Immunoassay based on antigen-antibody reactions is a highly sensitive and specific method, and is frequently used in various fields as an indispensable tool to detect and quantify various substances. In particular, an enzyme immunoassay is widely used because of its safety and comparable sensitivity to a radioimmunoassay. Nowadays numerous enzyme or fluorescent dye labeled monoclonal and polyclonal antibodies are commercially available for this purpose. The enzyme-labeled primary or secondary antibodies can be prepared by several methods as shown in the table. Enzyme-labeled secondary antibodies have been preferably used in the laboratory due to the following reasons: 1) High level skills are required to prepare the enzyme or fluorescent dye-labeled antibodies, 2) The specificity of the antibody is significantly reduced or is lost during the preparation procedure, 3) Milligram order of antibodies are necessary for the preparation, 4) Various enzyme or fluorescent dye-labeled secondary antibodies are commercially available. In particular, the activity loss of antibody mentioned in reason 2) occurs in the case of preparing the enzyme-labeled monoclonal antibody. Therefore, direct labeling of enzymes to monoclonal antibodies is not a common method.




Table 1 Labeling Methods


The method for direct labeling of enzymes to primary antibodies, however, can simplify the process of the experiment and perform multiple color staining. In addition, it can also solve problems caused by using secondary antibodies. Therefore, simple and reliable direct enzyme labeling method that does not affect the activity of monoclonal antibodies has been requested.

Dojindo Peroxidase Labeling Kit-SH (referred to as SH kit) and Peroxidase Labeling Kit-NH₂ (referred to as NH2 kit) have the following characteristics: 1) The amount of antibody to make a conjugate with peroxidase (POD) is 50 - 200 ug, 2) Gel filtration and dialysis are not necessary (a single filtration tube included in these kits is used for both purification and concentration), 3) Only 3 hours are enough to prepare the conjugate, 4) Since the labeling conditions are comparatively mild, the activity loss of the antibody does not easily occur.

POD-labeled monoclonal antibodies were prepared with the SH kit, NH₂ kit, glutaraldehyde two-step method and modified periodate method, and then the activity of the enzyme-labeled antibodies were compared using ELISA to evaluate each method.

Materials and Methods

Antibody: Monoclonal antibody against blue-tongue virus antigen (8A3B.6, IgG2a) from ascites was purified and used for experiments.

Enzyme-labeled antibody: POD-labeled monoclonal antibodies were prepared by using glutaraldehyde two-step method,article number 5 modified periodate method, article number 7 and Dojindo SH kit and NH₂ kit. Preparation of the conjugate using SH kit and NH2 kit was carried out according to the kit manual.

The comparison of POD-labeled antibody activity: Each POD-labeled antibody solution was adjusted to the same concentration by measuring the absorbance at 280 nm. The prepared POD-labeled antibody was serially diluted to be 10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 0.625 ug/ml, 0.313 ug/ml, and 0.156 ug/ml for ELISA. Inactivated blue-tongue virus antigen was adsorbed on 96-well microplate (MAXISORP, Nunc) by incubating at 37 °C for 60 min, and then blocking procedure was carried out with 20% BlockAce (Dai Nippon Sumitomo Pharmaceuticals) at 37 °C for 60 min. Each diluted POD-labeled antibody solution was added to the plate, and then the plate was incubated at 37 °C for 60 min. The antigen-antibody reaction was monitored by adding POD substrate solution (ABTS, Sigma) and reading the absorbance at 405 nm. The plate was washed at each process.

Results and Discussion

Figure 1 shows activity of POD-labeled antibody prepared by SH kit, NH₂ kit and modified periodate method. No absorbance was measured in the case of POD-labeled antibody prepared by glutaraldehyde two-step method (not shown in the figure). The POD-labeled antibody prepared by SH kit shows high OD value even at a low concentration of the antibody. Since the OD exceeded the maximum detection limit at a protein concentration over 0.625 ug/ml, the OD value over the detection range was indicated as 2.0. It is suggested that immunoreactivity of the antibody and activity of POD were not impaired because maleimide groups of activated-POD in SH kit can be labeled to only hinge region of the antibody and little self-coupling occurred under mild labeling condition.



Figure 1: Comparison of enzyme activity of conjugates prepared by various methods


The OD value of the POD-labeled antibody prepared by SH kit was approximately double of that prepared by NH₂ kit at the same concentration of protein. In the case of SH kit, POD was labeled to the half molecular size of reduced IgG (molecular weight: 75,000) because maleimide groups in reactive-POD react with sulfhydryl groups of IgG. On the other hand, reactive-POD in NH₂ kit was reacted with whole IgG (molecular weight: 150,000). That is to say, the number of introduced POD against IgG prepared by SH kit was nearly double of the one prepared by NH₂ kit. In addition, there is possibility that POD could be labeled around the antigen recognition site (FAB) of IgG, and immunoreactivity of IgG to the antigen may decrease partially. The OD value of POD-labeled antibody prepared by the modified periodate method was extremely low compared to the SH kit and NH₂ kit as shown in the figure. Since POD kept its enzyme activity, it was speculated that the immunoreactivity of the monoclonal antibody to the antigen was greatly reduced during the labeling procedure. In the case of polyclonal antibody, the activity of the enzyme labeled antibody prepared by the glutaraldehyde two-step method or periodate method is mostly sufficient for the following assay. However, it is known that pH change causes a reduction or loss of activity of a monoclonal antibody. The monoclonal antibodies used in this experiment almost lost their activities by the reaction with glutaraldehyde two-step method, and the activity of the antibodies was remarkably lowered by the reaction with modified periodate method. The degree of deactivation of the antibody differs depending on the type of the monoclonal antibody, however, glutaraldehyde two-step method and modified periodate method are not appropriate for making enzyme-labeled monoclonal antibodies. SH as well as NH₂ kits are excellent kits because they do not affect the immunoreactivity of monoclonal antibodies to the antigen, but allow for fast and easy labeling. In addition, the POD labeled antibodies prepared by these kits did not lose any activity at 4 °C for 4 months when the antibodies were stored in the storage buffer included in these kits. Fluorescein-labeled antibody was prepared in an easy and quick manner using Fluorescein Labeling Kit-NH₂ from Dojindo without losing the immunoreactivity. (Other labeling kits are available from Dojindo)


Authors:
Shinya SHIMIZU, Ph.D.
Senior researcher, Research Team for Advanced Biologicals
National Institute of Animal Health, National Agriculture and Food Research Organization
3-1-5 Kannondai Tsukuba
Ibaraki, 305-0856, Japan

Jiro Hirota, M.Sc.
Researcher Associate, Research Team for Advanced Biologicals
National Institute of Animal Health, National Agriculture and Food Research Organization
3-1-5 Kannondai Tsukuba
Ibaraki, 305-0856, Japan

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