Subfemtomole Enzyme Immunoassay for Human Growth Hormone Using Affinity Chromatography and Enzyme Amplified Detection R. Lejeune,* L. Thunus,*
F. Gomez,? F. Frankenne,?
J.-L. Cloux,$ and G. Hennent
*Service de Chimie analytique, Universite’ de Lb&e, Institut de Pharmacie, rue Fusch, 3, B-4000 LiGge, Belgium; TService d%ndocrinologie expbrimentale et clinique, Universite’ de Litige, Centre hospitalier universitaire B 23, Sart-Tilman, B-4000 Liege, Belgium; and SBiocode, Passage Lemmonier, 34, B-4000 LiGge, Belgium
An immunometric assay is described which allows fast detection of attomole amounts of an antigen. The sensitivity is 100 to 1000 times better than that of classical sandwich immunometric assays. Our system allowed the measurement of human growth hormone in the range of 0.1 amol to 100 fmol in a 4-h time period overall. A chromatography column is sequentially filled with two immunoaffinity resins: SP-MI--E,-Ab, in the upper half and SP-M,--E,-Ab, in the lower half, where Ab, and Ab, represent complementary antibodies reacting with the antigen to be assayed, E, and E, represent enzymes, MI and M, represent substances reacting reversibly with E, and E,, respectively, and SP represents the chromatographic solid phase; the sign - represents covalent linkages and the sign -- reversible linkages. The sample solution is passed through the column, resulting in binding of the antigen to the first encountered antibody, yielding the immobilized complex SPMI--E,-Ab,--Ag. The M, bound is then destabilized by washing with solution of agonist to M,. The freed complex is immediately trapped by the second antibody in the lower part of the column, resulting in the entity SPM,--E,-Abz--Ag--Ab,-E, . After a washing step, an amplified detection allows the measurement of the antigen through the activity of the enzyme E, . The antigen-antibody reactions occur in the presence of a very large excess of antibody. The continuous equilibrium displacement due to the chromatographic procedure enhances the yield of complex formation. These factors explain the extremely low levels (subattomole) capable of being detected with this original technique. o 1990 Academic
The sandwich assay, remarkable for its high sensitivity and good precision (l), necessitates prolonged incu0003-2697190 $3.00 Copyright 0 1990 by Academic Press, All right,s of reproduction in any form
bation and critical manipulations. Especially when assays are carried out in antibody-coated plastic tubes, slow protein leakage as well as irreproducible adsorption behavior is often encountered (2,3). An advantageous alternative is to perform immunological reactions on the solid phase of an affinity chromatography column (4), which results in significantly reduced incubation times and improved reproducibility. However, the advantage of the chromatographic process appears only at the immunoassay first step. Consequently, several additions of the labeled antibody are needed for reproducible binding (5). Competitive immunoassays have recently been improved by using a reversible capture of the specific antibody onto an electrode membrane which is used as a detector (6). This procedure allows the use of only one detector for assaying different antigens but does not improve the sensitivity and the speed of the assay. The method proposed here uses affinity chromatography and reversible capture of enzyme antibody conjugates in an original way to obtain a significant increase in noncompetitive immunoassay efficiency. The originality of the method is to preserve the efficiency of affinity chromatography during both steps of immunoassays. Affinity chromatography and reversible capture of antibodies being the main characteristic of the new method, we have tentatively named our method reversible antibodies capture immunoassay (RACIA).’ The measurement of human growth hormone concentration has been used as a model to check the performances of the RACIA in terms of speed, reproducibility, and sensitivity. ’ Abbreviations noassay.
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order to get the highest sensitivity possible. During the elution step, the affinity column goes back to a native state and is again ready to receive antibody-enzyme conjugates directed at the same antigen or another antigen provided that specific antibodies are conjugated to identical enzymes.