Anal. Chem. 1995, 67, 3051 -3056
Thin-Layer lmmunoaffinity Chromatography with Bar Code Quantitation of C-Reactive Protein Staffan Nilsson,*lt Catharina Lager,* Thomas Laurell,a and Staffan Bimbaumi Department of Technical Analytical Chemistry, Chemical Center, P.0. Box 124, and Department of Electrical Measurements, P.O. Box 118, University of Lund, 221 00 Lund, Sweden, Aghardsgatan, 5,223 51 Lund, Sweden, and Pharmacia AB, 112 87 Stockholm, Sweden
A rapid thin-layer immunoafhity chromatographic methbd for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacterial infections, is described, where material and reagent costs are minimal. The analysis is based on the “sandwich” assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaflkity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoafhity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membranebound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectricdriven ink-jet printer. The time required for the analysis is less than 10 min. Quantitationis achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector. C-reactive protein (CRP, 220 kDa) was first described in 1930 as a factor found in plasma from patients with pneumonia. The protein was found to complex the C-polysaccharide in the membrane of pneumococcus. CRP consists of five identical peptide units which are associated to form a dish with pentameric symmetry. CRP is produced by the liver and is also called an acute phase reactant which increases in the case of tissue injury, infection, or inflammation. The increase can be in the magnitude of 100-1000. The rise is dependent upon the type of the disease and its complications. The concentration of CRP in serum rises quickly after stimuli but also falls off quickly and can fluctuate during an illness. In healthy individuals, the serum level is below 10 mg/L.’ Increased serum levels of CRP are seen 6-12 h after onset of the inflammatory reaction, and maximum levels are reached within 48-72 h. CRF’ levels will return to normal 5-10 days after remission of inflammation. For differentiation between viral and bacterial infections, low or moderately elevated CRP ’ Department of Technical Analytical Chemistry, University of Lund.
* Aghardsgatan.
Department of Electrical Measurements, University of Lund. Pharmacia AB. (1) Ledue, B.;Poulin, E.; Leavitt, F.; Johnson, A.Clin. Chem. 1989,35,20012002.
0003-2700/95/0367-3051$9.00/0 0 1995 American Chemical Society
levels indicate viral infection. In general, bacterial infections will cause higher CRP levels (>40 mg/L) than viral infections (
