Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to thyroglobulin

We used a computer programmed standard IgG curve for computer-assisted quantification of assay results for autoantibodies to thyroglobulin (Tg) by quantitative enzyme-linked immunosorbent assay (ELISA). Specific antibody levels in unknowns were quantified by comparison of their optical density readings with a standard curve of absorbance vs concentration obtained with dilutions of the reference serum. Anti-Tg antibodies were detected in 80% of the patients with chronic thyroiditis and 90% of those with Graves' disease. Anti-Tg antibodies were also detected in 14.3% of the healthy controls. The titer of anti-Tg antibodies detected by tanned red cell hemagglutination correlated well with that detected by ELISA, although, the sensitivity of the ELISA was higher. By our computer-assisted conversion method, the anti-Tg antibody can be readily and reliably quantified and low titer antibodies to Tg can be detected with adequate precision.     

Enzyme-linked immunosorbent assay (ELISA) for human epidermal growth factor (hEGF)

An enzyme-linked immunosorbent assay for human epidermal growth factor (hEGF) by the sandwich method with the use of orthophenylene diamine (OPDA) for the substrate of the enzyme reaction was developed. This assay showed high sensitivity, and there was no problem with the recovery rates even without any pretreatment of samples. The changes of hEGF levels in plasma and serum depend on the condition of blood storage from sampling until separation of the cell content. The levels of hEGF markedly increased with time in serum, especially when stored at room temperature but less so in plasma. Changes of hEGF levels in plasma were negligible for up to 3 h if the blood was stored at 4 degrees C until separation.     

Enzyme-linked immunosorbent assay (ELISA) for quantitation of blood group A antibodies in intravenous immunoglobulin

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure antibody to human blood group A utilizing porcine substance A as the coating antigen. The anti-A ELISA assay was compared to the Coombs antiglobulin test using anti-A containing samples of intravenous immune globulin. ELISA assay results were more reproducible and demonstrated greater precision than Coombs assay results. The correlation between ELISA and Coombs potencies is highly significant in the case of dilutions within one lot but not for different lots at a fixed dilution. The ELISA precision as well as the ability to produce test values in a linear progression rather than the doubling dilution system of the Coombs assay provides the potential for sensitive studies involving anti-A.     

Enzyme-linked immunosorbent assay (ELISA) for the measurement of small quantities of alpha 2-macroglobulin

A simple, sensitive and precise enzyme-linked immunosorbent assay for the quantitation of alpha 2-macroglobulin (alpha 2M) in supernatants of cell cultures was constructed. All reagents apart from the alpha 2M standard were commercially available. The assay range was 2.0-500 micrograms/l. The intra-assay coefficient of variation (CV%) was 4.6%, and the imprecision between runs was 8.9% at 10 micrograms/l and 9.0% at 110 micrograms/l. Recovery of alpha 2M, added to cell culture medium free of serum, was 97.5 +/- 7.2% (mean +/- SD) and the recovery of alpha 2M added to pooled human serum was 101 +/- 6.0%. There was no significant difference between the recovery of alpha 2M-standard and alpha 2M-trypsin complexes, whereas the dose response of a commercial alpha 2M-standard was lower than expected (81.1 +/- 7.5%), indicating a lower purity and/or conformational changes in the epitopes of this reagent. As expected, supernatants of mononuclear lymphocyte cultures enriched in monocytes contained significantly higher concentrations of alpha 2M than supernatants of cell cultures depleted in monocytes. Our results indicate that the ELISA method could be useful tool in the study of the alpha 2M turnover in all cell cultures in vitro.     

Enzyme-linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone

The development of non-competitive microtitre plate enzyme-linked immunosorbent assays for steroids was investigated using immobilised steroid as immunosorbent and enzyme-labelled second antibodies. An assay for testosterone with polyclonal anti-testosterone immunoglobulins was optimised with respect to a number of parameters but remained unsatisfactory for clinical assays. The results were applied to developing an aldosterone assay using monoclonal anti-aldosterone immunoglobulins. The latter method was used for the determination of urinary aldosterone and the results are compared with those obtained by a classical radioimmunoassay. Problems concerned with the use of sulphur-containing proteins and with the presence of low affinity antibodies in polyclonal preparations are discussed.     

Enzyme-linked immunosorbent assay for chromogranin A

This is an enzyme-linked immunosorbent assay (ELISA) for determining chromogranin A (CGA) with use of a monoclonal antibody. CGA was isolated from bovine chromaffin granules. The analytical ELISA procedure for bovine CGA was developed and optimized. Typical standard curves ranged from 500 pg to 500 ng of CGA. We then studied human plasma CGA-immunoreactivity as measured by this assay. The curve for dilutions of human plasma paralleled the standard curve for bovine CGA. The intra-assay coefficient of variation for determination of human plasma CGA was 4.56%, indicating that reliable determinations can be performed for human plasma. However, further study revealed the presence of two CGA-immunoreactive substances in human plasma, one of which corresponds to the native CGA. The nature of the second immunoreactive substance still remains unknown. Nevertheless the measured CGA concentrations (ranging from 0.19 to 0.35 mg/L) in plasma are comparable with previously reported values.     

Semi-automated enzyme-linked immunosorbent assay (ELISA) for the quantification of apolipoprotein B using monoclonal antibodies

A semi-automated competitive, double-antibody, solid-phase enzyme-linked immunosorbent assay for apolipoprotein B (Apo B) has been developed which utilizes microtiter plates with commercially available monoclonal antibodies and alkaline phosphatase-conjugated second antibody. The working range of the assay is 20–200 ng. The concentration of plasma Apo B was 0.88 ± 0.20 g/l (n = 40) for a random sample of normal adults. The correlation coefficient for this assay, compared to a radial immunodiffusion assay, was 0.95 (slope = 1.13, INTERCEPT = −15). The quantification of the samples was not influenced by freezing and thawing, storage at −20°C for up to 9 mth, or the lipoprotein particle on which the Apo B was present. The method is suitable for measurement of apolipoprotein B in either normal or pathological plasma, lipoprotein density classes, and is sensitive enough to quantify Apo B in cell biological and molecular biological investigations.     

Enzyme-linked immunosorbent assay for apolipoprotein C-I

A non-competitive sandwich enzyme-linked immunosorbent assay for apolipoprotein C-I was developed. Sheep antibody to this apolipoprotein C-I, purified by affinity chromatography, was used for coating the wells of a microtiter plate and as a conjugate with alkaline phosphatase. The linear range of the assay was from 80 ng to 15 ng. It was sensitive down to 5 ng. The intra-assay variation coefficient was 2.8%, and the inter-assay variation coefficient 5.3%. The mean concentration of apolipoprotein C-I was 61 +/- 20 mg/l in healthy normal males, and 65 +/- 19 mg/l in females. Apolipoprotein C-I levels were positively correlated with the total cholesterol concentration in both sexes (p less than 0.002). A significant correlation with triacylglycerol was only observed in males (p less than 0.05). A significant increase of apolipoprotein C-I was observed in type V hyperlipoproteinaemia, and in the only studied case of type III.     

Enzyme-linked immunosorbent assay (ELISA) of changes in specific IgG antibodies to five venoms during venom immunotherapy

An enzyme-linked immunosorbent assay (ELISA) was developed that could measure titres of human IgG antibodies to five different venoms (honeybee, yellow jacket, yellow hornet, white-faced hornet, and wasp), and to honeybee phospholipase A. Changes in specific IgG anti-venom titres were measured in twenty patients that had systemic anaphylactic reactions to insect stings, and ten non-allergic controls. After being stung and prior to treatment all patients had anti-venom IgG titres greater than controls. Treatment with small doses of venom over 1-2 months resulted in prompt rises in anti-venom IgG titres that may represent secondary anemnestic responses primed by prior stings. All patients undergoing venom immunotherapy showed at least 2-fold increases in IgG antibody to the venoms they were treated with by the time maintenance doses of 100 mcg were achieved, with one exception. Significant cross-reactive increases in anti-vespid IgG antibodies to venoms not used for treatment occurred in nine of eighteen treated patients. Overall, ELISA of IgG antibodies to five venoms allowed clear evaluation of the considerable variation of IgG responses among different patients. We conclude that serial determination of venom-specific IgG titres by ELISA offers an important adjunct to evaluating the results of venom immunotherapy.     

Enzyme-linked immunosorbent choriomammotropin assay.

We have devloped an enzyme-linked immunosorbent assay for determining choriomammotropin (human placental lactogen) in serum. Unlabeled hormone competes with choriomammotropin-beta-galactosidase conjugate for antibody bound to polystyrene tubes. The entire assay can be performed in 2.5 h with good precision. The coefficient of variation for one sample with a mean concentration of 5.6 mg/L, assayed 10 times on the same day, was 5.7%. The coefficient of variation for nine samples (3.5 to 9.0 mg/L) assayed on five different days was 7.9%. Forty-eight clinical samples were assayed (y) and compared with results obtained by radial immunodiffusion (x). The resulting regression equation was: y = 1.05x + 0.78; r = 0.91.     

Enzyme-linked immunosorbent assay for alpha-fetoprotein in human serum

A simple and inexpensive enzyme-linked immunosorbent assay for determination of serum alpha-fetoprotein concentration, with results available within 4 h, is described. The assay has a working range of 2-100 ng/ml, with within- and between-batch coefficients of variation (n = 24) at different concentrations between 8.9 and 12.7%. Results on 80 patients' sera (AFP concentrations 0-175 ng/ml) correlate well with those by radioimmunoassay. Turn around time (4 h) is shorter than with similar immunoassays currently available.     

Enzyme-linked immunosorbent microtiter assay for red cell serology

Enzyme-linked immunoabsorbent assays (EIA) permit quantitation and automation of reactions. Previous reports have also shown improved sensitivity for red cell antibodies. We have developed a microtiter technique for EIA, thus decreasing the amounts of reagents required. EDTA was used rather than NaOH to stop the enzyme reaction allowing more precise timing of reactions and improved quantitation of large batches of tests. As in prior reports, the sensitivity was slightly greater than the antiglobulin test, but occasional red cell antibodies failed to show specificity by EIA. The use of microtiter plates and EDTA decreased the steps and reagents required and has the potential for automation of at least the optical reading of such assays in the blood bank.     

Enzyme-linked immunosorbent assay of lipoprotein(a) in serum and cord blood

We have developed a new sensitive method for quantifying lipoprotein(a) (Lp(a] in human serum, using a 'sandwich' type noncompetitive enzyme-linked immunosorbent assay (ELISA). The solid-phase used was a polystyrene plate. The anti-Lp(a) antibody-enzyme conjugate was labelled by linking Fab' fragments to peroxidase (EC 1.11.1.7) by the maleimide method. The minimum detectable concentration was 0.5 ng/well. Routinely, the assay was carried out with 1,000-fold diluted serum, and Lp(a) was quantified between 4.0 and 500 mg/l. Within-run coefficients of variation (CVs) ranged from 3.5% to 10.4% and between-run CVs from 5.0% to 11.1%. Results by the ELISA were in good agreement with those by radial immunodiffusion (r = 0.955). The distribution of Lp(a) in serum from 820 healthy donors was highly skewed: mean 141.1 mg/l, medium 97.9 mg/l. In cord blood, the mean and median were 15.6 and 9.8 mg/l, respectively. This ELISA for Lp(a) has the advantages of being highly sensitive and specific, simple to perform, and does not use radioisotopes.     

Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)

This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors.     

Enzyme-linked immunosorbent assay of serum pepsinogen I

A hybridoma monoclonal antibody against human pepsinogen I was used to develop an enzyme-linked immunosorbent assay for pepsinogen I in serum. In the two-step competitive procedure using antimouse immunoglobulin F(ab')2 fragment coupled to alkaline phosphatase, the measurable assay range was 8-256 micrograms/l. No cross-reactivity with rat pepsinogen 1, human pepsinogen II, gastrin I, bombesin, somatostatin and peptide YY was shown. However, there was slight cross-reactivity (0.09%) with porcine pepsinogen. The coefficients of variation within and between series were 7.6% and 13.0%. This enzyme-linked immunosorbent assay for serum pepsinogen I correlated positively with radioimmunoassay (r = 0.87, n = 92). The concentration range of serum pepsinogen I in 354 healthy controls was 15-100 micrograms/l with a lognormal distribution. Serum pepsinogen I levels were significantly higher in the subjects who developed active duodenal ulcer or active gastric ulcer, but significantly lower in those who had gastric cancer, than in control subjects.     

One-step enzyme-linked immunosorbent assay (ELISA) for measurement of serum free leptin

In man, circulating leptin levels are increased with obesity and are regulated by a complex of hormonal, feeding and body-weight changes. Accurate and precise methods to quantitate circulating serum free leptin (f-leptin) concentrations are needed for physiological and clinical studies. We developed a one-step enzyme immunoassay to measure human f-leptin in serum. The detection limit was 0.40 ng/ml. The recovery of leptin added to serum was 90.8-102.8%. The within-run and between-day coefficients of variation (C.V.) ranged from 2.8 to 7.7 and 5.7 to 9.7%, respectively, and the immunoassay had an overall recovery rate for serial dilution in the range of 94. 0-109.9%. Measured serum f-leptin concentrations in 201 adults correlated (r=0.449, P<0.001) directly with body mass index (BMI kg/m(2)), particularly when results were separated by gender (r=0. 709 for male, P<0.001; r=0.643 for female, P<0.001). We conclude that this one-step enzyme immunoassay is accurate for measuring f-leptin in human serum.     

Enzyme-linked immunosorbent assay for ileal human goblet cell mucin

A sandwich ELISA for measuring ileal human goblet cell mucin has been developed with a linear response range 0.2 to 1.5 ng mucin protein. It can be used to quantitate the mucin present in dilute and unpurified samples without interference from other glycoproteins and proteins. Reduction of the mucin decreased the reactivity by only 14% indicating that the assay reacts almost as well with mucin glycopeptides as with native mucin. The assay has the advantages over previously described immunoassays for mucin of giving a result in 6 h, detecting slightly lower concentrations of mucin, and is more sensitive, quantitative and specific than the traditional protein or periodic acid-Schiff assays used for glycoproteins.