A general guideline for validation of immunoassays for animal, various biological fluids, and/or conditioned media specimen analysis.

 

Validation of an immunoassay for a particular animal specimen should be undertaken prior to analysis of experimental animal samples. It is vital that the validation procedure is completed with the same specimen type as that used or planned in the experiment. The suitability of an immunoassay for a particular animal specimen will depend upon (1) the compatibility between the assay matrix and that of the specimen type; (2) the cross-reactivity or the analyte with the antibody components of the assay; (3) the sensitivity of the assay. In some cases, a kit may perform satisfactorily without modification, while others may require modest or extensive modification of validation with an appropriate calibrator for optimal performance. Ansh Labs evaluates species cross-reactivity by observing linear and parallel dilution. Investigators should use a neat (pure sample) and conduct serial dilutions using the “A” or zero Standard of the kit as the diluent. The results should be graphed with the dilution and standard curve appearing on the same set of axes.

 

Parallelism
As a matter of design, the assay matrix is typically similar to human serum. However, the composition of another species’ serum or plasma may be substantially different from the kit matrix, which may interfere with the detection of the analyte in the animal’s sample. Therefore, the first step in the validation of an assay for use with an animal specimen type is parallelism. Each species’ serum or plasma should be screened for parallelism, with the kit standard curve. A pool from several specimens may be used. The specimen should be diluted with the kit’s matrix (i.e., the “A” or zero Standard supplied in the kit), in a serial manner. The series of three to five dilutions, along with the neat specimen, are assayed with the kit. The dilutions are plotted with the standard curve and tested for parallelism. If the serial dilution runs parallel to the standard curve, the assay matrix is compatible. If there is no parallelism, the specimens may need to be extracted in a suitable solvent and reconstituted in the kit’s matrix.

 

Spike and Recovery
Spike and recovery is another experimental method that is extremely helpful for assessing and validating the accuracy of analytical techniques for species immunoreactivity or different biological fluid sample types. Spike-and-recovery assesses analyte detection and potential differences between the calibration matrix and the biological sample matrix. In Spike and recovery experiments, a known amount of recombinant or native protein is “spiked” into the species sample or intended sample type and run in the assay. The resulting concentration, or “recovery” of the spiked material ([observed]/[expected]*100 = %Recovery), demonstrate if the expected value of the experiment sample type can be measured accurately. %Recovery differing significantly from 100% (± the expected amount) is an indication that some interfering factor in the sample matrix may be causing a falsely high or low value. We can adjust the matrix to optimize an ELISA method for a particular species when the spike-and-recovery test demonstrates a matrix interference.

 

Dynamic Range of the Assay
Hormone levels in animals may differ substantially from hormone levels in humans. The range of calibrators used for an assay may require adjustment. Generally, lower ranges are required for animal samples as compared to human specimens. It is necessary to create a “normal” range for that particular species, which may necessitate the addition of calibrators with lower concentrations for optimal measurement of animal sample levels. The sensitivity of an assay may be improved by altering incubation time and/or temperature, sample volume or altering the dilution of certain reagents (antibody, conjugate, etc.) in the assay.

 

Cross-Reactivity
Finally, if the purified animal antigen is available, another method for immunoreactivity validation is cross-reactivity of the target analyte with the immunoassay’s detection antibody. Steroid hormone structure is conserved across species, but, proteins are complex and can differ substantially in primary, secondary, and tertiary structures between species. The best case scenario is if the particular animal’s hormone shares common amino acid sequences and similar detection and capture antibody epitopes with the human protein and thus cross-react sufficiently. Immunoblot techniques can be used to test cross-reactivity. However, the tests described above for parallelism and spike and recovery studies offer convenient alternatives for validation.