and anti-cellular activity we found that a significant number of supernatants which react with the Falcon-polyvinyl chloride immunoassay plate used in an enzyme-linked immunosorbent rheumatoid factor assay also react with intracellular intermediate filaments.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

A double antibody radioimmunoassay for mouse hemoglobins: use of polyethylene glycol in conjunction with the second antibody.

A double antibody radioimmunoassay (RIA) system is described for detection of small quantities of hemoglobins. Mouse (C57BL/6) hemoglobin and horse anti-mouse hemoglobin antiserum were used to develop the system.The first phase of the RIA, i.e., the initial reaction between the antigen and the antibody, was found to be complete within 24 h. The reaction proceeded better at 4°C than at 25°C. The second phase, i.e., separation of bound from unbound antigen, was achieved by precipitation with a second antibody (goat anti-horse IgG) and polyethylene glycol (PEG). A 50 g/l concentration of PEG was found to be best suited for the assay. Mixing of all the reagents together was found to decrease the binding as compared to the system in which second antibody and PEG were added after completion of the first phase. Maximum precipitation of the complex took place within 30 min after the addition of the second antibody and 1 h after the addition of 50 g/l PEG.The RIA system described here combines the conventional double antibody RIA with the PEG method. This method has decreased the amount of time necessary for precipitation from 24 h (or longer) to 1 h. Large molecular weight antigens could not be estimated in the conventional PEG method because of their insolubility in 200 g/l PEG utilized in the assay. The use of a low concentration of PEG along with the second antibody in the method described here allows the estimation of large molecular weight antigens. This double antibody-PEG method has a general applicability for small as well as large molecular weight antigens.     

A statistical approach to determine monoclonality after limiting cell plating of a hybridoma clone

One of the standard methods to isolate a hybridoma clone producing a monoclonal antibody requires successive steps of limiting dilution. The probability of obtaining a monoclonal antibody increases with the number of limiting dilution steps. However, without meticulous visual screening monoclonality is hard to prove. Here we describe a statistical analysis, based on Poisson's approximation, which allows one to calculate the number of hybridoma cells at a given plating efficiency so that when seeded a predictable number of mono-, bi-, etc.-clonal cultures are obtained after the first step of limiting cell plating.     

An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.     

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.     

Interaction of carboxypeptidase A with monoclonal antibodies

Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinc-containing exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of approximately 10(6) M-1 were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.     

An ELISA assay for murine amyloid a and serum amyloid a utilizing monoclonal antibodies

An enzyme-linked immunoassay has been developed to quantitate the amyloid A (AA) and serum amyloid A (SAA) proteins of mice. The assay utilizes monoclonal rat anti-mouse AA as the antibody. The principle advantage of this method is that it avoids the need to denature the mouse sera before its SAA content can be measured.