A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

https://www.pnas.org/content/pnas/76/9/4350.full.pdf

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

In 1982, the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology (ACR)published revised criteria for the classification of systemiclupus erythematosus (SLE) (1). During the ensuing decade several investigators, including Drs. Graham Hughes and Donato Alarcon-Segovia, among others, have described the presence and clinical associations or antiphospholipid antibodies in patients with SLE, as well as the occurrence of theprimary antiphospholipid syndrome (2-5). In 1992, Piette and colleagues suggested that the ACR revised criteria be reevaluated in light of the above discoveries (6).

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780400928

Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.

For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780290816

Development of criteria for the classification and reporting of osteoarthritis: Classification of osteoarthritis of the knee

https://www.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1538-7836.2006.01753.x

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

The New York and the Rome diagnostic criteria for ankylosing spondylitis (AS) and the clinical history screening test for AS were evaluated in relatives of AS patients and in population control subjects. The New York criterion of pain in the (dorso) lumbar spine lacks specificity, and the chest expansion criterion is too insensitive. The Rome criterion of low back pain for more than 3 months is very useful. Our study showed the clinical history screening test for AS to be moderately sensitive, but it might be better in clinical practice. As a modification of the New York criteria, substitution of the Rome pain criterion for the New York pain criterion is proposed.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780270401

Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria.

Autoantibodies in the diagnosis of systemicrheumatic diseases

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle.

Antinuclear antibodies wer demonstrated in the sera of 43 of 45 (96%) patients with progressive systemic sclerosis (22 of 24 with diffuse scleroderma and 21 of 21 with the CREST syndrome variant). This high percentage of positive reactions resulted from the use of a tissue culture substrate (Hep-2) to detect antinuclear antibodies by the indirect immunofluorescent method. Patterns of nuclear staining included diffuse fine speckles, large coarse speckles, nucleolar and centromere staining. When organ sections such as mouse kidney were used as substrate for the detection of antinuclear antibodies, nucleolar staining and centromere staining were the two patterns most frequently overlooked. Three types of antibodies appeared to be highly specific for scleroderma: antibody to Sc1-70 antigen, antibody to centromere, and antinucleolar antibody. The anti-centromere antibody appeared to be highly selective for the CREST variant of progressive systemic sclerosis.

Diversity of antinuclear antibodies in progressive systemic sclerosis

An extract of human lymphocytes from continous cell culture was used as the antigen source to detect antibodies in sera of patients with Sjogren's syndrome (SS). Using double diffusion in agarose, 85 per cent of a selected group of patients had precepating antibodies. Three precipitating antigen-antibody systems were detected and were shown to be different from those described previously in othe systemic rheumatic diseases. The SS precipitating antibodies were temporarily classified as precipitins, A, B, and C. SS patinets with sicca syndrome but without clinical rheumatoid arthritis had precipitin systems A and/or B, and SS patients with associated rheumatoid arthritis had precipitin system C. Serum reactants were demonstrated by immuno-electrophoresis to migrate in the gamma globulin region. The precipitating activity of the serum factors was not destroyed by treatment with 2-mercaptoethanol and was not removed by absorption of rheumatoid factor from the sera. The reactivity of the lympuocyte antigens was destroyed by treatment with trypsin but not by deoxyribonuclease or ribonuclease.

/pdf/antibodies-to-cellular-antigens-in-sjogren-s-syndrome-a4dwghzo21.pdf

Antibodies to cellular antigens in Sjögren's syndrome.

Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti-60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti-52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.

/pdf/a-52-kd-protein-is-a-novel-component-of-the-ss-a-ro-3ooli66g7u.pdf

Eng M. Tan

Papers

Autoantibodies in the diagnosis of systemicrheumatic diseases

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle.

Diversity of antinuclear antibodies in progressive systemic sclerosis

Antibodies to cellular antigens in Sjögren's syndrome.

A 52-kD protein is a novel component of the SS-A/Ro antigenic particle.