Microtiter-latex test for C-reactive protein, for screening large numbers of samples.

The slide-latex test for C-reactive protein (CRP) takes a few minutes per sample, but the critical timing involved makes it unsuited for use with large numbers of samples. In addition, almost 40% of CRP-positive samples give negative results by either nephelometry or CRP enzyme-linked immunoassay (1). By using microwells instead of slides, increasing the time of the reaction from 3 to 60 mm, and increasing the serum dilution without introducing new steps, we have facilitated the processing of samples and increased the test’s specificity. A local hospital classified serum samples as CRP-positive or -negative by doing an anti-CfiP slide-latex test (Lab. Diag., Morganville, NJ). Anti-CRP antiserum from rabbits was obtained from Llorente, Madrid, Spain. Polyvinyl wells (MRC-96) were from Limbro, Handen, CT. Wash about 600 j.zL of 0.8-zL particles of latex (#{176}Estapor K109”, 10 g/L latex suspension; Rhone-Poulenc, Coubevoie, France) with 10 mL of glycine buffer (50 mmol/L, pH 8.6) containing 30 mmol of NaC1 per liter. Resuspend in 20 mL of the same buffer and add 150 L of CRP antiserum. Incubate at 37 #{176}C for 3 h, with agitation, then wash the latex suspension in the same buffer and resuspend it in 5 mL of dilution buffer (per liter: 200 mmol of borate, 75 mmol of NaCl, 2 mmol of CaCl2, 10 g of bovine serum albumin, 0.1 g of thimerosal, and 0.5 mL of Tween 20, pH 8) and store at 0#{176}C. Before use, dilute this suspension fourfold in dilution buffer. Pipette 5 jzL of serum into each well, add 50 L of anti-CRP-bound latex, and gently agitate all samples by moving the plates horizontally. Let the samples stand at room temperature for 60 mm. Homogeneously dispersed latex corresponds to negative CRP samples; coarse pellets appear in the positive CRP samples. Different anti-CRP latexes must be titrated to ascertain the correct amount of sample and latex, to adjust sensitivity. We use a cutoff for positive CRP of 6 mg/L. We tested several types of wells in different shapes and materials; polyvinyl Vshaped wells over black backgrounds gave the greatest contrast, the differences between positive and negative wells being easily visible. The percentage of false positives was decreased from 38.8% in the slide-latex method (1) to 6.2% in the microtiter-latex test. The percentage of true values increased from 61.2% to 76.3% (n = 80). The increased specificity can be due to avoidance of the artefacts caused by reading-time differences from sample to sample when a large number of samples are involved. Evaporation artefacts are also avoided. Interference from rheumatoid factor and highly lipemic sera is minimized by diluting the sample 10fold (done simultaneously with the addition of latex) compared to the 1:1 dilution factor in the slide-latex test. Prozone phenomena were also decreased because of this higher latex/serum ratio. By using automatic pipettes we could dispense 100 samples and latex suspensions in 11.4 s per sample. This time could be shortened if multichannel pipettes are used. Working time per sample is decreased mainly because no stick mixing of sample and latex is required. To check for prozone effect, one can re-assay the negative samples after 1 h by adding another 50 pL of latex per well to the negative samples,agitatingthe wells, and waiting another hour before reading the results. This method is useful for screening large number ol samplesrequiringonly qualitative (yes/no) answers.