Enzyme immunoassays with special reference to ELISA techniques.

In this review outlines are given on various types of enzyme immunoassay. The applications to such enzyme immunoassays, especially ELISA, are dealth with in detail. It is concluded that these techniques have high sensitivity and will be suitable in due course as routine laboratory tests.

A comparison of three tests for rubella antibody screening.

Sera from 1000 women attending antenatal clinics were screened for the presence of rubella antibody by haemagglutination inhibition (HI), single radial haemolysis (SRH), and enzyme-linked immunosorbent assay (ELISA). With 925 sera concordant results were obtained with all three tests. There were 46 false-negative HI tests due to the necessity of allowing for residual non-specific inhibitors. With SRH there was one false positive and three that were negative by SRH but HI- and ELISA-positive. By ELISA 14 sera gave a false-positive result (OD 405 nm greater than or equal to 0.2). Ten of these could be eliminated by taking an OD 405 nm of 0.5 as the threshold but then another 10 sera became false negatives.

Diagnosis of human toxocariasis by antigen capture enzyme linked immunosorbent assay.

AIMS: To evaluate an antigen capture enzyme linked immunosorbent assay (ELISA) which detects a carbohydrate epitope on the excretory-secretory (ES) antigens of Toxocara canis in clinical practice. METHODS: Serum specimens from healthy adults, patients with acute visceral larva migrans, ocular and inactive toxocariasis, and with other helminth infections were examined by two site antigen capture ELISA. RESULTS: Over half of the patients (19/28) with acute toxocariasis had a positive result in contrast to a small proportion of those with inactive disease (1/10) or ocular infection (2/7). False positive reactions, however, were found in 25% of the patients with serologically confirmed schistosomiasis and filariasis. CONCLUSIONS: This assay is useful in confirming the diagnosis of acute visceral larva migrans but could not be used alone in diagnosis because of false positive reactions in patients with other helminth infections.

A microplate enzyme-immunoassay for toxoplasma antibody.

A new test for the detection and measurement of toxoplasma antibody is described. Test sera are reacted with antigen-sensitized wells in micro-haemagglutination plates. Any attached antibody is shown by the addition of an enzyme-labelled antiglobulin followed by assay of the enzyme reaction with its substrate. The test is easy to carry out on a large scale, and there is a positive correlation between the results and dye test and haemagglutination test titres.

IgM and antibody measurement in the diagnosis and management of Gambian trypanosomiasis.

Serum and cerebrospinal fluid (CSF) IgM were measured in 182 patients at various stages of Gambian sleeping sickness and correlated with antibody levels measured by enzyme-linked immunosorbent assay (ELISA). Each of these tests in serum gave a 30% false negative result, but when both were used this fell to 12%. Measurements of IgM in CSF were raised in 87% of patients with advanced disease and in none of the early cases. The IgM levels fell slowly to normal by 12 months after treatment. A high level at this time, or a rise after treatment, was helpful in diagnosing relapsed patients. Antibody levels in CSF were of no use in diagnosis or prognosis, and were raised in many controls.

Epitope mapping of HBsAg using a panel of human anti-HBs antibodies.

Mapping of B cell epitopes on HBsAg was performed using a panel of human anti-HBs antibodies. Synthetic peptides representing different regions of HBsAg failed to inhibit the binding of two antibodies which recognized non-conformational HBsAg determinants in dot-blot ELISA and HBsAg polypeptide bands in immunoblot analysis. Cross-inhibition studies using five of the antibodies conjugated to horseradish peroxidase suggested that at least three different epitopes are recognised by the panel of antibodies, two of which are within the 'a' group determinant.

Comparison of an EBV transformed cell line and an EBV hybridoma cell line producing the same human anti-HBs monoclonal antibody.

A stable human-mouse heterohybridoma secreting human anti-HBs monoclonal antibody in continuous culture for 12 months was generated. It grew faster than the parent EBV transformed lymphoblastoid cell line (LCL) but produced the same level of specific antibody. The LCL was positive for the Epstein-Barr Virus Nuclear Antigen (EBNA), human CD 23 and contained a diploid number of human chromosomes. The heterohybridoma was negative for EBNA, CD 23 and mouse Ly-1 mouse, despite retaining a full complement of diploid mouse chromosomes and a limited number of human chromosomes.

Development and characterization of human anti-HBs antibodies.

Peripheral blood mononuclear cells (PBMC) from 13 healthy hepatitis B vaccines were transformed with the Epstein-Barr virus (EBV) and lymphoblastoid cell lines (LCL) producing antibodies to hepatitis B surface antigen (anti-HBs antibodies). Seven LCL and two clones secreting human anti-HBs monoclonal antibody were generated and their antibodies purified. One clone was fused with a mouse myeloma and the antibody from a cloned anti-HBs secreting heterohybridoma purified. One of the 10 purified human anti-HBs antibodies was characterized as IgG4, the remainder were IgG1. The antibodies had either kappa or lambda light chains. Five of the antibodies which were conjugated to horseradish peroxidase recognised the "a" group determinants.

Human erythrocyte superoxide dismutase in adults, neonates, and normal, hypoxaemic, anaemic, and chromosomally abnormal fetuses.

An enzyme-linked immunosorbent method is described for the assay of human erythrocyte superoxide dismutase (SOD). The method was specific and precise as well as simple, economical, and reliable. The mean levels of adult SOD with SD were 652 +/- 122 mg/kg hemoglobin (Hb) for males and 635 +/- 100 mg/kg Hb for females. In neonates the levels were 528 +/- 92 mg/kg Hb. Normal fetal levels at 20-36 weeks gestation were 421 +/- 90 mg/kg Hb. Mean SOD levels in hypoxemic growth-retarded fetuses and in anemic fetuses from red cell iso-immunised pregnancies (before their first intrauterine blood transfusion) were the same as those in normal fetuses. However, in a case of trisomy 21 the fetal level of SOD was significantly increased.

The measurement of human erythrocyte carbonic anhydrase I by the enzyme linked immunosorbent assay (ELISA).

A method is described for the assay of human erythrocyte (RBC) carbonic anhydrase I (HCAI) by the enzyme linked immunosorbent assay. The method was found to be a simple, reliable and precise technique and gave a mean C.V. for 40 samples, assayed in quadruplicate, of 3.32% and a range of 0.84-5.9. The mean erythrocyte HCAI value and standard deviation for 20 normal men and women were respectively 16.9 +/- 3.4 and 15.4 +/- 2.1 mg HCAI/g haemoglobin. The use of heparin as an anticoagulant interfered with the assay resulting in apparent HCAI concentrations as low as 60% of those obtained using EDTA.

ELISA--a possible alternative to establish a therapeutic drug monitoring system in severe and complicated falciparum malaria.

A modification of an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of plasma quinine levels is described. The test is rapid, sensitive and reproducible.

A comparison of isotopic and enzyme-immunoassays for tropical parasitic diseases.

A comparison is made of enzyme-immunoassay and radio-immunoassay for the detection of antibody in Chagas's disease, sleeping sickness, malaria, schistosomiasis ans invasive amoebiasis. Both assays were sensitive and reproductible and gave comparable results.

A comparison of chloroquine and pyrimethamine as malaria chemoprophylactics in young Nigerian children.

The efficacy of chloroquine and pyrimethamine as malaria chemoprophylactics was investigated in young Nigerian children. Chloroquine resistance had not been documented in the study area; pyrimethamine resistance was probably present but uncommon. Children who received weekly chemoprophylaxis with pyrimethamine had a lower prevalence of malaria parasitaemia and malaria antibodies than children who received weekly chemoprophylaxis with chloroquine. Pyrimethamine given monthly gave a comparable degree of protection to chloroquine given weekly. Chloroquine frequently induced vomiting in young children and this may have impaired its efficacy as a prophylactic. We conclude that, in an area where neither chloroquine nor pyrimethamine resistance is prevalent, pyrimethamine is a better chemoprophylactic for young children than chloroquine.

A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite.

The enzyme-linked immunosorbent assay (ELISA) has been the most widely used serological test in snake bite immunodiagnosis and epidemiology. The technique has been applied, however, without due consideration of the many factors which would affect an inherently sensitive test system, especially in tropical rural areas where large scale snake bite studies are usually carried out. This review discusses the effects of non-specific reactivity, cross reactivity and the quality of reagents on both the sensitivity and specificity of venom antigen and antibody detection assays. Simple laboratory modifications to optimize the assays are described. The importance of using the predictive value to assess the validity of applying the same test system in different circumstances is stressed. To fulfil its potential as the most versatile immunoassay technique in snake bite research, the test conditions of the ELISA will have to be much more stringently controlled in future.

Viscus perforation in peritoneal dialysis patients: diagnosis and outcome.

OBJECTIVE: To determine the incidence and outcome of spontaneous viscus perforation in peritoneal dialysis (PD) patients and which factors could facilitate early diagnosis. DESIGN: A retrospective chart review was done on all patients with viscus perforation and on a control group with peritonitis secondary to gram-negative organisms. SETTING: A tertiary care University Hospital Peritoneal Dialysis program. PATIENTS: All patients with surgically proven spontaneous viscus perforation from 1978 to June 1992 (n = 15). A group of control patients (n = 15) with gram-negative bacterial peritonitis was also reviewed for comparison. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Hospital days, patient survival after perforation, and return to peritoneal dialysis were the main outcomes measured. Peripheral white blood cell (WBC) count, PD fluid WBC count with differential, PD fluid cultures, radiologic information, and surgical intervention were also evaluated. Data were analyzed using the Mann-Whitney test to determine significant differences between the two groups. RESULTS: Viscus perforation occurred in 15 of the 431 patients on PD from 1978 to June 1992 (3.5%). In comparison to the control group, patients with viscus perforation had a significantly higher peripheral WBC count (p = .016), a higher mean PD fluid WBC count (p = .006), and a higher mean percentage of polymorphonuclear cells in the PD effluent (p = .038). Multiple organisms on PD fluid cultures were noted in 12 of 15 patients with perforation and in only 3 control patients. Pneumoperitoneum was seen on abdominal or chest radiograph or computerized tomographic (CT) scan in 10 of 15 patients with perforation and in only 1 of 15 patients in the control group. All patients with viscus perforation required surgery and 6 expired. Only 1 death occurred in the control group. Only 1 of the 9 patients surviving perforation was able to resume PD, in contrast to 13 of 14 surviving control patients. CONCLUSION: We conclude that viscus perforation is associated with high morbidity, mortality, and technique failure. Diagnosis may be made by repeatedly searching for intraperitoneal free air on radiograph or CT scan in patients with persistently elevated peripheral and PD fluid WBC count, and for multiple organisms on PD fluid culture.

Long-term outcome of diabetic patients receiving peritoneal dialysis.

OBJECTIVE: Data from the United States Renal Data Systems (USRDS) suggest that older diabetic patients with end-stage renal disease will have improved survival if they receive hemodialysis versus peritoneal dialysis. Younger diabetic patients have equal survival on either treatment modality. To address more specifically the risk factors for long-term survival of diabetic patients receiving peritoneal dialysis, we analyzed the long-term outcome of 118 diabetics receiving peritoneal dialysis over a decade and compared them to 165 nondiabetic patients. DESIGN: Retrospective analysis utilizing the Cox proportional hazards model to identify risk factors for survival of both diabetic and nondiabetic patients. SETTING: An experienced, single-center, university-based dialysis program. PATIENTS: All patients receiving home peritoneal dialysis for at least one month from 1 January 1981 to 31 December 1990. Diabetics were classified as type I or type II, in addition to age stratification. Most type I diabetic patients used insulin via the intraperitoneal route. MAIN OUTCOME MEASURES: Patient survival and technique survival. RESULTS: The most significant risk factor for diabetic patient survival was diabetes type (relative risk type I to type II 0.14, p < 0.0001). On treatment serum albumin, predialysis blood urea nitrogen and predialysis serum cholesterol were also significant risk factors (p < 0.01). For nondiabetic patients, age, on treatment serum albumin, and current smoking were significant survival risk factors. Survival of patients 55 years or less was not significantly different between diabetic and nondiabetic patients. Survival of patients greater than 55 years was better in nondiabetic patients. CONCLUSION: These findings of a long-term follow-up period suggest a good survival for younger type I diabetic patients receiving peritoneal dialysis. Reasons other than age for the discrepancy in survival of young versus old diabetics receiving peritoneal dialysis should be sought.

Serological response of mice to Fusobacterium necrophorum.

Mice immunised with killed or living Fusobacterium necrophorum, by five different regimens, almost invariably failed to produce antibodies demonstrable by a passive haemagglutination test. An enzyme-linked immunosorbent assay (ELISA), however, usually demonstrated a serum antibody response. This suggested that F necrophorum was not in fact immunosuppressive in mice--a possibility that had been entertained to explain the great difficulty in protecting mice immunologically against challenge with F necrophorum.

Comparisons of serological tests for Babesia in British cattle.

A comparison was made between the microplate enzyme linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) and complement fixation (CF) tests for the detection of antibodies in the serum of cattle experimentally infected with Babesia divergens and B major. Antibodies were detected using all three tests but they were detected earlier using the CF test. However CF titres were consistently lower than those obtained using the other tests. Although there was little to choose between the IFA and ELISA tests, it was suggested that the ELISA may be preferable since it is less subject to operator error and operator stress, and can be adapted readily to field use.