Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA)

Kendall, Cynthia; Ionescu-Matiu, Irina; Dreesman, Gordon R.

doi:10.1016/s0022-1759(83)80022-2

Cited by 254 publications

(105 citation statements)

References 18 publications

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“…Columns were washed with 10 ml of phosphate-buffered saline, and eluted with 750 l of 0.1 M glycine (pH 3.0), dripping directly into 750 l of 0.1 M Na 2 HPO 4 (pH 9.2) for neutralization. Affinity purified antibody was dialyzed into phosphate-buffered saline to remove glycine, and biotinylated as described (43). This protocol was adapted for purification and biotinylation of the antisera against intact C/EBP␣, kindly provided by Pernille Rorth, and C/EBP␣ short proteins.…”

Section: Methodsmentioning

confidence: 99%

ATF-2 and C/EBPα Can Form a Heterodimeric DNA Binding Complex in Vitro

Shuman

Cheong

Coligan

1997

Journal of Biological Chemistry

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We screened an expression cDNA library with a radiolabeled C/EBP␣ fusion protein and isolated three independent cDNAs encoding ATF-2, a bZIP protein that binds cAMP response elements (CRE). This interaction requires the respective bZIP domains, which form a typical bZIP heterodimer with altered DNA binding selectivity. C/EBP␣ and ATF-2 homodimers bind CRE sites, but ATF-2:C/EBP␣ heterodimers do not. Heterodimers bind an asymmetric sequence composed of one consensus half-site for each monomer, and may thus have a unique regulatory function. As predicted, co-transfection of ATF-2 with C/EBP␣ results in decreased activation of transcription driven from consensus C/EBP-binding sites. In contrast, C/EBP␣ and ATF-2 function cooperatively to activate transcription driven by the asymmetric sequence. Both factors are expressed in liver, where immunoprecipitation experiments show that ATF-2 co-precipitates with C/EBP␣. These results are consistent with the interpretation that C/EBP␣ and ATF-2 can associate in vivo. Moreover, the formation of ATF-2:C/EBP␤ heterodimers suggests that cross-family dimerization with ATF-2 may be a general property for C/EBP family proteins.

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Section: Methodsmentioning

confidence: 99%

ATF-2 and C/EBPα Can Form a Heterodimeric DNA Binding Complex in Vitro

Shuman

Cheong

Coligan

1997

Journal of Biological Chemistry

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“…Only neo selection was used, because under double selection for neo and gpt, cells showed retarded growth. Clones producing H and L chains were identified by screening culture supernatants in an avidin-biotin enzyme-linked immunosorbent assay (24). Plates were coated with either 10 Ag of antigen (antibody S43.…”

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confidence: 99%

Stepwise intraclonal maturation of antibody affinity through somatic hypermutation.

Kocks

Rajewsky

1988

Proc. Natl. Acad. Sci. U.S.A.

148

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Using recombinant DNA techniques, we reconstructed a genealogical tree (Sablitzky, F., Wildner, G. & Rajewsky, K. (1985) EMBO J. 4,[345][346][347][348][349][350] that connects three clonally related B cells producing somatically mutated antibodies to a progenitor cell expressing a germ line-encoded antibody. The somatic mutants had been isolated from an in vivo immune response. The germ line-encoded progenitor antibody bound the antigen with high affinity. Intraclonal affinity maturation occurred stepwise over a 15-fold range.Affinity maturation of antibodies in the course of immune responses is a classical phenomenon in immunology (1). In the frame of the clonal selection theory (2), it would reflect the 'selection and expansion of B-cell clones that existed before immunization and are committed to the production of high-affinity adtibodies. However, it recently has become clear that affinity maturation involves a process of intraclonal diversification through somatic hypermutation. This process seems to be set in motion after immunization and leads to the accumulation of memory B cells expressing high-affinity antibodies (for a review, see ref.3).A useful >approach to study somatic hypermutation in B-cell differentiation pathways is the molecular analysis of antibody genes expressed by clonally related cells (4-8). We present here a direct demonstration of step-wise affinity maturation of a germ line-encoded antibody specificity through somatic hypermutation within a single B-cell clone. MATERIALS AND METHODSVariable (V)-Region Gene Cloning from Hybridoma A20/44. A 7.15-kilobase (kb) EcoRI fragment containing the active V-diversity (D)-joining (J) segment (7) was subcloned into pBR328 (pA20/44). A 2.65-kb BamHI/EcoRI fragment containing the V-D-J segment, -0.5-kb 5' region, and the immunoglobulin heavy (H) chain enhancer was isolated from pA20/44. The 5' BamHI site was converted to an EcoRI site by a BamHI-EcoRI adapter (Amersham) (9) and cloned into PUC19 (pA20/44R1). For cloning of the light chain V region (VL), size-enriched EcoRI-digested DNA of cell line A20/44 was ligated to phage arms of Charon 35 (10) and packaged in vitro. Recombinant phages-hybridizing to a K light chain J region (JK)-specific probe (a 2.7-kb HindIII fragment derived from the immunoglobulin K chain locus including JK1-5) were isolated and characterized by restriction mapping. From phage P8, which contains the active VK-JK and CK (K chain constant region) gene on a 14-kb EcoRI fragment, the VK-JK gene was subcloned as an EcoRI/HindIII fragment into PUC19 (pVKA20/44). The CK fragment was subcloned as a 4.5-kb HindIII fragment into PUC19 (pCK). The sequence of the genomic clones containing the active V regions was determined from M13mp18/19 subclones (11, 12) to confirm the mRNA sequence previously described (6). Two positions that represent ambiguities in the mRNA sequence of the heavy chain V region (VH) at position 100 could be determined as two guanosines; the resulting triplet encodes a glycine. Nucleotide 11 3' Of JH3 is ...

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“…Biotin can be covalently coupled to antibody in a high specific activity without affecting the antigen-binding capacity. Thus, this technique enhances the assay sensitivity (Kendall et al 1983), and the use of this avidin-biotin system in the present quantitative method increases the sensitivity of assay and relatively shortens the processing time. Thus we were able to develop a noncompetitive sandwich-type ELISA of high specificity and sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…Biotin labeling of monoclonal antibody was performed as described previously (Kendall et al 1983). Briefly, 1 ml of a 2 mg/ml solution of SZ-2 in 0.1M NaHCO 3 was mixed with 14 μ l of an 11.76 mg/ml solution of N-hydroxysuccinimidobiotin (Pierce Chemical Co., Rockford, IL) in dimethylsulfoxide.…”

Section: Biotin Labeling Of Monoclonal Antibodymentioning

confidence: 99%

A High-Throughput Biotin-Avidin-ELISA for Studying Expression of Platelet Membrane Glycoproteins and Its Clinical Application

Zhang

Zhao

et al. 2010

Tohoku J. Exp. Med.

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Platelet membrane glycoproteins (GPs) are critical for normal platelet adhesion, activation and aggregation. To define the abnormalities in surface GP expression on circulating platelets and provide a better biomarker of bleeding and thrombotic disorders, we have developed a accurate, time-saving and high-throughput biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) with the monoclonal antibodies (mAbs), 7E3 against the complex of GPIIb and GPIIIa (GPIIb/IIIa), SZ-51 against P-selectin, and SZ-2 against GPIb, respectively. The levels of P-selectin and GPIIb/IIIa were measured in patients with acute myocardial infarction (AMI), intracerebral hemorrhage (ICH), or diabetes mellitus (DM)) and healthy subjects. Inhibition of GP expression was evaluated with SZ-21, an inhibitory mAb against GPIIIa and aspirin, respectively. The sensitivity of BA-ELISA is high enough to detect platelet count as low as 3.13 × 10 9 /L in platelet-rich plasma (PRP). Both the inter-assay and intra-assay coefficient variation are less than 10%. Adenosine diphosphate (ADP)-induced or non-ADP-induced expression of P-selectin and GPIIb/IIIa was significantly higher in AMI, ICH or DM than that in controls (P < 0.01 for each). Either SZ-21 or aspirin can inhibit the ADP-induced expression of P-selectin and GPIIb/IIIa. Importantly, a high correlation was detected between BA-ELISA and flow cytometry methods. These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet GP expression. The newly developed BA-ELISA can be popularized in community hospitals, because it does not require sophisticated equipments and reagents. This method is suitable for screening inhibitors of platelet activation and has a potential in use for diagnostic purposes.

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Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA)

Cited by 254 publications

References 18 publications

ATF-2 and C/EBPα Can Form a Heterodimeric DNA Binding Complex in Vitro

ATF-2 and C/EBPα Can Form a Heterodimeric DNA Binding Complex in Vitro

Stepwise intraclonal maturation of antibody affinity through somatic hypermutation.

A High-Throughput Biotin-Avidin-ELISA for Studying Expression of Platelet Membrane Glycoproteins and Its Clinical Application

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