Points to remember before opting Sandwich ELISA
It is also known by the name of capture ELISA. The process follows coating of an antibody on the microtiter well followed by addition of sample containing antigen forming antigen-antibody complex. Then a second enzyme-linked antibody specific for a different epitope on the antigen is added which is subsequently developed to form a colorimetric reaction.
Few important consideration are there before choosing this technique for particular experiment. The capture antibody, which is used for coating microtiter plate and detection antibody, which is second antibody binding to the protein of interest, must recognize two different non-overlapping epitopes of protein of interest. Also, the binding of the capture antibody to the epitope must not alter the structure of epitope recognized by the detection antibody. Matched pairs of antibodies that do not interfere with one another and can bind simultaneously are used as capture and detection antibodies. Antibody suppliers must provide information about epitopes and indicate pairs of antibodies that have been validated in ELISA as matched pairs.
Cost is also an important consideration which depends on the requirement of the experiment. To get specificity, Monoclonal antibody can be utilized as both capture and detection antibody. However it must be weighed against the cost and time required for producing two monoclonal antibodies. In comparison to monoclonal antibody, polyclonal is less expensive (~5 fold) to produce. Also sometimes people use the same antibody for the capture and detection, which can limit the dynamic range and sensitivity.
During assay reference standards, positive and negative controls, are always put up along with the test samples in the same test run. Using ODs of reference standards (controls) a cut off value (COV) is then determined for each test run. Commonly COV is calculated by taking average absorbance values (ODs) of 3 known negative controls from within the test run plus a constant factor, which is usually 3 or 4 standard deviations of the mean absorbance values from a large number of known negative samples (usually > 100). In an alternate method, the COV is calculated from a large number (statistically significant) of known negative samples by determining the highest absorbance obtained within the distribution curve. It is necessary to re-calculate the constant factor whenever a new batch of reagents are used. Ideally, the COV is about twice the OD given by the negative reference standard. Values of the test samples above the COV are read as positives and those below are considered negative. Each test run is put through validation criteria (pertaining to the expected ODs of the reference controls). Any test run failing to meet the validation criteria is null and void and must be repeated.
This ELISA format is a good option to achieve sensitivity and specificity together. Sensitivity is defined as the ability of a test system to detect the minimal possible quantity of the analyte in a test sample. Whereas, specificity connotes the ability of a test to detect none other than the analyte of interest. Sandwitch ELISA has the sensitivity in the range of 98-100 per cent, and specificity of 95-98 per cent.
This format of ELISA is useful in detecting the antibody presence along with identifying the class of the antibody. For example, in IgM class capture and detection, the wells gets coated with anti-μ chain (M antibody capture ELISA or MACELISA) and for IgG class capture, anti-gamma chain is coated (GACELISA). This ELISA-based technique of a pair of antibodies sandwiches the target compound and specifically quantifies it among other compounds. In one of study, authors utilized this ELISA format for quantifying cellobiohydrolase I (CBH I), in an effort to degrade crystalline cellulose to glucose. This is important in the line of search for methods to employ biomass as renewable source of energy. In this study, authors quantified CBH I in the crude preparations of the cellulase complex from Trichoderma reesei crude culture broth with minimal interference from other enzymes or other materials present in the broth . They reported that an improved specificity of the assay was achieved using MAb as the coating antibody and PAb as the second, detecting antibody.
Similarly, in another study Buhler et al. optimized a double-antibody sandwich ELISA for endoglucanase I (EG-I) in a culture broth of Trichoderma reesei using MAb as the coating antibody and PAb as the detecting antibody. They found the test to be specific for EG-I and no interfrence from endoglucanase II nor cellobiohydrolase I or II, which were also present in broth. As little as 20 pg of EG-I protein was reported to be detected. They were able to show that the assay was both sensitive and specific .
For Tuberculosis detection the conventional smear examination for the bacilli is the main test. The available studies, however, give an indication for ELISAs potential as an adjunct to conventional means of diagnosis. ELISA has played a role in the antigen detection in sputum, bronchial lavage, serum and other body fluids. Further advances in ELISA formats may prove beneficial in areas where specimen collection is difficult, e.g. childhood tuberculosis, or where bacterial identification difficult such as in inaccessible walled-off lesions and in tuberculosis of CNS, bone, gastrointestinal tract and genitourinary tract. Few studies have tried to detect antigen in CSF using antibody enzyme conjugates against M. tuberculosis H37RV and M. bovis BCG strains. Both gave specificity of 100 per cent but positivity was 79.7 per cent and 67.5 per cent respectively. Another study estimated mycobacterial Ag A60 specific immunoglobulins IgM, IgA and IgG in the sera . Their conclusion was that a to detect cases of adult tuberculosis combined IgA and IgG antibody titres gives high sensitivity (91.6%) and specificity (90.0%). Also it has been found that IgM estimation can be utilized to detect cases of reactivation of tuberculosis.
- Riske FJ, Eveleigh DE, Macmillan JD. Double-antibody sandwich enzyme-linked-immunosorbent-assay for cellobiohydrolase-i. Appl Environ Microbiol. 1990;56(11):3261–3265.
- BUHLER R. Double-antibody sandwich enzyme-linked-immunosorbent-assay for quantitation of endoglucanase-i of trichoderma-reesei. Appl Environ Microbiol. 1991;57(11):3317–3321.
- Desai T, Gogate A, Deodhar L, Todddywalla S, Kelkar M. Ind. J Pathol Microbiol. 1993;36:348–355.
- Gupta S, Kumari S, Banwalikar JN, Gupta SK. Diagnostic utility of the estimation of mycobacterial antigen A60 specific immunoglobulins IgM, IgA and IgG in the sera of adult human tuberculosis cases. Tuber Lung Dis. 1995;76:418–424.