Whole-blood agglutination assay for on-site detection of human immunodeficiency virus infection

Abstract

Simple and rapid diagnostic tests are needed to curtail human immunodeficiency virus (HIV) infection, especially in the developing and underdeveloped nations of the world. The visible-agglutination assay for the detection of HIV with the naked eye (NEVA HIV, which represents naked eye visible-agglutination assay for HIV) is a hemagglutination-based test for the detection of antibodies to HIV in whole blood. The NEVA HIV reagent is a cocktail of highly stable recombinant bifunctional antibody fusion proteins with HIV antigens which can be produced in large quantities with a high degree of purity. The test procedure involves mixing of one drop of the NEVA HIV reagent with one drop of blood sample on a glass slide. The presence of anti-HIV antibodies in the blood sample leads to clumping of erythrocytes (agglutination) that can be seen with the naked eye. Evaluation with commercially available panels of sera and clinical samples has shown that the performance of NEVA HIV is comparable to those of U.S. and European Food and Drug Administration-approved rapid as well as enzyme-linked immunosorbent assay kits. The test detects antibodies to both HIV type 1 (HIV-1) and HIV-2 in a single spot and gives results in less than 5 min. The test was developed by keeping in mind the practical constraints of testing in less developed countries and thus is completely instrument-free, requiring no infrastructure or even electricity. Because the test is extremely rapid, requires no sample preparation, and is simple enough to be performed by a semiskilled technician in any remote area, NEVA HIV is a test for the hard-to-reach populations of the world.

FIG. 1.

Diagrammatic representation of various fusion proteins. Recombinant Fd of an MAb or its fusion protein makes an interchain disulfide bond with the corresponding light chain. These fusion proteins are obtained by assembling the light chain with Fd-Fd fusions containing HIV-derived peptide and by using a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation under proper redox conditions to assemble the functional Fab. The monomeric Fab fusion proteins containing HIV-derived peptides are purified to a high degree and free of aggregates by multistep column chromatographic procedures. The intrachain disulfide bonds of the light chain and Fd are not shown. Fd, antibody fragment containing the variable domain and the first constant domain of the heavy chain; LC, light chain of antibody; env13, 31 amino acids (590 to 620) of HIV-1 envelope gp41 (AVERYLKDQQLLGIWGCSGKLICTTAVPWNA); env24, 31 amino acids (581 to 611) of HIV-2 envelope gp36 (AIEKYLQDQARLNSWGCAFRQVCHTTVPWVN); c-myc, decapeptide recognized by MAb 9E10; p246, amino acids 8 to 153 of HIV-1 capsid protein p24; C, cysteine. The yield corresponds to the amount of Fab obtained from 8 liters of renatured material containing 800 mg of total protein.

FIG. 2.

Evaluation of various mixtures of Fab fusion proteins. Mix A contains B6Fabenv13 and B6Fabenv24 each at 10 μg/ml; mix B contains B6Fabenv13, B6Fabenv24, 10F7 Fabenv13 and 10F7Fabenv24 each at 10 μg/ml; and mix C contains A41Fabp246 (15 μg/ml) in addition to the components of mix B. All three mixtures were tested with the samples of panel PRB202 by a slide assay. Twenty microliters of a 1:1 mix of a serum sample from panel PRB202 and RBCs from an individual with type O RhD-negative blood was added to 20 μl of each reagent on a glass slide, and the agglutination reaction was read visually on an arbitrary scale of 0 to 4, with 0 indicating no visible agglutination and 4 indicating strong agglutination with large clumps.