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Nephritis - Nephritogenic Monoclonal Antibodies

Goodpasture’s syndrome is a severe renal disease associated with pulmonary hemorrhage mediated by autoantibodies against the glomerular basement membrane (GBM). Chondrex, Inc. provides unique rat monoclonal antibodies against the non-collagenous domain (NC1) of the alpha 4 chain of type IV collagen of GBM, which induces nephritis similar to Goodpasture’s syndrome in Wistar Kyoto (WKY) and the sub-strains of rats. Futhermore, rat models of glomerulonephritis and diabetic nephropathy demonstrate comparable albuminuria with human patients, suggesting that rat urinary albumin levels are useful for evaluating disease severity and treatment efficacy. 

Chondrex Inc.'s rat urinary protein assay kit uses the turbidity method in a 96-well flat bottom plate, which offers a solution for assaying a large number of rat urine samples within a short time and a wide assay range (Link). This kit requires a minimal volume (less than 10 μl) of urine sample, and is ideal for studying nephritis in various strains of rats that do not secrete a sufficient volume of urine during a 16-hour collection period.  Assay results are good.

Nephritogenic Monoclonal Antibodies

Product Quantity Catalog # Price (USD)
Nephritogenic Monoclonal Antibody, Clone 114, 1 mg 1 mg/ml x 1 ml 70201 699.00
Nephritogenic Monoclonal Antibody, Clone 114, 5 mg 1 mg/ml x 5 ml 70205 3400.00
Nephritogenic Monoclonal Antibody, Clone a84, 1 mg 1 mg/ml x 1 ml 70211 699.00
Nephritogenic Monoclonal Antibody, Clone a84, 5 mg 1 mg/ml x 5 ml 70215 3400.00
Nephritogenic Monoclonal Antibody, Clone b35, 1 mg 1 mg/ml x 1 ml 70221 699.00
Nephritogenic Monoclonal Antibody, Clone b35, 5 mg 1 mg/ml x 5 ml 70225 3400.00

Rat Albumin Detection Kit

Product Catalog # Price (USD)
Rat Albumin Detection Kit 3020 299.00

Rat Urinary Protein Assay Kit and Standard

Product Catalog # Price (USD)
Rat Urinary Protein Assay Kit 9040 90.00
Rat Urinary Protein Assay Standard 90401 56.00

For Further Information: Rat Nephritis Protocol

Individual monoclonal antibodies induce mild to severe nephritis in rats within 1-2 days by a
single IP or IV injection.
Monoclonal antibody, clone b35 (IgG2b), induces severe nephritis associated with hematuria and pulmonary hemorrhage.
Monoclonal antibody, clone a84 (IgG2a), induces severe nephritis associated with hematuria.
Monoclonal antibody, clone 114 (IgG1), induces mild nephritis at the same dose of b35 and a84.

Susceptible and Non-Susceptible Strains of Rats
For unknown reasons, the susceptibility to nephritis is very limited to WKY and its sub-strains.
In addition, the susceptibility varies significantly among these sub-strains and the breeders as
shown in the table.

 

For example, WKY/NCrlCrlj (Charles River Japan) develop severe nephritis by a single IP or IV injection of anti-alpha 4 (IV)NC1 monoclonal antibodies (Figure 1). On the other hand, SHR/Crl rats (Charles River USA) are also susceptible to anti-alpha 4 (IV)NC1 monoclonal antibody-induced nephritis, but the severity is much lower than observed in WKY/NCrlCrlj as shown in Figure 2.

Figure 1 - Dose effect of nephritogenic monoclonal antibody on a) pulmonary hemorrhage and b) proteinuria
in WKY/NCrlCrlj (Charles River Japan)

Figure 2 Nephritis in SHR/Crl rats (Charles River USA)
Nephritis induced by a single IP injection of b35, a84, and 114 (100 mg/rat) in SHR/Crl (Charles River USA) was
much milder than the nephritis in WKY/NCrlCrlj (Charles River Japan).

Figure 3 – Histological changes in the kidney after injection of nephritogenic monoclonal antibodies
Apparent histological changes can be observed in the kidneys after injection of our nephritogenic monoclonal
antibodies such as enlarged glomeruli with severe endocapillary hypercellularity and extracapillary changes such as
capsular adhesion and crescent formation.

Turbidity Assay and Bradford Assay

The turbidity assay method has been widely used to determine urinary protein levels in human specimens, because it is accurate, easy to use, and economical. However, the current turbidity method requires large sample volumes, and cannot be used for assaying urine specimens from certain strains of rats, because of low urine volumes. Please note that the16-hour urine volume varies significantly from 0.1 to 20 ml depending on rat strains. Furthermore, reading turbidity (OD 450 nm) of individual test tubes using a spectrophotometer is time consuming. 

Protein concentrations in urine samples can be determined by Bradford assay methods.  However, the turbidity assay method using 3% sulfosalicyclic acid dihydrate is more convenient than the Bradford method because of the wider range (0.5 – 4.0 mg/ml) of the dose response curve. Importantly, regardless of which assay method is used, bovine serum albumin (BSA) cannot be used as a standard. The dose response curve of BSA significantly differs from the dose response curve of serum proteins in the turbidity assay. More importantly, the OD value of globulins by the Bradford assay method is only 70% of the value of BSA. Therefore, a standard protein solution prepared from normal rat serum is ideal for assaying urinary protein levels regardless of the assay method.