Detection of Antibodies and Soluble Antigen-Antibody Complexes by Precipitation with Polyethylene Glycol

Creighton, W D; Lambert, Paul Henri; Miescher, P. A.

doi:10.4049/jimmunol.111.4.1219

Cited by 361 publications

(2 citation statements)

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“…ICs can be precipitated from serum using PEG [13], and it was later confirmed that low concentrations of PEG selectively precipitate ICs with minimal free IgG, probably by steric hindrance effects [23,24]. Large proteins or complexes are precipitated by PEG by steric exclusion only if they are surrounded by linear PEG chains [25,26].…”

Section: Introductionmentioning

confidence: 90%

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A Proteomics-Based Method for Identifying Antigens within Immune Complexes

Menikou,

McArdle,

et al. 2023

Advances in Medical Imaging, Detection, and Diagnosis

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A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

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Section: Introductionmentioning

confidence: 90%

“…Large proteins or complexes are precipitated by PEG by steric exclusion only if they are surrounded by linear PEG chains [25,26]. Although numerous methods are available for recovery of ICs by PEG precipitation [5,7,[23][24][25][26][27][28][29][30][31][32], very few studies have identified the antigen within ICs.…”

Section: Introductionmentioning

confidence: 99%

A Proteomics-Based Method for Identifying Antigens within Immune Complexes

Menikou,

McArdle,

et al. 2023

Advances in Medical Imaging, Detection, and Diagnosis

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Blocking factors in sera of breast cancer patients

Tanaka

Yonemoto

Waldman

1979

Cancer

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In previous studies, we have demonstrated that serum blocking factors (SBF) from sera of breast cancer patients was specific in our Leukocyte Adherence Inhibition assay (LAI).34 To elucidate the nature of SBF, we have investigated the antigen‐antibody complex by precipitation of immune complexes (I.C.) with polyethylene glycol (PEG). Specific and nonspecific blocking activities of sera obtained from breast cancer patients were investigated using the LAI and PHA‐induced lymphocyte blastogenesis (PHA Blastogenesis) assays. I.C. precipitated in PEG, were utilized to test simultaneously the blocking capacity of these complexes and the corresponding serum. Blocking activity of sera and corresponding I.C. correlated in 20 out of 24 (83%) experiments in the LAI assay and 16 out of 19 (84%) experiments in the PHA Blastogenesis assay. When immune complexes from breast cancer patients were quantitatively compared with normal donors and benign breast patients, a significantly higher yield of protein concentration was obtained. Furthermore, I.C. of benign breast patients was also higher than normal controls. When 10 of the 28 I.C. were further fractionated into Ig (Retentate) and antigen containing component (Filtrate) by ultrafiltration after acid treatment, neither of these components manifested blocking. When reconstituted at a ratio of Ig:antigen = 1:1, blocking activity was again restored in 3 out of 3 in LAI and in 3 out of 4 in blastogenesis. In two experiments, I.C. with negative blocking was readily converted to a positive blocking state with the addition of a small amount of antigen. I.C. also demonstrated specificity in blocking activity and the degree of blocking activity depended on the concentration of I.C. in cultures. Evidence obtained suggests that specific and nonspecific serum blocking factors are related to I.C. but not to the fractionated components of Ig nor antigen. Cancer 43:838–847, 1979.

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Bioanalytical Strategy to Support Clinical Immunogenicity Assessment

Wang¹,

Luong²

2022

Bioanalytical Aspects in Biological Therapeutics

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Detection of Antibodies and Soluble Antigen-Antibody Complexes by Precipitation with Polyethylene Glycol

Cited by 361 publications

References 0 publications

A Proteomics-Based Method for Identifying Antigens within Immune Complexes

A Proteomics-Based Method for Identifying Antigens within Immune Complexes

Blocking factors in sera of breast cancer patients

Bioanalytical Strategy to Support Clinical Immunogenicity Assessment

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