A modified ELISA that selectively detects monoclonal antibodies recognizing native antigen

A modified ELISA procedure is described which permits selective detection of monoclonal antibodies reactive with the native antigen. The essence of the method is the use of immobilization methods that do not result in denaturation of the antigen during adsorption to the microtiter plates. This was accomplished by using polyclonal antibodies, adsorbed to the wells of the microtiter plate, as the immobilizing agent.     

Mapping epitopes for 20 monoclonal antibodies to CR1

Complement receptor type one (CR1; CD35) binds and processes C3b and C4b opsonized immune complexes and regulates complement activation. We have characterized the epitopes of 13 previously reported and seven new MoAbs to human CR1. The MoAbs formed seven groups based on their reactivity with a panel of deletion forms of CR1. Seventeen of the MoAbs reacted with CR1 at more than one site, a consequence of its repetitive sequence. All five of the MoAbs recognizing epitopes in the nearly identical repeats 3, 10, and 17, as well as one MoAb which reacted with repeats 8 or 1/2 of 9 and 15 or 1/2 of 16, blocked cofactor activity for C3b. Knowledge of the repeats bearing the epitopes for these MoAbs should facilitate the further characterization of CR1.     

A simple method for ranking the affinities of monoclonal antibodies

Measurement of the binding of constant trace amounts of labelled antigen by increasing dilutions of culture supernatant allows the ranking of monoclonal antibodies in the order of their affinity for the antigen. The theoretical basis for this method is discussed and it is illustrated with data from a set of anti-alphafoetoprotein monoclonal antibodies. Hybridomas secreting antibodies of desired affinity for immunoassay, histochemistry or antigen purification can thus be selected at an early stage after fusion.     

ELISA tests and monoclonal antibodies for EBV

Monoclonal antibodies have now been prepared against a number of the major polypeptides associated with the different EBV-induced antigenic complexes. These antibodies have made it possible to purify and characterize most of these polypeptides and to identify the viral genes encoding for these proteins. In addition, new ELISA tests have been developed which measure antibodies in different immunoglobulin classes against the purified polypeptides. These assays have been useful in the diagnosis of EBV-associated diseases in clinical laboratories. The development of these antibodies and their application to the development of the new ELISA tests will be reviewed in this paper.     

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.     

Competitive ELISA employing monoclonal antibodies specific for dothistromin

Dothistromin (DOTH) is a fungal toxin occurring in Pinus radiata needles contaminated with Dothistroma pini. Monoclonal antibodies (MAbs) of high affinity were prepared against DOTH and incorporated into competitive ELISAs. MAbs were secreted by hybri‐doma prepared from mice immunized with DOTH conjugated, through the aromatic ring of the anthraquinone, to bovine serum albumin. DOTH could be quantitated at 2–60 ng ml^‐1 (0.1–2 ng/assay) and at 8–300 ng ml^‐1 (0.4–15 ng/assay) using peroxidase‐labelled MAb 10C12A5 and MAb 6A2D4 respectively in indirect competitive ELISA. The corresponding limits of detection of DOTH were < 300 pg and 1 ng ml^‐1 respectively. The furan ring was an important structure in the epitope recognized by both MAbs.     

A simple guide to the terminology and application of leucocyte monoclonal antibodies

This review aims to provide a simple guide and quick reference to the terminology and diagnostic applications of leucocyte monoclonal antibodies. The differentiation cluster terminology, where applicable, is used throughout.     

A simple, rapid ELISA method for the detection of DNA antibodies.

Employing an enzyme-linked immunosorbent assay (ELISA) technique the serum antibodies against native (double stranded) and denatured (single stranded) deoxyribonucleic acid (DNA) have been measured in various disease groups and a group of blood donor sera. The ELISA method has been compared with a radioimmunoassay method using native (double stranded) DNA is substrate antigen and a latex-fixation technique using particles coated with soluble deoxyribonucleoprotein (SNP). It is concluded that ELISA offers an economic and reliable alternative to isotope techniques for the assessment of antibody content in systemic lupus erythematosus (SLE) and related disease states for the clinical laboratory.     

Loss of ELISA specificity due to biotinylation of monoclonal antibodies

A significant degree of nonspecificity was found in ELISA determinations of soluble urokinase receptor (suPAR) in human blood plasma when biotinylated monoclonal antibodies (Mabs) were used for the detection layer. Surface plasmon resonance studies using both nonbiotinylated and biotinylated antibodies demonstrated that biotinylation reduced specific binding of the antibodies to their target antigen, suPAR. Furthermore, biotinylation produced a new interaction with unknown human plasma protein(s), unrelated to suPAR. Nonspecific interaction with plasma protein(s) was also observed after biotinylation of a Mab having no specific target antigen in human plasma and, in both cases, the level of nonspecific interaction was directly related to the degree of antibody biotinylation. These results reinforce earlier observations that biotinylation of antibodies can reduce the affinity of antibodies, but also indicate that, in addition, biotinylation can reduce the specificity of immunoassays for plasma proteins.     

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.     

Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA

We found in preliminary studies using 125I-labelled antibodies that an antibody bound to a solid-phase antigen was recognized more efficiently than an antibody adsorbed directly to the solid phase. The present study was designed therefore to quantitate the differential recognition of an antibody adsorbed directly to the solid phase and an antibody bound to antigen on the solid phase using the amplified enzyme-linked immunosorbent assay (a-ELISA), and to compare results with the amounts of specific antibody determined by quantitative immunoprecipitation. The degree of differential recognition was quantitated for rabbit IgG and SIgA anti-ovalbumin (anti-OA) and anti-fluorescein, and was found to be dependent upon the isotype of the antibody and not its specificity. The ratio describing the differential recognition of SIgA antibodies (1.8) was much less than for IgG antibodies (greater than 30) and remained constant over the titration range analyzed while the ratios obtained for IgG varied substantially (25-60) over the same range. These ratios of differential recognition were used to estimate rabbit IgG antibody levels to OA, bovine serum albumin, ferritin and alpha-lactalbumin. The estimates obtained were consistently much less than total antibody levels measured by quantitative precipitation. The use of glutaraldehyde-aggregated OA in the ELISA, however, increased the amount of IgG anti-OA and SIgA anti-OA capable of recognizing OA adsorbed on plastic from 12 to 50 and from 30 to 80%, respectively.     

Mapping epitopes on the insulin molecule using monoclonal antibodies

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.     

Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product

An enzyme-linked immunosorbent assay (ELISA) has been described for serological determination of hepatitis B virus genotypes, using monoclonal antibodies (mAb) against seven distinct epitopes (b, m, k, s, u, f and g) on the preS2-region products of hepatitis B surface antigen (HBsAg). The usefulness of this method for serological detection of genotype E, however, was theoretical, because no HBsAg samples of this genotype were included in the original test panel. Moreover, the predicted serotype of genotype E (bksufg) closely resembled that of genotype D (bksu, bksuf or bksug). Four HBsAg samples of genotype E were tested by the original described ELISA. The epitope g, predicted to be present in these samples by amino acid sequences, was not detected when HBsAg of genotype E was captured on a solid phase by mAb to the common determinant 'a' of HBsAg and then reacted with mAb to g (5156) labeled with horseradish peroxidase. However, the four examples of HBsAg of genotype E were captured by mAb 5156 to g on a solid phase; they were then detected by labeled mAb to the common determinant 'a'. Since epitopes f and g co-occurred on HBsAg of genotype E, HBsAg samples of this genotype were also detected, by 'sandwiching' them between immobilized mAb to g and labeled mAb to f. By contrast, HBsAg of genotype D in 90 sera was not reactive when sandwiched between mAb to f and g. Thus, this modified ELISA enables the serological determination of all six genotypes of HBsAg and, by inference, of hepatitis B virus.     

An ELISA to detect monoclonal antibodies specific for lipid determinants of Mycoplasma pneumoniae

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of monoclonal antibodies directed towards lipid determinants of Mycoplasma pneumoniae. Chloroform-methanol lipid extracts as well as chromatographed lipid fractions of M. pneumoniae were bound to polyvinylchloride microtiter wells. Of 293 clones positive to M. pneumoniae as detected by a whole cell ELISA, a total of 78 clones produced antibodies which selectively bound to lipid extracts of M. pneumoniae. The simplicity and reproducibility of the assay permit rapid screening for detection of antibodies directed against non-protein cellular antigens.     

ELISA for detection of Mycoplasma pneumoniae antigens using monoclonal antibodies

Seven monoclonal antibodies directed against major antigens of Mycoplasma pneumoniae were selected for the development of an antigen detection assay. Three of these were directed to the 170,000-dalton adhesin of M. pneumoniae. The test was an antigen-capture enzyme immunoassay using the different monoclonal antibodies for capture of antigen and a polyclonal rabbit antiserum as detection reagent. With three of the monoclonal antibodies a detection limit of approximately 2 ng M. pneumoniae protein was obtained, as determined by titration of M. pneumoniae organisms in buffer. The detection limit of the assays was only slightly less when the other four monoclonal antibodies were used. In artificially infected nasopharyngeal aspirates the detection limit was approximately 10 times lower. The fact that no significant differences in the detection limit of the assays were recorded using monoclonal antibodies directed against different antigens indicates that these antigens were available for reaction with antibodies irrespective of their location in intact M. pneumoniae cells. In the assay there were no significant cross-reactions with a number of bacterial species potentially colonizing the respiratory tract, except for a protein A-positive strain of Staphylococcus aureus. Our test is equally sensitive to another recently described ELISA using polyclonal antibodies. In comparison with other recommended methods such as immunoblot and culture-amplified antigen detection assays, the ELISA is more rapid and less laborious.     

A simple, rapid method for screening hybridomas producing monoclonal antibodies to platelet proteins

Several parameters influence the outcome of somatic cell fusions based on the K?hler and Milstein technology, and a number of steps are of critical importance, including the screening strategy. The procedure chosen, appropriate for the type of antibody required, should be rapid and sensitive, in order to clone the relevant hybrids as quickly as possible. A simple and quick dot blot-based method is reported, suitable for screening hybridoma culture supernatants in order to identify clones producing monoclonal antibodies to platelet constituents.     

Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation

A method is described for determining whether particular monoclonal antibodies are specific for carbohydrate or non-carbohydrate antigenic determinants. In a model system consisting of the Lewis a human blood group determinant attached to either protein or lipid, mild periodate oxidation destroyed the carbohydrate determinant without altering protein or lipid epitopes. The technique was readily applied to antigens bound to plastic wells for ELISA, to nitrocellulose sheets for Western blots, and to thin layer chromatography (TLC) plates for TLC immunostaining. Mild periodate oxidation can prove useful during the early stages of hybridoma screening in order to select for or against anti-carbohydrate antibodies.     

人脱嘌呤脱嘧啶核酸内切酶单克隆抗体抗原表位的鉴定及应用

目的:研究鉴定人脱嘌呤脱嘧啶核酸内切酶单克隆抗体(hAPE1 mAb)的抗原表位,并建立定量检测hAPE1的ELISA一步法。方法:设计并合成APE1-15肽阵列,鉴定hAPE1 mAb 2-G1和4-F6的抗原表位,应用三维立体结构观察软件Molsoft.ICM-Pro模拟hAPE1 mAb抗原表位的立体结构;采用改良的过碘酸钠法标记抗体,以hAPE1 mAb为捕获抗体和酶标抗体,建立hAPE1的ELISA一步检测法。结果:APE1-15肽阵列检测结果和抗原表位三维结构显示,2-G1mAb的抗原表位对应为hAPE1天然蛋白氨基酸残基序列的76-90位和109-123位,位于氧化还原区域,为构象型抗原表位;4-F6 mAb的抗原表位对应为hAPE1天然蛋白氨基酸序列的109-147位,位于DNA修复内切酶活性区。ELISA一步法检测hAPE1蛋白的线性范围为8.0~200μg/L,最低检测限为2.0μg/L。平均批内变异系数为8.67%,平均批间变异系数为12.45%,平均回收率为105.47%。结论:hAPE1 2-G1 mAb和4-F6 mAb具有不同的抗原表位,成功建立的hAPE1 ELISA一步法为简便、快速、准确检测血清中hAPE1的含量奠定了基础。     

A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.     

Development of monoclonal antibodies and ELISA specific for the mouse vascular endocan

Human vascular endocan is a proteoglycan exhibiting tumorigenic activity through both its glycan and protein cores. Endocan mRNA is identified as being one of the most significant molecular signatures defining a poor prognosis in lung, breast, kidney, prostate, and thyroid malignancies. The survival inversely correlates with endocan expression in tumor tissue from hepatocarcinoma, and in serum from lung cancer. In mouse, endocan mRNA is also increased in tumor vessels. However, mouse endocan has not yet been fully characterized. Here, we produced a panel of rat monoclonal antibodies directed against mouse endocan, leading to the development of a specific mouse/rat endocan ELISA. Mouse endocan serum level was measured at a median of 0.96 ng/mL and 1.08 ng/mL in 129Sv mice and C57bl6, respectively. These results also provide new tools to characterize and explore the role of endocan in mouse and rat models of human diseases. These results present mouse vascular endocan as a circulating molecule similar to human endocan.