Why are immunochemical methods important?
A large proportion of major crops currently grown in the US have been enhanced with one or more traits through the use of modern biotechnology methods. These crops are the result of the incorporation of specific agronomic, crop protection or quality traits into plants grown for agricultural production. These modified or transgenic plants differ from conventional plants in that they contain additional genes which produce a corresponding number of new proteins, the presence of which give the modified plants the desired beneficial traits. A gene for a specific trait is introduced by insertion of the gene DNA fragment into the plant genome via recombinant DNA methods. The introduced DNA fragment produces a protein which is expressed in the plant tissues. A particular introduced protein may produce a desired agronomic trait in the plant, or it may confer insect, disease or herbicide resistance to the plant, thus improving crop production. An introduced protein may also produce a quality trait such as enhanced flavor or nutritional value of the plant. With the introduction of transgenic plants, analytical technology is rapidly evolving to measure the introduced proteins. Among the most useful techniques for ag biotech applications are immunoassays. Immunoassays offer simple, specific and sensitive protein detection to address a wide range of needs of technology providers, food and commodity distribution channels, and regulatory bodies.
What are some of the relevant terms used in discussion of immunoassays and immunochemical methods?
Accuracy – The closeness of individual predicted concentrations to the true concentration in a sample. Note: this definition combines the idea of bias (systematic error) and precision (random error).
Assay – Qualitative or quantitative analysis of a substance.
I. Recovery bias (absolute) – Bias with respect to the actual concentration of analyte – the degree to which the predicted concentration of an analyte differs, on the average, from the actual (or true) concentration.
II. Method bias (relative) – Bias with respect to another method – the degree to which the predicted concentration in a sample differs, on the average, from the predicted concentration determined by another method.
False Positive – The percent test positively in a population of true negatives.
False Negative – The percent test negativity in a population of true positives.
Interferences – Effects on the analytical performance of an immunoassay caused by substances other than the analytes(s) of interest.
Kit – A test kit is a packaged system of the key or principal components to detect or measure a specific analyte(s) in a given matrix(ces) within a laboratory or non-laboratory environment. The key components include antibodies, enzyme conjugates, etc. that may only be readily prepared by the purveyor of the kit. Test kits include directions for use and are often self-contained, complete, analytical systems, but they also may require supporting supplies and equipment.
Method – The entire system of procedures that describe how a final reassure of analyte concentration is obtained for a target matrix.
Precision – The extent to which replicate analyses of a sample agree with each other. Usually expressed as the standard deviation or percent coefficient of variation (%CV) of a population of values: %CV = (Standard deviation / Mean) X 100.
Reproducibility – The ability of the analytical method to yield the same result within analyses, between analyses and between operators.
I. Detection Sensitivity (Limit of Detection) – The smallest concentration of analyte that can be statistically significantly distinguished from zero for a given sample matrix with a stated degree of confidence.
II. Limit of Quantitation – The smallest concentration of analyte that can be measured in samples and yield a predicted concentration with stated relative precision or accuracy (or both).
1. Kit Stability (Shelf Life) – The length of time that a complete kit can be stored under given conditions and still yield results that are within the stated performance parameters.
2. Sample Stability – the length of time that a target analyte level remains stable in a specific matrix under a given set of storage conditions.
Why is polymerase chain reaction (PCR) an important analytical method in agriculture?
The agricultural industry applies PCR at numerous points in the development process of a genetically-modified (GM) crop product such as gene discovery, cloning, vector construction, transformant identification, screening of plants, characterization and seed quality control. Commodity crop companies, food companies and third-party diagnostic testing companies also rely on PCR for a number of purposes such as verifying the presence or absence of GM material in a product, quantify the amount of GM material in a product, and to certify compliance contracts between buyer and seller of commodity grain.
What is the PCR process?
The PCR process mimics in vitro the natural process of DNA replication occurring in all cellular organisms in which the DNA molecules of a cell are duplicated prior to cell division. DNA reproduction during PCR does not cover the entire sequence of the original DNA molecules but is restricted and targets a specific, relatively short region of the template DNA molecule. Short, single-stranded, synthetic DNA molecules call primers give the specificity of the reaction. Primers are designed to be complementary to their intended binding site. A single cycle of the PCR reaction and the corresponding temperature profile are divided into three phases: denaturation, annealing and elongation. At the end of these phases, the targeted DNA region has been replicated into two copies of the original double-stranded DNA molecule. This process of selective duplication is repeated multiple times in a cyclic reaction. DNA replication is catalyzed by heat-stable DNA polymerase enzymes. The kinetics of the DNA reproduction resemble an exponential amplification in which the replicas of distinct length (amplicons) accumulate quickly and outnumber the original template molecules.
What are some good references for use in learning more about immunochemical methods and PCR methods?
1. Immunoassay as an Analytical Tool in Agricultural Biotechnology. Grothaus, G.D., Bandla, M., Currier, T., Giroux, R., Jenkins, G.R., Lipp, M., Shan, G., Stave, J.W., and Pantella, V., Journal of AOAC International, Vol. 89, No. 4, 2006, pp. 913-928.
2. Polymerase Chain Reaction Technology as an Analytical Tool in Agricultural Biotechnology. Lipp, M., Shillito, R., Giroux, R., Spiegelhalter, F., Charlton, S., Pinero, D., Song, P., Journal of AOAC International, Vol. 88, No. 1, 2005, pp. 136-155.
3. New Frontiers in Agrochemical Immunoassay, Kurtz, D.A., Skerritt, J.H., Stanker, L. (eds), AOAC International, Arlington, VA, 1995
4. Immunoanalysis of Agrochemical: Emerging Technologies, Nelson, J.O., Karu A.E. and Wong R. (eds.), ACS Symposium Series No. 586, American Chemical Society, 1995
5. Immunoassay for Trace Chemical Analysis: Monitoring Toxic Chemical in Humans, Food and the Environment, Vanderlaan, M.V. Stanker, L.H., Watkins B.E. and Roberts D.W. (eds.), ACS Symposium Series No. 451, 1991
6. Mihaliak, C.A. and Berberich S. A. “Guidelines to the Validation and Use of Immunochemical Methods for Generating Data in support of Pesticide Registration”, in: Nelson, J.O., Karu A.E. and Wong R.B. (eds.), Immunoanalysis of Agrochemical: Emerging Technologies, ACS Symposium Series No. 586, American Chemical Society, 1995, pp. 288 – 300.
7. Rittenburg J. and Dautlick J. “Quality Standards for Immunoassay Kits” in: Nelson, J.O., Karu A.E. and Wong R.B(eds.), Immunoanalysis of Agrochemical: Emerging Technologies, ACS Symposium Series No. 586, American Chemical Society, 1995, pp. 301-307.
8. IUPAC Pesticides Report #33. Immunoassays for Residue Analysis ofAgrochemicals: Proposed guidelines for precision, standardization, and quality control. Pure & Appl. Chem., Vol. 67, No. 12, pp. 2065-2088, 1995.
9. Lipton, C.R., Dautlick, J.X., Grothaus, G.D., Hunst, P.L., Magin, K.M., Mihaliak, C.A., Rubio, F.M. and Stave, J.W. 2000. Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients. Food and Agricultural Immunology 12: 153-164.