Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR

Abstract

A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase AmpliTaq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth. The inhibitory effect was reduced when PIgG was heated at 95 degrees C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95 degrees C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads.

FIG. 1

SDS-PAGE of PIgG in comparison with SDS-PAGE of MIgG1 and MIgG3. Electrophoretic separation was carried out by SDS-PAGE with 12% polyacrylamide gels. Proteins were detected with Coomassie brilliant blue. Lane 1, low-molecular-mass protein standard (Amersham Pharmacia Biotech); lane 2, PIgG; lane 3, MIgG1; lane 4, MIgG3.

FIG. 2

Inhibitory effects of different concentrations of PIgG, MIgG1, and MIgG3 on a PCR mixture containing AmpliTaq Gold and 1 ng of L. monocytogenes DNA. Lanes 1 and 8, 100-bp molecular mass marker (Amersham Pharmacia Biotech); lane 2, negative control; lane 3, positive control; lane 4, 0.82 μg of PIgG; lane 5, 0.41 μg of PIgG; lane 6, 0.082 μg of PIgG; lane 7, 0.041 μg of PIgG; lane 9, 0.68 μg of MIgG1; lane 10, 0.34 μg of MIgG1; lane 11, 0.068 μg of MIgG1; lane 12, 0.034 μg of MIgG1; lane 13, 0.77 μg of MIgG3; lane 14, 0.39 μg of MIgG3; lane 15, 0.077 μg of MIgG3; lane 16, 0.039 μg of MIgG3.

FIG. 3

Removal of the inhibition of human plasma by DNA-agarose beads. Lane 1, 100-bp molecular mass marker (Amersham Pharmacia Biotech); lanes 2 to 6, different dilutions of human plasma molecules that did not bind to DNA-agarose beads (undiluted and diluted 1:5, 1:10, 1:50, and 1:100, respectively); lanes 7 to 11, different dilutions of human plasma molecules that did bind to DNA-agarose (undiluted and diluted 1:5, 1:10, 1:50, and 1:100, respectively).