Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.     

A dot-immunobinding assay for monoclonal and other antibodies

A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Applications for screening supernatants of hybridomas making monoclonal antibodies, and the use of such antibodies in the determination of the tissue distribution of the corresponding antigens, are described. An application for the screening of human pathological sera for multiple antibodies in one operation is also described.     

Antibodies specific to a deoxyribodinucleotide sequence.

Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the antisera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.     

Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

The use of a nitrocellulose-enzyme immunoassay for the rapid screening of monoclonal antibodies to human enteroviruses

A simple, indirect enzyme immunoassay using purified enterovirus adsorbed on to nitrocellulose has been developed for screening monoclonal antibodies to enteroviruses. The sensitivity of the assay ranged from 10 ng to 1 microgram of viral protein and was 10- to 50-fold more sensitive than conventional EIA on microplates. This simple, sensitive and specific assay proved to be a useful and practical tool for detecting monoclonal antibodies which would not be found in a conventional EIA screening procedure.     

The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax

Dextran (molecular weight, 71,200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonal antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay.     

Sequence specific antibodies to a deoxyribodinucleotide

Antibodies were elicited in rabbits to the dinucleotide, dpTpA, conjugated to a carrier protein. Among a few deoxyribomono- and oligonucleotides tested for binding to the antibodies by nitrocellulose tilter binding assay, only dpTpA and dpTpA containing oligonucleotides showed binding. The inhibition of the binding of 3H-dpTpA to the antibodies by various nonradioactive mono- and oligonucleotides showed that the antibodies were sequence specific and recognized the whole molecule of dpTpA. dpTpA specific antibodies were purified by a two step affinity chromatography. By competition studies, it was found that the purified antibodies bound denatured DNA at dpTpA sequences. The antibodies did not bind RNA.     

Antibodies specific to two deoxyribotrinucleotide sequences.

Antibodies to the deoxyribotrinucleotides dpApTpA and dpApApT were prepared by injecting the bovine serum albumin conjugates of the respective haptens in rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of the tritiated haptens to the immunoglobulins by various nonradioactive mono- and oligonucleotides, using nitrocellulose membrane binding assay. Anti-dpApTpA and anti-dpApApT antisera were found to contain antibodies which were highly specific to the respective hapten sequence.     

Immunoprofiling of pectic polysaccharides

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.     

Negative purification method for the selection of specific antibodies from polyclonal antisera

We developed a protocol to remove non-specific antibodies from polyclonal antisera by adsorption on non-target antigens immobilized on nitrocellulose membranes. This "negative" purification method is simple and provides better immunoreagents than the blocking of nonspecific antibodies in solution or the enrichment of specific antibodies on nitrocellulose membranes. For routine applications, this method is quicker and cheaper than the purification protocols based on selective precipitations and affinity chromatography.     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

A simple immunological detection method for the direct screening of genes from clone banks

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.     

Development of a simplified method for subclass isotyping and screening monoclonal antibodies

A simplified dot-blot procedure for screening and subclass isotyping of monoclonal antibodies is described in which only 0.5-50 ng of antigen and 1 microliter of antibody are needed to perform the test. The results on the nitrocellulose membrane can be stored indefinitely for future reference. This method is less expensive, uses smaller quantities of antigen and antibody, and is faster than presently used enzyme-linked immunosorbent assay techniques or other dot-blot methods. Monoclonal antibodies against Mycoplasma gallisepticum were screened and isotyped using this technique.     

Quantification of DNA-dependent RNA polymerase subunits and initiation factor(s) by antibody-linked polymerase assays

The antibody-linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS-denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates.     

Monoclonal antibodies against eucaryotic ribosomes. Use to characterize a ribosomal protein not previously identified and antigenically related to the acidic phosphoproteins P1/P2

Mice were immunized against chick ribosomes with the use of various protocols and immunogen preparations. Hybridomas were prepared, clones screened, and specific antibodies identified by reversible protein staining followed by immunoperoxidase staining on nitrocellulose blots. Clones were obtained which secreted specific antibodies against ribosomal proteins S6, L7, L18a, P1/P2, and also against ribosomal RNA. Antibodies were typed by means of a dot-binding assay with typing antibodies immobilized on a solid support of nitrocellulose, and also characterized by their species cross-reactivities. The common determinant on proteins P1 and P2 cross-reacted with proteins of similar molecular weight in all eucaryotes tested, and with a determinant in a previously uncharacterized 38,000-dalton protein of the large ribosomal subunit. We designate this protein P0. The determinant of P0 was also present in a protein of similar molecular weight in all eucaryotes tested. Unlike P1 and P2, P0 was not removable from ribosomes by an ethanol-NH4Cl washing procedure. No evidence for a precursor-product relationship between P0 and P1/P2 was found. P0, P1, and P2 were found in active polysomes and in the nucleolus. The molecular weights of the nucleolar forms were not identical with those of the cytoplasmic forms, suggesting some processing during ribosomal assembly and/or transport.     

Rapid isotyping of murine monoclonal antibodies by enzyme-linked immunofiltration assay

Summary As a part of the initial characterization of monoclonal antibodies, the isotype is routinely identified by enzyme-linked immunosorbent assay (ELISA). In the present study, we describe an isotyping methodology that uses the technique referred to as enzyme-linked immunofiltration assay (ELIFA) for greatly accelerating the assay compared with ELISA. In the ELIFA method, solutions are filtered through a nitrocellulose membrane using a controlled flow rate to bind proteins. By using this technology, the time required to isotype a monoclonal antibody was reduced from a minimum of 4 to 8 hr using a standard ELISA assay to 30 min with ELIFA.     

Dot immunodetection for sphingomyelinase with monoclonal antibody

An assay for the detection of sphingomyelinase with monoclonal antibodies is described. The assay takes advantage of nitrocellulose membranes as antigen adsorbent on which a dilute sample can be concentrated as a spot, using a specially designed 96-well filtration device which is commercially available. The method requires only 6 micrograms of the extracts from leukocytes and liver, and it is 10 times more sensitive than the colorimetric assay. This reduced amount of sample material also has the merit of requiring only a 0.5-ml blood sample from patients. The combination of this dot immunoassay with the monoclonal antibody allows a sensitive and a specific assay, and is also applicable as a screening test on a large number of samples. Furthermore, the possibility of differential diagnosis of Niemann-Pick disease types by detecting isoenzymes by this method was examined after isoelectric focusing of placental sphingomyelinase.     

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.     

Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immobilized on nitrocellulose

The nitrocellulose binding assay was used for quantitative studies on the cytochemical reactions for the three enzymes most frequently used in immunocytochemistry. The results show a linear relationship between the amount of enzyme immobilized on nitrocellulose and the amount of the enzyme reaction product. The similar course of the formation of the reaction product after DAB/H2O2 staining for peroxidase immobilized on nitrocellulose and for immunoperoxidase labeled cells indicates a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. Furthermore, a mild acid treatment for the abolition of endogeneous peroxidase activity in tissues and cells applicable to immunoperoxidase staining procedures is proposed.     

Quantitative assay of the binding of small molecules to protein: comparison of dialysis and membrane filter assays

A rapid assay involving filtration through a nitrocellulose filter is described for the quantitative detection of a protein which specifically and reversibly binds a small molecule. This assay is quantitatively characterized by direct comparison with equilibrium dialysis. The filtration assay is highly sensitive and reproducible when applied to the binding of histidine by the J protein, a component of histidine transport. The effects of several variables on this method are examined. Also, an equilibrium dialysis procedure designed for optimal sensitivity and range in the assay of proteins by binding activity is described.