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.

Toxocariasis: serological diagnosis by enzyme immunoassay.

An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in males as in females but showed no significant regression with age between 20 and 65 years. Of 62 patients with non-toxocaral helminthic infections, all had antitoxocaral antibody levels within the range of values observed in healthy controls and had a mean level which was not significantly elevated. All of 13 patients with clinical toxocariasis had enzyme-linked immunosorbent assay (ELISA) antibody levels above the 100th percentiles of both the healthy population and the helminth-infected group and had a significantly high mean value (p less than 0.001) more than 12 times that of the healthy or infected controls. The high degree of sensitivity and specificity of the toxocariasis enzyme-immunoassay indicates that this new test should be useful in reference immunodiagnostic applications and in large-scale seroepidemiological surveys.

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.

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.

An enzyme-linked immunosorbent assay (ELISA) for field diagnosis of visceral leishmaniasis.

A simplified enzyme-linked immunosorbent assay (ELISA) was evaluated as a diagnostic test for visceral leishmaniasis in the field on 222 individuals with splenomegaly and 110 controls. The test was shown to have a sensitivity of 98.4% and specificity of 100% when compared with parasite identification by splenic aspiration. The data indicate that the ELISA is an accurate, safe, and economical alternative to splenic aspiration for the diagnosis of visceral leishmaniasis.

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.

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.

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.

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.

The distribution of enzyme variation in populations of Plasmodium falciparum in Africa.

Isolates of Plasmodium falciparum collected from the east and west coasts of tropical Africa and also from several inland regions have been examined for electrophoretic forms of the enzymes GPI, LDH and 6PGD. Variation within and between isolates was found in the parasite forms of all three enzymes. While not disproving the existence of genetic divisions between P. falciparum populations in Africa the distribution of this variation provides no evidence for such a hypothesis.

A serological study on primary hepatocellular carcinoma in Zambia.

The enzyme linked immunosorbent assay (ELISA) was used to measure alpha-foetoprotein (AFP) levels and to detect Hepatitis B surface antigen (HBsAg) and antibody to Hepatitis B surface antigen (anti-HBs) in patients with primary hepatocellular carcinoma and in controls free of the disease in Zambia. Virtually all the patients with primary hepatocellular carcinoma had grossly elevated AFP levels and about two thirds of them were positive for HBsAg. All the controls had much lower AFP levels and one sixth were positive for HBsAg.

Serum C-reactive protein levels and falciparum malaria.

A microplate ELISA was developed to measure C-reactive protein (CRP) and it was used to establish the relationship between CRP levels and malaria. Highest serum CRP levels were found in African patients with high Plasmodium falciparum parastaemia. However, even African children with lower parasitaemia had higher CRP levels than others without parasitaemia. All African groups studied had CRP levels above those of a control UK group.

Detection of Neisseria meningitidis cell envelope antigen by enzyme-linked immunosorbent assay in patients with meningococcal disease.

Sera from various populations and cerebrospinal fluid (CSF) from meningitis patients were tested for Neisseria meningitidis cell envelope antigen by an enzyme-linked immunosorbent assay (ELISA) sandwich system. The minimum optical density (OD) for antigen detection in CSF was defined as the mean value obtained with specimens from a group of tuberculous meningitis patients plus two standard deviations. By this criterion, antigen was detected by ELISA in four of five CSF specimens from group A meningococcal meningitis patients. Very high ELISA values were obtained with sera from fulminating cases but control sera and sera obtained from meningococcal meningitis non-fulminating cases could not be clearly differentiated by this technique. The possible reasons for this are discussed. The results of this study show that as little as 15 ng/ml (protein) of cell envelope antigen can be detected by the ELISA sandwich test and suggest that the technique may be of value in clinical diagnosis of meningococcal disease.

The development and validation of a simple antigen detection ELISA for Plasmodium falciparum malaria.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is described for the detection of Plasmodium falciparum antigen. The test is based on an immunoglobulin (Ig) M capture monoclonal antibody on the solid phase and an IgG monoclonal antibody conjugated to peroxidase. The simple test takes about 2.5 h to complete and, because it uses whole blood with no prior treatment, it is possible to process batches of 50-100 samples simultaneously. The test is specific to P.falciparum and has a sensitivity close to that usually achieved with Giemsa-stained blood films. The reagents employed are stable at refrigerator temperatures for over 6 months, and as the test is compatible with human immunodeficiency virus and hepatitis B surface antigen ELISAs it could be suitable for blood transfusion screening.

A comparison of thin layer immunoassay (TIA) and enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Trypanosoma cruzi.

The enzyme-linked immunosorbent assay (ELISA) was compared with a new solid phase method, thin layer immunoassay (TIA) for the serodiagnosis of Chagas's disease. 156 sera from Brazilian adult patients who were subject to investigation for chronic Chagas's disease, and 100 sera from healthy Swedish blood donors were analysed. There was a very good agreement between the methods with regard to positive and negative results and it is concluded that either method could be used for sero-diagnosis of Chagas's disease depending upon the choice and facilities of the particular laboratory.

Standardization of the indirect fluorescent antibody test for malaria.

Methods are described whereby results of malarial immunofluorescence tests can be evaluated objectively. The IFA test was quantitated by standardizing the physical system against a fluorescent standard and preparation of biological standards of malarial antisera and fluorescein labelled conjugates. Using these known standards the reactivity of antigens was characterized. It was found that antigen preparations are best when they include mature schizonts, and keep best when they are stored in a dry condition at or below -20 degree C. However, even under carefully controlled conditions of storage, antigens showed considerable variation of reactivity between individual batches.

Detection and measurement of species-specific malarial antibodies by immunofluorescence tests.

Species-specific serodiagnosis of malaria could be made by means of the standardized indirect fluorescent antibody test, either by determination of the usual end-point titres or by fluorescent intensity measurements on antigens. The malarial antibody levels could also be measured by the fluorescent intensity measurements at a single serum dilution. Thus the fluorescent intensity measurements could effectively replace the end point titre determination, with the advantages of standardization and saving in technician time.

An enzyme-linked immunosorbent assay for the detection of Entamoeba histolytica antigens in faecal material.

This paper describes a method for the detection of Entamoeba histolytica antigens in stool samples using a multi-layer ELISA. The method is sensitive and specific, showing no interference with other intestinal parasites, e.g. E. coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii, Hymenolepis nana, Giardia lamblia, Trichomonas and Ascaris. The method provides a rapid and simple screening assay for E. histolytica infections and should assist in diagnosis and epidemiological studies of the disease.