American Association for Clinical Chemistry
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NACB - Scientific Shorts
NACB - Scientific Shorts (formerly NACB Blog)
By Frederick G. Strathmann, PhD, DABCC
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​The use of mass spectrometry for clinical diagnostics has grown considerably due in large part to the rapid advancements with liquid chromatography-tandem mass spectrometry (LC-MS/MS). More in-depth discussions of clinical mass spectrometry principles and its uses in the clinical laboratory can be found elsewhere.(1,2) Instead, the reemergence of using antibodies for sample extraction (referred to as immunoextraction or immuno-enrichment) will be discussed.
Current sample preparation methods range from the relatively simple to significantly more complex. Regardless of what method is used, the end goal is to remove as much of the unwanted components present in the original sample while preserving the analyte or analytes of interest. The complexity of the matrix and the properties of the antibodies themselves can influence greatly the degree of sample preparation needed prior to immunoextraction. For more complex matrices such as blood or serum/plasma, protein precipitation is commonly used as it helps to disrupt protein-analyte interactions, provides an initial though crude first separation, and affords an opportunity for sample concentration if needed.
Antibodies can be immobilized to a solid phase such as a chromatographic column or polystyrene beads. The sample is incubated with the immobilized antibody, unwanted components are washed away, and the enriched analyte is eluted. Most often, the sample is concentrated by drying, followed by final resuspension and preparation for injection into the liquid chromatography system.
The combination of antibodies and a mass spectrometer is not a new concept. In the mid 1980s, publications using antibodies to extract analytes of interest from complex matrices such as serum and plasma for analysis by mass spectrometry began to emerge. In the clinical diagnostics field, Henion and his colleagues published numerous articles throughout the 1990s and early 2000s using immunoextraction methods to dramatically lower detection levels for various classes of compounds. Recently, laboratories at the Cleveland Clinic and the University of Washington have published methods using immunoextraction for the analysis of 1,25 dihydroxy vitamin D by mass spectrometry for use in the clinical laboratory. In clinical protein analysis, antibody enrichment methods, such as SISCAPA®, are arguably becoming the gold-standard method for peptide and protein quantitation.
The major benefit of using immunoextraction is the drastic reduction in the complexity of the final sample. Immunoassays have adequate sensitivity but often lack specificity, and using LC-MS/MS as the final detection method couples the sensitivity of an immunoassay with the specificity of an LC-MS/MS method. In addition, analytes that appear identical to a mass spectrometer and prove challenging to separate chromatographically may be resolvable based on differing degrees of cross-reactivity with the antibody.
Despite the initial sticker shock of a clinical mass spectrometer, consumable costs are typically less when compared to platform specific immunoassays. In conjunction with better overall patient care, cost savings over the long term is a major driving force behind the successful expansion of mass spectrometry in the clinical laboratory. Adding an antibody to an LC-MS/MS method not only adds complexity to the sample preparation but can add a substantial cost per test. Standardization of results remains a challenge as well, with each unique source of antibody having independent and often considerably different performance characteristics.
Interest is once again growing regarding the use of immunoextraction as a sample preparation method for LC-MS/MS assays in the clinical laboratory. With relative ease, new analytes can be measured using substantially the same analysis with substantially similar methods and accomplishing similar results as found in previous publications. Even a mass spectrometry purist has to admit that an antibody can look awfully nice on the seat of a bicycle built for two, even if a fish is steering.

 

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Posted by Andrew Hoofnagle
On 3/28/2012

I think that is supposed to refer to the age-old adage that a mass spectrometer needs an antibody like a fish needs a bicycle. Thanks for an interesting read Fred.

Posted by
On 3/12/2012

Purification of anlalytes using high affinity antibodies is an age old and well accepted method in biochemical analysis. By obtaining highly purified form of analyte through immunoextraction it may be easier to choose the ionic fragment that is unique to the analyte, while standardizing the methods for LC/MS, rather than looking for the unique ionic fragment in the sea of fragments obtained when complex matrix systems are used. The LC/MS scientist need not spend too much of his/her energy to find out the correct combination of settings to get that unique ionic fragment

Posted by
On 3/12/2012

Using affinity column isn't much different from solid phase or liquid phase extraction and they are all up-stream sample preparation methods to clean up the sample or concentration the compounds of interest before you inject them to the mass spec. I'd like to add another use of affinity column which is to remove the proteins that you don't want which are nonetheless abundant, such as Albumin or globulin. This technique has been widely used in proteomics related research world, although it's still kind of new for clinical mass spec. Dr. Strathmann provided a great discussion on this topic to ease the fear and/ or embarrassment. I'll say just think it as another way to clean up your sample. Yan Zhang, Ph.D.

Posted by
On 3/8/2012

It is usually productive to preconcentrate an analyte and simply a sample before introduction to any instrument. There is more liklihood that the use of antibodies could even replace the LC in LC/MSMS, especially as ambient ionization methods become better understood and high mass resolutions add a lot of qualitative confidence in avoiding interferences.

Posted by
On 3/6/2012

You could also measure the mass/charge of an antibody-antigen complex rather than just use it as an improved cleanup/extraction/selection step. Still great point - enjoyed article...but .... the fish analogy? Is that based on that old image of a fish out of water for LC MS analysis? Not sure non-MS types would get it. Not even sure I get it :)

About the Author
Frederick G. Strathmann, PhD, DABCC
Frederick G. Strathmann, PhD, DABCC 
 

​References:

1. Chace D, Mass spectrometry in the clinical laboratory. Chemical reviews, 2001. 101(2): p. 445-772.

2. Strathmann FG and Hoofnagle AN, Current and future applications of mass spectrometry to the clinical laboratory. Am J Clin Pathol, 2011. 136(4): p. 609-16.

Conference
September 6-7, 2012
Chicago, IL