Component Description
IgG autoantibody testing of a subsample stored serum of specimens from NHANES (1999–2004) was conducted to estimate the prevalence and specificities of selected autoantibodies in the US population in 1999-2004.
Eligible Sample
One third subsample of participants aged 12 years and older from NHANES 1999-2004 in the Dioxin subsample who had stored sera (total of N = ~5,000 from NHANES 1999-2004).
Description of Laboratory Methodology
Immunofluorescence analysis
Testing of IgG autoantibodies to human cellular antigens was performed by the HEp-2 cell immunofluorescence assay using slides from INOVA Diagnostics, San Diego, CA (Cat # 508100) following the manufacturer’s instructions.
The cellular pattern of staining and its staining intensity from 0 to 4+ was described using a reference gallery per below. The determination of autoantibody titers from 1:80 to 1:1280 was performed by serial dilution on samples that showed a 3+ or greater nuclear and/or cytoplasmic immunofluorescence pattern.
Immunoprecipitation analysis
Testing of IgG autoantibodies to human cellular antigens was performed on samples that showed a 3+ or greater immunofluorescence staining using immunoprecipitation assays by a standard method (Reeves WH,et al., 2006)
Sera that showed known autoantibody specificities based on visible immunoprecipitated protein bands consistent with the combination of bands and their estimated size per the table below were recorded. Immunoprecipitated ribonucleic acid bands on stained gels were also recorded when they were needed to confirm the specificity. When necessary, the size and the pattern of migration were compared with reference sera (Reeves WH,et al., 2006).
Data Processing and Editing
Data Access: All data are publicly available.
Laboratory Quality Assurance and Monitoring
Immunofluorescence analysis
Serum samples were tested using established protocols outlined by manufacturer. A set of CDC autoimmune reference sera (12 different specificities) (Chan EKL et al., 2007) and 2 healthy control sera were coded and tested in a blinded fashion two times to verify consistent results throughout screening test: towards the beginning and when approximately half of the samples were tested.
Samples that showed discrepant results were repeated.
More than every 50th sample (2% of the total from NHANES 1999-2004) was automatically repeated to assess assay precision issues. Additional samples were repeated for the following reasons:
1) Titering of 3+/4+ positive samples (repeat, n=692)
2) Randomly selected samples for titering (and repeat, n=1035)
3) Other repeats are performed whenever there is any doubt for accuracy or technical issues. Samples repeated with 2 independent staining = 895; 3 independent staining = 194; 4 independent staining = 4.
Immunoprecipitation analysis
Serum samples were tested using the standard immunoprecipitation protocol (Reeves WH,et al., 2006). A set of CDC autoimmune reference sera (12 specificities) and 2 healthy controls were tested in a blinded fashion two times to verify consistent results throughout screening test: towards the beginning of the study and when approximately half of the samples were tested.
Samples that showed common bands were repeated in parallel side-by-side fashion for comparison with each other and with known controls as needed.
Assay reproducibility was initially assessed by random selection of the ANA 3+ or 4+ sera number of serum specimens tested and repeating the immunoprecipitation assay. The percentage of samples that were repeated was actually 15% (103/692) of the sera tested by IP.
Analytic Notes
The NHANES Survey Design:
The analysis of NHANES laboratory data should be conducted using the key survey design variables. The NHANES Demographic data files for each 2-year survey cycle contain core information on the survey and the survey design variables for strata and PSU (SDMVSTRA and SDMVPSU, respectively) which are recommended to be used in variance estimation. This laboratory data files may be linked to other NHANES data files using the unique survey participant identifier SEQN.
Weighting:
Special subsample sample weights WTANA6YR in the file are required to analyze the NHANES 1999-2004 ANA data. This is because the ANA data were analyzed using stored serum samples obtained from survey participants who had been selected from NHANES 1999-2004 Dioxin subsamples. The NHANES 1999-2004 ANA sample and the subsample weights were created for 6 years analysis and should be used to analyze all six years together.
Immunofluorescence analysis
A standard gallery of images showing the intensity of staining and the cellular patterns of reactivity is shown below and the following patterns were reported:
Nuclear patterns
1) homogeneous 2) speckles 3) centromere
4) nuclear dots 5) nuclear envelope 6) nucleolar
Cytoplasmic patterns
1) homogeneous 2) speckles 3) mitochondria-like
4) Golgi complex 5) centriole 6) GW Body
7) cytofilament 8) rods/rings 9) lysosomes
Mitotic cell patterns
1) homogeneous 2) speckles 3) spindle apparatus
4) mid-body 5) chromosomes
Nuclear Patterns
Nuclear staining |
Images |
Description of staining patterns |
Homogeneous |
|
Uniform diffuse staining of the nucleus in interphase cells |
Speckles |
|
Speckles in the nucleus of interphase cells |
Centromere |
|
Discrete speckles (40-80/cell) in interphase cells and on metaphase plates |
Nuclear dots |
|
Countable discrete speckles PML bodies, Cajal bodies (coiled bodies) |
Nuclear envelope |
|
Homogeneous nuclear staining with greater intensity at its outer rim |
Nucleolar |
|
Homogeneous or speckled staining of the nucleolus |
Cytoplasmic Patterns
Cytoplasmic staining |
Images |
Description of staining patterns |
Homogeneous |
|
Uniform diffuse staining of the cytoplasm in interphase cells |
Speckles |
|
Speckled staining of the cytoplasm |
Mitochondria-like |
|
Coarse granular filamentous staining |
Golgi complex |
|
Discontinuous speckled or granular perinuclear staining |
Centriole |
|
Distinct dots (2-4/cell) in cytoplasm and at the poles of mitotic spindle |
GW body |
|
Discrete dots in cytoplasm of interphase cells |
Cytofilament |
|
Cytoskeleton staining with variable intensity interphase cells |
Rods/Rings |
|
Distinct rod and ring structures throughout the cytoplasm |
Lysosomes |
|
Distinct and bright speckled staining irregularly distributed in the cytoplasm |
Mitotic cell patterns
Mitotic cell staining |
Images |
Description of staining patterns |
Homogeneous |
|
Uniform diffuse staining of the mitotic cytoplasm |
Speckles |
|
Numerous speckles in cytoplasm of mitotic cells |
Spindle Apparatus |
|
The spindle fibers between the poles are stained in mitotic cells, associated with cone-shaped decoration of the mitotic poles |
Mid-body |
|
Staining of the intercellular bridge that connects daughter cells by the end of cell division, but before cell separation |
Chromosomes |
|
Diffuse staining of condensed chromatin in mitotic cells |
Immunoprecipitation analysis
A standard set of images defining the immunoprecipitation patterns of reactivity is shown below and the specific reactivities that are reported are shown in the table below. Those samples that did not show any of these reactivities were reported as negative.
Analysis of protein components of autoantigens (12.5% SDS-PAGE). 35S-methionine labeled K562 cell extract was immunoprecipitated by prototype human autoimmune sera or normal human serum (NHS). Components of each autoantigen are indicated by red arrowheads.
Analysis of protein components of autoantigens (8% SDS-PAGE). 35S-methionine labeled K562 cell extract was immunoprecipitated by prototype human autoimmune sera or normal human serum (NHS). Components of each autoantigen are indicated by red arrowheads.
Analysis of RNA components of autoantigens. K562 cell extract from 107 cells was immunoprecipitated by prototype sera for anti-U3RNP, Jo-1, and normal human serum (NHS). RNA component was extracted and analyzed by urea-PAGE and silver staining. RNA components of each autoantigen are shown with arrowheads. Total RNA and the pattern by anti-Sm serum are shown as references.
Identification of autoantibodies by protein and RNA immunoprecipitation
Autoantibody Specificity |
35S-methionine IP protein analysis |
IP-RNA analysis silver staining |
Definition |
U1RNP |
Y |
ND |
A characteristic set of UsnRNPs proteins, U1-70k, A (33kD), B’/B (29/28kD), C (22kD), D1/D2/D3 (16kD). E,F, and G without U5-200kD doublet |
Sm |
Y |
ND |
A characteristic set of UsnRNPs proteins as above with U5-200kD doublet |
Ribosomal P |
Y |
ND |
A characteristic set of proteins P0 (38kD), P1 (19kD), and P2 (17kD) |
Ro60 (SS-A) |
Y |
ND |
60kD protein |
La (SS-B) |
Y |
ND |
48kD protein |
Su (ago2) |
Y |
ND |
A set of 100/102kD doublet and 200kD proteins |
Replication protein A |
Y |
ND |
A set of 70kD, 32kD, and 14kD proteins |
Jo-1 (histidyl-tRNA synthetase) |
(Y) |
Y |
50kD protein and tRNA by RNA analysis |
PL-7 (threonyl-tRNA synthetase) |
Y |
ND |
80kD protein |
PL-12 (alanyl-tRNA synthetase) |
Y |
ND |
110kD protein |
EJ (glycyl-tRNA synthetase) |
Y |
ND |
75kD protein |
OJ (isoleucyl-tRNA synthetase) |
Y |
ND |
130kD, 150kD, 170kD proteins |
SRP (signal recognition particle) |
(Y) |
Y |
72/68kD, 54kD, 19kD, 14kD, 9kD proteins and 7SL RNA by RNA analysis |
Ku |
Y |
ND |
70kD and 86kD proteins |
PM-Scl |
Y |
ND |
110kD, 75kD, and a set of other proteins |
Mi-2 |
Y |
ND |
240kD, 150kD proteins and several other proteins |
Topoisomerase I (Scl-70) |
Y |
ND |
110kD protein with a smear toward higher molecular weight due to phosphorylation |
RNA polymerase I/III |
Y |
ND |
A characteristic set of proteins. Two largest subunits of each (RNAP I: 190, 120kD, RNAP III (155, 135kD) |
U3RNP (Fibrillarin) |
(Y) |
Y |
34kD protein, nucleolar staining, and U3RNA by RNA analysis |
NOR-90 |
Y |
ND |
93/95kD thick protein doublet |
IP: immunoprecipitation; Y: Identity can be confirmed; (Y) identity suggested but requires RNA-IP to confirm; ND: not done