4.39: Immunochemistry
- Page ID
- 123343
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)
A laboratory is interested in measuring albumin in urine at concentrations > 10 mg/L (\(\mu\)g/mL). One approach being considered is to use an immunochemical assay.
QUESTIONS
- Which immunoassay would be most suitable for the desired assay?
- The laboratory currently has no analyzer to which reagent for urine albumin can be adapted. After reviewing the specifications of the available assays, the laboratory decides to concentrate the urine one-hundredfold using an Amicon B-15 concentrating apparatus. Which of the immunoassays is now most suitable for urinary albumin measurements?
Questions to Consider
- Which immunoassays are sensitive enough to measure as little as 10 mg/L of antigen?
- Which of these assays are able to quantitate urine albumin?
- Which of these assay are technically complex or require dedicated, expensive instrumentation?
- Which additional immunoassays are sensitive enough to quantitatively measure antigens present at a level of 1 mg/mL?
- Answer
-
- There is not one best assay for this situation. The available equipment and experience of the laboratory must be considered, and can often be limiting factors. The most critical factor may have to do with WHICH instruments are currently available in the laboratory.
- Since the RID assay is the easier assay to set up and requires no expensive equipment, this is the most suitable technique for the desired assay. In addition, RID plates for measuring albumin are commercially available.
Answers to Questions to Consider
- As shown in Table 12-1 (p. 242-243), assays sensitive enough to measure 10 mg/L include counterimmunoelectrophoresis, immunonephelometry, complement fixation, fluoroimmunoassay, ELISA, and competitive ELISA.
- Counterimmunoelectrophoresis and complement fixation are semi-quantitative techniques (Table 12-1), and therefore are not useful for the desired assay. Imunonephelometry, turbidometry, immunometric assays using a wide variety of labels (such as fluoresence, chemiluminescence, enzymes), ELISA, and competitive ELISA are all quantitative assay methods. See also pp 239-240).
- Automated immunometric or immunonephelometric analyzers are costly, dedicated instruments. The immunometric analyzers usually have fixed menus and may or may not have the ability to measure a new analyte. The nephelometric analyzers are more felixible with respect to adding a new test. Tubidimetric analysis for proteins in the mg/L range can be accomplished on the newer random access, 'chemistry' analyzers (see Chapter 16) and it is relatively easy to add a new test. Most technologists are familiar with these routine instruments.
ELISA and competitive ELISA are assays that are less frequently used in non-specialty labroatories and the laboratory may not have sufficient experience with these techniques to be able to use them for the assay. - Radial immunodiffusion (RID) (p 225 and Figure 11-7) is suitable for the measurement of proteins in the range of 1 mg/mL. RID is relatively easy to do and has a coefficient of variation on the order of 10%, which is suitable for this type of measurement.