Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis

Science

Volumn 279, Issue 5353, 1998, Pages 1052-1054

  Clynes, Raphael ^a   Dumitru, Calin ^a   Ravetch, Jeffrey V ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AUTOANTIBODY; FC RECEPTOR;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; ANTIBODY RESPONSE; ANTIGEN ANTIBODY COMPLEX; ARTICLE; AUTOIMMUNE DISEASE; COMPLEMENT ACTIVATION; CONTROLLED STUDY; GENE DISRUPTION; GLOMERULONEPHRITIS; IMMUNE COMPLEX DEPOSITION; INFLAMMATION; KIDNEY INJURY; LUPUS ERYTHEMATOSUS NEPHRITIS; MOUSE; NEPHRITIS; NONHUMAN; PRIORITY JOURNAL;

ANIMALS; ANTIBODIES, ANTINUCLEAR; ANTIGEN-ANTIBODY COMPLEX; COMPLEMENT ACTIVATION; COMPLEMENT SYSTEM PROTEINS; CROSSES, GENETIC; DISEASE MODELS, ANIMAL; GLOMERULAR MESANGIUM; KIDNEY GLOMERULUS; LUPUS NEPHRITIS; MICE; MICE, INBRED C57BL; MICE, INBRED STRAINS; RECEPTORS, IGG;

ANIMALIA;

EID: 0032512855     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5353.1052     Document Type: Article

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45. +/- B/W female offspring mice. Mice were genotyped by polymerase chain reaction of tail-tip DNA samples. (Primer sequences: neo: CTCGTGCTTTACGGTATCGCC; gamma 1: ACCCTACTCTACTGTCGACTCAAG; gamma 2:TCACGGCTGGCTATAGCTGCCTT. Annealing temperature was 62°C. Knockout and wild-type amplified products were 260 and 224 base pairs, respectively).
46. Urine was collected at 2-week intervals and read by dipstick (2GP Chemstrip, Boehringer-Mannheim, Indianapolis, IN). We scored 3+ determinations (5 mg/ml) as positive for proteinuria. Using metabolic cages (Fisher, Pittsburgh, PA), we obtained, 24-hour urine collections from all mice with 3+ proteinuria. Statistical analysis of proteinuria and survival data were done with the StatSoft software package (Tulsa, OK).
47. +/- C57BI/6 kidneys as negative controls (28).
48. +/- mice were added to enzyme-linked immunosorbent assay (ELISA) plates coated with C1q (Sigma, St. Louis, MO) for detection of ICs and to dsDNA-coated plates (United Biotech, Mountain View, CA) for detection of antibodies to chromatin. After washing away unbound serum, the appropriate rat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (Pharmingen, San Diego, CA) was added. Alkaline-phosphatase-conjugated AKP polyclonal antirat IgG (Pharmingen, San Diego, CA) was used as secondary antibody. After incubation with para-nitrophenyl phosphate substrate, the samples were read spectrophotometrically at 405 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA). Pooled serum from 7-month-old wild-type B/W female mice was used to generate a reference standard curve.
49. We thank the Ravetch lab and M. Madaio for helpful discussions and F. Vital, D. White, and C. Ritter for technical and administrative assistance. Supported by NIH grants to J. V. R. and R. C.