How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate

Love, Robert A.; Villafranca, J. E.; Aust, Robert M.; Nakamura, Katsuyuki; Jue, R.A.; Major, J.G.; Radhakrishnan, R.; Butler, W.F.

doi:10.1021/bi00092a004

Cited by 65 publications

(55 citation statements)

References 48 publications

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“…A comparison of the sequence in the complementarity determining regions of CHA255 and 2A81G5 showed very little homology, except for a histidine in the CDR3 region of the heavy chain. This histidine was of interest because in the crystal structure of the CHA255⅐In(III)-EDTA complex, this histidine provided the only direct coordination between the antibody and the chelated metal (11). Molecular models were subsequently constructed of 2A81G5 to determine if the His-96 in the CDR3 region of the heavy chain could assume the conformation necessary to provide such a coordination site for the cadmium ion.…”

Section: Discussionmentioning

confidence: 99%

“…The initial model was energyminimized to relax any constraints that may have been introduced during the building process. It was then superimposed onto the structure of monoclonal antibody CHA255, whose structure has been solved to 2.2-Å resolution (11). Superpositioning of the heavy chain framework regions 1-3 gave a r.m.s.…”

Section: Materials-balb/cmentioning

confidence: 99%

“…The The curves drawn through the data points were generated using a one-site model, and the values for the dissociation constants are given in Table I. monoclonal antibody CHA255, whose crystal structure has been solved to 2.2-Å resolution (11), was raised to a hapten structurally related to the Cd-EDTA-KLH antigen used in the present study. The possible antigen binding site of the 2A81G5 antibody was examined by comparing the model with the structure of CHA255 bearing an indium-loaded EOTUBE tetraacetic acid in the binding pocket.…”

Section: Molecular Modeling Of Heavy and Lightmentioning

confidence: 99%

“…The molecular features responsible for an antibody's ability to differentiate among metals have been explored in some detail with CHA255 (11). Binding studies have shown that CHA255 was highly specific for the indium chelate; the antibody's affinity decreased from 24-to 20,000-fold when other metals were substituted for In(III) in the chelate complex.…”

mentioning

confidence: 99%

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Metal Binding Properties of a Monoclonal Antibody Directed toward Metal-Chelate Complexes

Blake

Chakrabarti

Khosraviani

et al. 1996

Journal of Biological Chemistry

151

143

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Monoclonal antibodies directed toward metal chelating agents or metal-chelate complexes have many potential uses both in medicine and in environmental analysis. In medicine, chelator-linked antibodies have been used in vivo to transport and deliver radioisotopes to specific target sites, such as tumors (1-3). The availability of monoclonal antibodies to the chelating agent has permitted researchers to investigate the biodistribution of these "magic bullets" in cancer chemotherapy (4). In environmental analysis, the availability of monoclonal antibodies that can distinguish among different metals provides a basis for rapid, sensitive immunoassays that can be used onsite to assess heavy metal contamination (5, 6). Monoclonal antibodies that bind to chelators such as diethylenetriaminepentaacetic acid and 1,4,7,10-tetraazacyclododecane-N, NЈ, NЉ, Nٞ-tetraacetic acid have been reported (7, 8); however, these antibodies were directed primarily toward the chelate portion of the molecule and did not demonstrate the ability to differentiate among various metals bound in the chelate complex. Two hybridomas that synthesized monoclonal antibodies showing metal ion specificity have also been reported. The first, CHA255, was the result of immunization with a derivative of In(III)-EDTA coupled to a carrier protein by a thioureido-L-benzyl group (4). The monoclonal antibody synthesized by CHA255 bound to a variety of chelate complexes but bound to indium-chelate complexes more tightly than it did to other chelated metals (4, 9). Monoclonal antibodies directed toward mercuric ions have also been elicited by immunization of animals with a glutathione-Hg derivative (10). These antibodies, which bind to mercury with high affinity, have been used to detect mercuric ions in aqueous samples (6) and are the basis for a commercially available immunoassay to monitor mercury contamination in environmental samples.The molecular features responsible for an antibody's ability to differentiate among metals have been explored in some detail with CHA255 (11). Binding studies have shown that CHA255 was highly specific for the indium chelate; the antibody's affinity decreased from 24-to 20,000-fold when other metals were substituted for In(III) in the chelate complex. The structural basis for this fine discrimination was investigated by x-ray crystallographic analyses of the antigen-binding fragment complexed with either In(III) or Fe(III) chelates of thioureido-L-benzyl-EDTA. A notable feature of the antibodyIn(III)-EDTA complex was the additional coordination of the chelated metal by a histidine residue from the heavy chain's third complementarity determining region. This histidine coordination did not occur in the corresponding Fe(III) complex, due to a slightly different hapten coordination that reduced access to the metal. It was concluded that the absence of the histidine ligation was largely responsible for the 24-fold lower binding of the iron hapten to CHA255 relative to that of the indium hapten. The larger differences in the binding a...

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Section: Discussionmentioning

confidence: 99%

Section: Materials-balb/cmentioning

confidence: 99%

Section: Molecular Modeling Of Heavy and Lightmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Metal Binding Properties of a Monoclonal Antibody Directed toward Metal-Chelate Complexes

Blake

Chakrabarti

Khosraviani

et al. 1996

Journal of Biological Chemistry

151

143

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“…Since then, immunoassays have been widely applied in biology, medicine, agriculture and environment sciences. In the field of heavy metal ion analyses, immunoassays have had a long development process [12][13][14][15][16][17]. In the 1980s, Meares et al [18][19][20][21][22] first developed a series of methods for synthesizing complexes of metal-chelate-antibodies for clinical medical radio-imaging analysis.…”

mentioning

confidence: 99%

Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions

Xie

Fan

et al. 2011

Sci. China Life Sci.

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In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid (CMPA) derived from penicillin. Results from tests with immune rabbits indicate that: (i) A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; (ii) the immunogenic antigen (CMPA-BSA) can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and (iii) antibodies in antisera showed higher affinity to OVA-GSH-CuCl than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples. Heavy metals are a class of persistent toxic contaminants and residues found at various levels in the environment and in agricultural and industrial products [1]. Unlike carbon-based contaminants that can be completely degraded to relatively harmless products, metal ions can be transformed in only a limited number of ways through chemical or biological remediation processes [2]. What is worse, through biomagnifications, heavy metal ions existing in the environment can invade the human body and remain there for a long time, posing a serious threat to human health [3].Copper is an essential element of some metalloenzymes necessary for the normal course of biochemical processes but, like other heavy metals, it is toxic at high concentration [4,5]. All of these factors pose potential risks for the environment and human health [6] and therefore copper and other heavy metals must be monitored and identified in the environment. The traditional technologies for detecting heavy metals are instrument-based measurements such as atomic absorption spectrometry [7], inductively coupled plasma spectroscopy (ICP-AES and ICP-MS) [8] and stripping voltammetry [9]. Although these conventional instruments accurately detect the amount of metal in an individual sample, they all require expensive laboratory equipment in a centralized facility and sample turnaround is slow. In addition, instrumental analyses provide no information about the metal oxidation state.In this case, immunoassays based on the affinity and specificity of an antigen-antibody reaction offer an alternative approach in the field of metal ion measurement. Com-

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Unusual amino acid usage in the variable regions of mercury-binding antibodies

Westhoff¹,

Lopez²,

Goebel³

et al. 1999

Proteins

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Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region , which is unusual for murine antibodies. Muta-genesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens. Proteins 1999;37:429-440.

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How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate

Cited by 65 publications

References 48 publications

Metal Binding Properties of a Monoclonal Antibody Directed toward Metal-Chelate Complexes

Metal Binding Properties of a Monoclonal Antibody Directed toward Metal-Chelate Complexes

Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions

Unusual amino acid usage in the variable regions of mercury-binding antibodies

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