Affinity immunoblotting

Knisley, Keith A.; Rodkey, L. Scott

doi:10.1016/0022-1759(86)90320-0

Cited by 44 publications

(5 citation statements)

References 12 publications

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“…For visualization of anti-A antibody spectrotypes we used a modification of the method described by Knisley et al. [20]. Briefly, 2 .…”

Section: Lsoelectric Focusing and Affinity Immunoblottingmentioning

confidence: 99%

“…For visualization of anti-A antibody spectrotypes we used a modification of the method described by Knisley et al [20]. To be sure that specific antibodies were detected on the blots, we performed absorption experiments using red blood cells (RBC) and A antigen of different sources coated on microtiter plates.…”

Section: Lsoelectric Focusing and Affinity Immunoblottingmentioning

confidence: 99%

“…Along the same lines a study on anti-tetanus toxoid antibodies, has demonstrated idiotype fluctuations after booster immunization of healthy individuals [ 171. However, these findings are contrasted by others, using (auto-)antibodies against DNA, histidyl-transfer RNA synthetase and thyroglobulin, revealing different pol yclonal patterns for each individual that remain unchanged over long periods of time [6, 18, 191. Direct analysis of the clonal distribution of specific antibodies, without the need for prior purification steps, has lately become possible through the technique of isoelectric focusing (IEF) and subsequent affinity immunoblotting [20]. We have used a modification of this technique for spectrotype analysis of naturally occurring ABO antibodies in healthy individuals.…”

Section: Introductionmentioning

confidence: 99%

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Naturally occurring ABO antibodies: Long‐term stable, individually distinct anti‐A IgG spectrotypes

1992

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Spectrotypes of naturally occurring IgG antibodies to histo-blood group substance A were analyzed by isoelectric focusing and a newly developed, non-radioactive affinity immunoblotting assay. Specificity was tested by preabsorption of sera on red blood cells and on four different types of blood group substance A. Clonotype-banding patterns of sera from 18 different blood donors were individually distinct, most of them showing an oligoclonal pattern with less than 15 visible bands. To analyze the influence of IgG subclass composition on spectrotype patterns, IgG subclasses were measured by ABO enzyme-linked immunosorbent assay (ELISA) and were shown to be mostly IgG1 and IgG2. Anti-A IgG1 spectrotypes could be detected with four sera and were also individually distinct. A series of six and seven serum samples, respectively from two donors was used to analyze the time-related changes of anti-A IgG spectrotypes. Banding pattern of both individuals remained stable during the whole observation period of 167 and 82 days, respectively, and no differences between follow-up samples could be detected. This long-term stability of individual anti-A IgG spectrotypes is backed by constant serum levels of anti-A IgG and -IgM antibodies during the observation period as measured by ABO ELISA.

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“…For visualization of anti-A antibody spectrotypes we used a modification of the method described by Knisley et al. [20]. Briefly, 2 .…”

Section: Lsoelectric Focusing and Affinity Immunoblottingmentioning

confidence: 99%

Section: Lsoelectric Focusing and Affinity Immunoblottingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Naturally occurring ABO antibodies: Long‐term stable, individually distinct anti‐A IgG spectrotypes

1992

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“…Our modification of the IEF/AIB method, based on the AIB method originally described by Knisley and Rodkey [16] and adapted for CSF analysis by Kaiser [17] and Sindic and Laterre [18], uses a glutardialdehyde fixation step to increase sensitivity [19]. The method was further modified by using alkaline-phosphatase instead of peroxidase-based detection.…”

Section: Discussionmentioning

confidence: 99%

Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with Hevylite™ antibodies. comparison with the free light chain oligoclonal pattern

Zeman

Hradílek

Švagera

et al. 2012

Fluids Barriers CNS

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Background Oligoclonal IgG bands in cerebrospinal fluid that are absent in serum indicate intrathecal IgG synthesis and are a sensitive marker of CNS inflammatory diseases, in particular multiple sclerosis. It may be of interest to determine whether these bands are predominantly IgGκ or IgGλ. Methods We have used Hevylite™ antibodies and developed a technique for detection of oligoclonal IgGκ and IgGλ bands by means of isoelectric focusing followed by immunoblotting. The same technique was used for oligoclonal free κ and free λ detection. Among several techniques tested, affinity immunoblotting appears to be the most sensitive; it can detect less than 1 ng of IgGκ or IgGλ paraprotein. We compared oligoclonal IgG profiles with those of oligoclonal IgGκ and IgGλ. There was good agreement concerning the presence or absence of intrathecal synthesis. We observed the ratios between oligoclonal IgGκ and IgGλ bands, and they did not always match the ratios between free κ and free λ bands. We were also able to detect antigen-specific CSF-restricted oligoclonal IgGκ and IgGλ bands in neuroborreliosis. It remains to be determined subsequently by a clinically-oriented prospective study, whether predominant IgGκ/IgGλ or free κ/free λ can be observed more frequently in particular diseases with oligoclonal IgG synthesis. Discussion Very sensitive detection of oligoclonal IgGκ and IgGλ bands in cerebrospinal fluid with Hevylite antibodies is feasible; detection of antigen-specific IgGκ or IgGλ is possible as well. In particular situations, e.g. when difficulties arise in distinguishing between oligoclonal and monoclonal pattern, the test may be of considerable clinical value.

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“…The analysis covcrs a continuous pH spectrum and is not limited by the availability of appropriately spaced p l marker proteins or carbamylated standards [23] to assess thc stepless characteristics of the carrier ampholyte mixture. The development of carrier ampholytes with such characteristics is extremely important in the analysis of protein mixtures such as polyclonal antibody preparations [5,25], which are vastly heterogeneous with respect to their isoelectric points. Carrier ampholytes which have many species within a narrow pH range are particularly attractive for preparative I E F applications.…”

Section: Discussionmentioning

confidence: 99%

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation

Knisley

Rodkey²

1992

Electrophoresis

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A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

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Affinity immunoblotting

Cited by 44 publications

References 12 publications

Naturally occurring ABO antibodies: Long‐term stable, individually distinct anti‐A IgG spectrotypes

Naturally occurring ABO antibodies: Long‐term stable, individually distinct anti‐A IgG spectrotypes

Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with Hevylite™ antibodies. comparison with the free light chain oligoclonal pattern

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation

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