Theoretical analysis of the sensitivity of the solid phase antibody assay (ELISA).

The sensitivity of the solid phase ELISA antibody assay was analyzed from a theoretical perspective. Results show that the investigator may choose either affinity dependent or affinity independent performance by adjusting the assay conditions. High antigen concentration (concn) and small interepitope distance favor affinity independence, while low antigen concn and large interepitope distance favor affinity dependent assay behavior. The maximum sensitivity will occur in the affinity independent region. Antibody titers obtained under affinity independent conditions will reflect true antibody concn, but measurements performed under affinity dependent conditions will underestimate antibody concn.

Theoretical analysis of the accuracy of calibrated immunoassays for measuring antibody concentration.

The accuracy of calibrated immunoassays for measuring antibody concentration was analysed from a theoretical perspective. The study shows that there are theoretical limits on the accuracy of antibody immunoassays, which are determined by the affinity of the standard and unknown antibodies and the conditions chosen for the assay. As a result of these limits, calibration of an immunoassay with a standard antibody does not automatically ensure accurate measurements of antibody concentration. Extremely large errors may develop in affinity-dependent assays when the affinities of the standard and unknown antibodies are different. Assay conditions and the affinity of the standardizing antibody must be chosen carefully to measure antibody concentration accurately.

A modified Farr technique for the detection of antibody in the presence of antigen-antibody complexes.

Antibody bound in an antigen-antibody complex was barely detectable by the standard Farr technique using 10 microgram/ml of radiolabeled antigen. When the Farr test was repeated using larger concentrations of antigen the ABC measurements increased several-fold. In separate experiments complexes were dissociated with low pH buffer. The radiolabeled antigen was then added and the mixture returned to neutral pH before the Farr assay was performed. Pretreatment of the complexes with citrate-buffered saline pH 3.1 enhanced antibody measurements 1.5-3.6-fold. Pretreatment with glycine-buffered saline pH 2.2 enhanced complexed antibody measurements 2.1-4.8-fold. The low pH buffers did not affect ABC values on free antibody. Antibody bound in immune complexes can be detected with a modified Farr technique using a high concentration of antigen and/or by pretreatment of the complexes with low pH buffers before the addition of radiolabeled antigen.

Neo-Mull-Soy metabolic alkalosis: a model of Bartter's syndrome?

A case of Neo-Mull-Soy-induced metabolic alkalosis occurred in an 8-month-old child. This child had hypochloremic hypokalemic alkalosis as well as hyperreninemia. Initially, a diagnosis of Bartter's syndrome was made and treatment consisted of KCl replacement, indomethacin, and aspirin. In retrospect, the diagnosis of Neo-Mull-Soy induced metabolic alkalosis could have been suspected on the basis of the low chloride concentration in his urine. Proposed mechanisms for the etiology of Bartter's syndrome are reviewed. Neo-Mull-Soy induced metabolic alkalosis simulates Bartter's syndrome and supports the concept that the primary abnormality in Bartter's syndrome is chloride deficiency. The chloride deficiency in Bartter's syndrome results from a defect in active chloride transport in the thick ascending limb of the loop of Henle.

Accumulation of aluminum in a nondialyzed uremic child receiving aluminum hydroxide.

A child with renal insufficiency was treated with the oral phosphate binder aluminum hydroxide from age 6 to 31 months. The prescribed dose of elemental aluminum varied from 31 to 108 mg/kg/d. Concurrently the patient developed vitamin D-resistant osteomalacia which failed to improve with parathyroidectomy. Encephalopathy with myoclonic seizures, loss of speech, and motor impairment also occurred. Serum and bone aluminum levels were elevated at 334 micrograms/L (normal 7 +/- 3 micrograms/L) and 156 mg/kg (normal 3.3 +/- 2.9 mg/kg), respectively. This case demonstrates that aluminum may accumulate in tissue of children receiving oral aluminum hydroxide. The accumulation of aluminum may have contributed to the vitamin D-resistant osteomalacia and the encephalopathy in this patient. Children receiving aluminum-containing antacids as phosphate binders should be monitored for aluminum accumulation and signs of aluminum intoxication.

The Cockayne syndrome: an evaluation of hypertension and studies of renal pathology.

Three children with renal disease, hypertension, and the Cockayne syndrome were evaluated. All patients had severe hypertension; peripheral vein renin was elevated in two patients. Renal biopsy specimens from two patients were studied by light microscopy, electron microscopy, and immunofluoresence. Immunohistologic studies demonstrated deposits of immunoglobulin and complement in the vessels and glomeruli of the first patient; deposits of immunoglobulin and complement were seen in the glomeruli of the third patient. Also electron-dense deposits were seen in the glomerular basement membrane of the third patient. Circulating immune complexes were detected by the Raji cell and Clq binding techniques in this child as well. Both hypertension and renal disease are frequent complications of the Cockayne syndrome.

A rapid method for the determination of antibody affinity.

A rapid method for estimating antibody affinity is described. To perform the assay fixed amounts of radiolabeled antigen and antibody are mixed in different total reaction volumes. The fraction of antigen bound to antibody is measured in aliquots from each reaction volume by the ammonium sulfate precipitation technique. Affinity constants can be estimated from these data graphically or by means of a microcomputer. This method is much quicker and easier to perform than traditional techniques for estimating antibody affinity.

Pulmonary vasculitis with hemorrhage in anaphylactoid purpura.

We report a 14-year-old female with anaphylactoid purpura (AP) who developed pulmonary hemorrhage with acute vasculitis on lung biopsy. She improved with pulse methylprednisolone, daily prednisone and ventilatory assistance. Pulmonary vasculitis is a rare but serious manifestation of AP.

Intravenous methylprednisolone and oral alkylating agent therapy of prednisone-resistant pediatric focal segmental glomerulosclerosis: a long-term follow-up.

UNLABELLED: Prednisone-resistant nephrotic syndrome (NS) due to focal segmental glomerulosclerosis (FSGS), the most common acquired disease requiring chronic dialysis and transplantation in children, has a low likelihood of response to alkylating agent therapy. This report summarizes the results of a 0.75-12.5 (average 6.33) year follow-up of 32 pediatric cases of prednisone-resistant FSGS treated with a regimen of high-dose intravenous methylprednisolone (M-P) and alternate-day prednisone, plus an alkylating agent in 25/32. On last followup: 21/32 were in remission [urine protein-to-creatinine ratios (Pru/Cru) < or = 0.2]; 3/32 had mild proteinuria (Pru/Cru > 0.2-0.5); 2/32 had moderate proteinuria (Pru/Cru > 0.5-1.9); and 6/32 remained nephrotic (Pru/Cru > or = 2.0). Of the incomplete or nonresponders; 3/11 progressed to end-stage renal failure; 5/11 had decreased creatinine clearances (CrCl): and 3/11 had persistent proteinuria with normal CrCl. All of the persistently nephrotic children, but none of the complete responders, developed decreased CrCl. All of the complete responders were able to stop treatment; four relapsed but responded well to retreatment. CONCLUSIONS: This regimen of methylprednisolone and alternate-day prednisone, with or without an alkylating agent, is effective in achieving sustained remissions and preserving normal renal function in the great majority of children with FSGS and prednisone-resistant NS.

Multiple thromboses in a premature infant associated with maternal phospholipid antibody syndrome.

Phospholipid antibodies (lupus anticoagulant, cardiolipin) are associated with a syndrome of repeated fetal loss. Mothers with phospholipid antibodies are currently being treated with either prednisone, aspirin, or heparin to prevent fetal death. We describe a neonate whose mother had cardiolipin antibody and recurrent fetal loss and was treated with prednisone and aspirin. Thrombosis was noted in placental fetal vessels. Thromboses developed in the infant's aorta, left renal artery, middle cerebral artery, and superior sagittal sinus. Infants of phospholipid-positive mothers may have vascular thrombosis and should be carefully monitored for signs of thromboembolism.

Follow-up of infants with obstructive uropathy detected in utero and treated surgically postnatally.

Thirty-four infants with obstructive uropathy detected by prenatal ultrasonography at 34 +/- 0.56 weeks (SE) had surgery during the first 2 years of life. The growth and renal function of the 25 children after being followed for over 1 year are described. Twelve children had unilateral disease, and 13 had bilateral disease. All 12 children with unilateral disease grew at or above the fifth percentile and had normal renal function (glomerular filtration rate, 101 +/- 7 mL/min/1.73 m2). Thirteen children with bilateral disease were followed for 26.1 +/- 3.4 months. Growth was good: 10 of the 13 grew at or above the fifth percentile. The serum creatinine was 0.7 +/- 0.2 mg/dL, and the glomerular filtration rate was 91 +/- 10 mL/min/1.73 m2. One child required chronic dialysis. The prenatal diagnosis of urinary tract anomalies followed by early intervention may improve the long-term outcome of children with obstructive uropathy.

Extracorporeal membrane oxygenation in the neonate with congenital renal disease and pulmonary hypoplasia.

Extracorporeal membrane oxygenation (ECMO) is an effective treatment modality for the newborn with refractory hypoxemia. Oligohydramnios can be associated with congenital renal disease (CRD) and can result in respiratory insufficiency from pulmonary hypoplasia, delayed lung maturation, and persistent pulmonary hypertension of the newborn. In this retrospective study, the authors reviewed the outcome of four children with CRD who required ECMO in the neonatal period. Between October 1987 and December 1995, ECMO was used in four newborns with CRD and pulmonary hypoplasia unresponsive to maximal medical management. The causes of CRD were urinary obstruction (2), renal dysplasia (1), and vesicoureteral reflux (1). Neonatal survivors of ECMO with CRD had regular follow-up with a nephrologist, urologist, and pediatrician. Developmental history, assessment of renal function, and a nutritional evaluation were recorded on each visit. The follow-up period ranged from 6 months to 5 years. All patients with CRD were successfully weaned from ECMO. One child died, at 1 month of age, because of renal failure. The estimated glomerular filtration rates in the three survivors were 20, 24, and 60 mL/min/1.73 m2. Growth and development have been delayed in two patients. Based on the author's experience, ECMO may improve the survival of neonates with pulmonary hypoplasia and CRD. Factors associated with successful long-term outcome include (1) renal disease amenable to surgical correction, (2) aggressive nutritional support, and (3) a reliable social support system.

Theoretical aspects of screening for high affinity monoclonal antibody to cell surface antigens.

The relative affinity of the cell-antibody interaction can be determined by finding the highest titer of one reactant which binds effectively to a fixed, dilute concentration of the other. There are two ways to perform these experiments: the antibody dilution method and the cell dilution method. The antibody dilution approach requires measuring the highest antibody dilution which can react with a small, fixed concentration of cells. If the endpoint antibody concentration is small, the antibody has a high binding constant. In the cell dilution method the investigator must find the highest cell dilution which can bind a small, fixed antibody concentration. If the antibody has a high affinity constant, the endpoint cell concentration will be small. When the antibody and cell receptor concentrations are known, functional binding constants can be calculated directly from binding data using equations presented in this paper. Binding constants measured by these methods will help predict the behavior of monoclonal antibodies in immunoassays and in experiments where monoclonal antibody is injected in vivo for imaging or pharmacologic delivery.

A computer program for calculating antibody affinity constants.

A program is presented for the calculation of antibody affinity constants which avoids the limitations of graphical plotting of titration data using the Sips or Scatchard equations.