Matrix metalloproteinase-2 (MMP-2), synthesized as a 631 amino-acid proenzyme, is activated by cleavage of the first 80 amino acids and naturally inhibited by tissue inhibitor of metalloproteinase-2 (TIMP-2). We report here the production of MAbs against MMP-2 and TIMP-2 and their use in localizing the respective antigens on tumor tissues. The anti-MMP-2 MAb recognized the latent and activated MMP-2 mutant protein (mutein) with C-terminal deletion at amino acid 425, indicating that both N- and C-terminal amino acids of MMP-2 are not important for its binding. The binding study of anti-TIMP-2 MAb, using several C-terminally truncated TIMP-2 muteins, showed that the amino acids 111-126 of TIMP-2 are essential for the binding of this antibody. Besides their respective antigens, both MAbs also recognized the MMP-2/TIMP-2 complex. On frozen sections of breast tumor, anti-MMP-2 MAb stained mainly tumor-cell cytoplasm with varying intensity, while anti-TIMP-2 MAb gave a stromal staining of varying intensity and a weak or absent staining of tumor-cell cytoplasm, suggesting different localization of the proteins in these tumors. In addition, in 1/3 of the breast cases both antibodies also localized on tumor-cell membranes. Similar cytoplasmic and stromal but not membrane staining patterns were observed in colon, gastric, endometrial, squamous-cell, prostatic and ovarian carcinoma as well. Since MMP-2 degrades type-IV collagen, the major component of basement membranes, the differences between MMP-2 and TIMP-2 levels and localization in individual tumors may relate to the invasiveness of the tumor and thus provide predictive information. However, this aspect could not be discussed in this study because no biological and clinical parameters such as lymph-node involvement or Dukes' stage of the tumors were available.

The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.

No abstract