Further Improvement of the Enzyme-Linked Antiglobulin Test (ELAT) for Erythrocyte Antibodies

A solid-phase kinetic enzyme-linked immunosorbent assay has been developed to measure binding of antibodies to purified or synthetic blood group antigens and tested in the Lewis blood group system. Chemically synthesized Lewis α antigen is used as the target for the binding of serum antibody in this sandwich-type assay. Kinetic, rather than endpoint, determinations are used to calculate the amount of specific antibody. Data are presented showing the assay to be quantitative, sensitive, and specific. It can separately quantitate the amount of IgG or IgM anti-Lewis a present in patient sera. The assay uses commercially available reagents and is semiautomated. Thus, it will be useful for studies in quantitative immunohematology as other blood group antigens become available in purified form.

Section: Discussionmentioning

confidence: 99%

A New Technique in Quantitative Immunohematology: Solid-Phase Kinetic Enzyme-Linked Immunosorbent Assay

Spitalnik¹,

Cowles²,

Cox³

et al. 1983

“…The detecting anti body was added after the removal of red cell proteins. This is a major advantage over ELAT, where the release of he moglobin from erythrocytes remains a problem, causing ve ry high blank values, despite several attempts to eliminate these interferences [7,18], The IgG-ELISA was adapted from an immunoassay used in our laboratory for the mea surement of very low concentrations of IgA [15]. A pair of polyclonal antibodies giving minimal cross-reactivity (i.e.…”

Section: Comparison Of Elisa and Automated Hemagglutinationmentioning

confidence: 99%

“…Previous studies have described quantitation of anti-D using radioisotopes [4,5] or an enzyme-linked antiglobulin assay (ELAT) [6,7]. The latter has been modified for the assess ment of anti-D immunoglobulin preparations on microtiter plates [8].…”

Section: Introductionmentioning

confidence: 99%

An Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of Anti-D in Plasma Samples and Immunoglobulin Preparations

Hirvonen¹,

Tervonen²,

Pirkola³

et al. 1995

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.

“…In contrast, figure4 shows that this test is less sensitive than the polybrene test of Lalezari [6] as the latter is capable of detecting an anti-D concentration alloantibodies. Markier et al [10] were able to detect 30 ng/ml of antibodies with the alkaline phosphatase technique and Gilman et al [4] went down to 1 ng/ml using the same method but corrected the high absorbance values by subtracting the hemolysis blank for each test.…”

Section: Comparison With the Indirect Coombs Test And The Polybrene Testmentioning

confidence: 99%

Improvement of Enzyme-Linked Antiglobulin Test by Using an Antiglobulin Linked to Glucose Oxidase: Description of the Technique

Rigal¹,

Monestier²,

Lafont³

et al. 1984

The classic enzyme-linked antiglobulin test (ELAT) used to detect and quantify the amount of IgG antibodies on red blood cells (RBC) is sensitive to hemolysis and erythrocyte enzymatic activities. We describe a new ELAT by using glucose oxidase (GO) linked to antihuman IgG. The optical density base line of GO-ELAT, alkaline phosphatase-ELAT and peroxidase-ELAT were, respectively, 0.180, 0.350 and 0.550. This very low baseline of GO-ELAT was due to the absence of hemolysis (the pH of the GO substrate is 6.5). This technique is ten times more sensitive than the indirect antiglobulin test and detects up to 1 ng/ml of anti-D alloantibodies. Additional advantages of the technique are (1) there is no intrinsic GO enzyme in RBC, and (2) it is not necessary to fix the RBC.