Additional Information on Direct ELISA

The fundamental of effective treatment and therapeutics of a patient is the rapid diagnosis of the disease. Rapid diagnosis is done based on the analysis of the clinical symptoms coupled with few laboratory tests. Today ELISA is an indispensible method for medical and research laboratories. Direct ELISA, invented in 1971 by Engvall and Perlmann (1) and by Van Weemen and Schuurs (2), has pioneered other ELISA types. Direct ELISA method is suitable for determining the amount of high molecule-weight antigens. The surface of the plate is coated directly with the antibody or antigen. An enzyme tagged antibody or antigen enables the measurement. Incubation is followed by washing which removes the unbound antigens or antibodies from the medium, followed by substrate addition to produce a signal through coloration. The signal is measured to determine the amount of the antigen or antibody (3,4). This ELISA technique is typically used when the immune response to an antigen needs to be analyzed.  It employs antibody-adsorbed wells, which capture the antigen (analyte) and the bound antigen is then detected directly by a secondary antibody labelled with enzyme. It is suitable for the detection of proteinaceous antigens and may require pre-purification of sample and performed when desired antibody is available in a pre-conjugated state.

Fewer steps involved in this technique makes it a much faster than other ELISA techniques. The assay is also less prone to error since fewer reagents and steps are needed, i.e. no potentially cross-reacting secondary antibody needed. Along with its simplicity it suffers from higher background noise in comparison to indirect ELISA as the antigen immobilization is not specific. This is primarily because all proteins in the sample, including the target protein binds to the plate. Flexibility of direct ELISA is also affected by requirement of a specific conjugated primary antibody for each target protein. Also with no secondary antibody assay sensitivity reduces due to no signal amplification. Direct ELISA technique is typically used when the immune response to an antigen needs to be analyzed.

            Direct ELISA has been utilized by several research groups to identify biomolecules. A rapid, sensitive, and reliable serological diagnosis method was developed for Mycoplasma bovis utilizing Direct ELISA. Mycoplasma bovis has been reported to cause clinical and subclinical mastitis which spreads quickly within a dairy herd. P48 protein present on the membrane of M. agalactiae is an immunodominant invariable lipoprotein. An Antibody response against M. bovis recombinant P48 protein was detected in experimentally as well as naturally infected animals, suggesting a stable expression of the corresponding genes. P48 protein is a useful marker for M. bovis infection and an alternative candidate for the development of specific serological test for M. bovis. A Direct competitive ELISA (Dc-ELISA) to detect M. bovis specific antibody in serum based on a P48 protein mAb 10E was developed by Wu et al. (5). This facilitated M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology. The Dc-ELISA showed a higher positive rate than commercially available i-ELISA kits. It was observed that a Dc-ELISA with micro plates coated with purified recombinant P48 protein was more specific than those coated with whole-cell lysates of M. bovis (5). They tested a total of 165 clinical bovine serum samples and Dc-ELISA (93/165) detected more samples than the two commercial i-ELISA kits (79/165) or (65/165) respectively.  M bovis mastitis has been eliminated from a number of dairy herds by using this approach. Identifying infected cows earlier and separating them from the rest of the herd helped to minimise the further spread of M bovis mastitis and reduced the time required to eliminate it from an infected dairy herd.

            Free 25-hydroxyvitamin D (25(OH)D) is currently being investigated by the research community for its potential value as a better marker of vitamin D status than total 25(OH)D. 25(OH)D is tightly bound in serum to VDBP and moderately to albumin. The bound form is in equilibrium with the free form. However, the equilibrium is largely shifted toward the protein-bound fraction. This happens due to the high-affinity binding of 25(OH)D to VBDP. The free 25(OH)D represents only about 0.04% of the total 25(OH)D concentration, depending on the VDBP levels. Different groups preferred for a direct measurement method and the development of an ELISA played an important role. The free 25(OH)D ELISA has been used as a valuable tool to establish the clinical relevance of free 25(OH)D. Also concentration and genotype of vitamin D binding protein (VDBP) are important factors that determine the bioavailability of 25-hydroxyvitamin D [25(OH)D] in blood. The ELISA is based on monoclonal anti-25(OH)D antibodies and uses a specific incubation buffer that enables the capture of the free fraction of 25(OH)D only. Free 25(OH)D requires the measurement of three or four parameters which includes assay of VDBP and albumin and total 25(OH)D. The genotyping of VDBP is also necessary in order to select the correct binding coefficient to be used in the equation. This methodology has been extensively used over the past 10 years. However, the accuracy of some of the VDBP assays has been questioned as recent studies have shown contradictory findings regarding the VDBP binding coefficient. VDBP exists in different forms, and the assays that are based on monoclonal antibodies are too specific toward certain isoforms and tend to underestimate the binding protein concentration in some of the samples. This led to a call for a direct measurement of free 25(OH)D by several research groups. Recently an ELISA was developed for the direct measurement of free 25(OH)D. A simple and direct ELISA was developed, based on a two-step immunoassay procedure performed in a microtiter plate by Nicolas in 2017  (6).

Direct ELISA is a fast method, but has low sensitivity. It is effective only when there is a high amount of antigens in the sample.


  1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871–4.
  2. Van Weemen BK, Schuurs AH. Immunoassay using antigen-enzyme conjugates. FEBS Lett 1971;15:232– 6
  3. Engvall E. The ELISA, enzyme-linked immunosorbent assay Clin Chem 2010;56:319–20.
  4. Hornbeck P. Enzyme-linked immunosorbent assays. Curr Protoc Immunol 2001; Chapter 2: Unit 2.1. doi: 10.1002/0471142735.im0201s01.
  5. Fu PSun ZZhang YYu ZZhang HSu DJiang FWu W1. Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein. BMC Vet Res.2014 Feb 18;10:42. doi: 10.1186/1746-6148-10-42.
  6. Heureux NLindhout ESwinkels L. A Direct Assay for Measuring Free 25-Hydroxyvitamin D. J AOAC Int.2017 Sep 1;100(5):1318-1322. doi: 10.5740/jaoacint.17-0084. Epub 2017 May 11.