Additional Information on Indirect ELISA

The technique was developed in 1978 Lindström and Wager (1), after getting inspired by the direct ELISA method. The researchers reported measuring porcine IgG using this method. The name indirect implies that what determines and separates the antigen to be measured is not the primary antibody, but another antibody. In this method, the diseased serum is added to the antigen-coated wells and the plates are incubated. During this incubation, the antibodies formed against the antigens in the diseased serum plaque produce an antigen-antibody complex. To observe the complex a secondary antibody tagged with the enzyme is added that recognizes the antibody in the serum. Subsequently substrate is added to the medium to produce color and the concentration is determined. This method is used more commonly in endocrinology. It is also used to quantify the amount of antibody or antigen with high sensitivity.

The indirect ELISA is suitable for determining total antibody concentration in samples. It is similar to direct ELISA assays only difference being it a two-step process. It was used to detect antibodies to Mycoplasma bovis in milk samples collected from a herd with M bovis mastitis (2). Mycoplasma bovis causes clinical and subclinical mastitis within a dairy herd, which can spread quickly (3). By the rapid identification of infected animals and their segregation it can be minimised. M bovis-specific immunoglobulins in the milk of infected cows within the first nine weeks of outbreak indicated that the indirect ELISA may be useful for the detection of cows which have recently got infected. M bovis mastitis has been eliminated from a number of dairy herds by using this approach, followed by the culling of all persistently infected cows. Antibodies were detected in samples from nine cows which had developed clinical M bovis mastitis. Milk from only three consistently antigen-negative cows tested positive for M bovis antibodies. These results indicated the potential value of the indirect ELISA for the detection M bovis mastitis during the early stages of an outbreak.

Indirect ELISA is a simple and rapid technique for detecting and quantitating antibodies or antigens attached to a solid surface. Being one of the most sensitive immunoassays, it offers commercial value in laboratory research, diagnostic of disease biomarkers, and quality control in various industries. Two types of indirect ELISA are performed e-ELISA and k-ELISA. More frequently it is run as endpoint ELISAs (e-ELISAs) however, it suffers from some disadvantages. Firstly, as the chemical reaction can continue without functional enzymes the addition of a stop solution does not necessarily arrest color change. Secondly, at high and low antibody concentration the relationship between endpoint color intensity and antibody concentration need not be linear. Lastly, the chemical reaction is only approximately linear with the enzymatic concentration in the well during a brief period at the initial phase of the reaction and provided there is an abundant amount of substrate. Therefore, an e-ELISA is incapable of distinguishing between a moderate and large increase in antibody concentration when it lies in the upper regions of the linear scale of the OD, unless the sample undergoes predetermined dilutions.

A study was performed to determine whether it was possible to run both k-ELISA and e-ELISA on the same plate as k-ELISAs have certain advantages over e-ELISAs (4). Also, an appropriate time interval for k-ELISA measurements was established and a normalization method for k-ELISA slopes (slope ratio) was proposed. They used an indirect e-ELISA test for measuring antibodies against Ostertagia ostertagi in milk from dairy cattle. They found that running a k-ELISA had no effect on optical density ratio results of an e-ELISA on the same plate. An e-ELISA can become a k-ELISA if the OD is recorded at regular short intervals (e.g., 45 s) as soon as the chromatogenic reaction starts and it does not require a stop solution. The measurements from a k-ELISA are taken in real-time, allowing the necessary information to be gathered much sooner than an e-ELISA. Theoretically, k-ELISA results can quantify the initial approximate linear enzymatic reaction and thus be a truly quantitative test. The strong agreement of k-ELISA results at various time points with e-ELISA demonstrated the validity of using k-ELISA and thus reduces the total processing time and offers better agreement with less variability in its results.

One variation of indirect ELISA is indirect competitive ELISA (indirect cELISA). In this technique, antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. The wells are washed to remove unbound antigen-antibody complexes and free antigen. A reporter-labeled secondary antibody is then added followed by the addition of substrate. Substrate hydrolysis yields a signal that is inversely proportional to antigen concentration within the sample. The higher the antigen concentration in the test sample, the weaker the resultant signal in the detection step. This happens because when antigen concentration is high in the test sample, most of the antibody is bound before adding the solution to the plate. As most of the antibody remains in solution as complexes they get washed away before the addition of the reporter-labeled secondary antibody and substrate. Thus, The indirect cELISA is often used for competitive detection and quantification of antibodies against viral diseases in biological samples. One study proposed use of indirect ELISA to rule out the presence of economically devastating foreign animal diseases. It is based on the detection of antibodies to Senecavirus A (SVA) which will help to differentially diagnose Idiopathic Vesicular Disease (IVD) due to SVA (5).


  1. Lindström P, Wager O. IgG autoantibody to human serum albumin studied by the ELISA-technique. Scand J Immunol 1978;7:419–25.
  2. Application of an indirect ELISA to milk samples to identify cows with Mycoplasma bovis mastitis. Byrne WJ, Ball HJ, Brice N, McCormack R, Baker SE, Ayling RD, Nicholas RA Vet Rec. 2000 Mar 25; 146(13):368-9.
  3. JASPER, D. E. (1981) Bovine mycoplasmal mastitis. Advances in Veterinary Science and Comparative Medicine 25, 121-159.
  4. Vanderstichel RDohoo IMarkham F. Applying a kinetic method to an indirect ELISA measuring Ostertagia ostertagi antibodies in milk. Can J Vet Res. 2015 Jul;79(3):180-3.
  5. Cheryl M. T. Dvorak, Zeynep Akkutay-Yoldar, Suzanne R. Stone, Steven J.P. Tousignant, Fabio A. Vannucci, and Michael P. Murtaugh. An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine. BMC Vet Res. 2016; 13: 50.