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Lyme disease (LD) is difficult to diagnose absent hallmark clinical symptoms. LD immunoassays are problematic because of cross-reactivities, false-positives, sequestration of antibody and antigen in immune complexes (IC) early after infection, and persisting antibodies. Demonstration of IC in LD (10) opened the possibility of developing assays to detect immunoglobulin M (IgM) in IC at a time when antibiotic therapy would be useful. Subsequently, we confirmed the presence or absence of LD antigens/antibodies in sera and other biological fluids from putative LD patients (1-4). Assay development used “gold standard” erythema migrans and/or culture-positive (from biopsy) samples (e.g., CDC) and endemic area controls. We compared our enzyme-linked immunosorbent assay and immunoblots with many types of commercial (e.g., MarDx) and laboratory (e.g., immunofluorescent antibody, enzyme immunoassays) tests. Using monoclonal antibodies (MAb), standard assay development protocols (antibody/antigen checkerboards to ascertain correct concentrations of primary and secondary antibodies), appropriate blocking reagents, and sensitive detection methods (e.g., biotin-avidin), we obtained superior sensitivity and specificity of IC over comparator tests (3, 4) or whole serum alone (corroborated in independent laboratories). IC allowed us to “see” more bands in immunoblots (2) than a comparator commercial test, and exhaustive absorption of IC along with a “good” MAb even allowed us to detect Borrelia burgdorferi antigen in early infection (1, 2).  Although a report by Marques et al. (9) refutes beneficial use of IC in LD assays, there are enormous differences between their work and mine.  To emulate our protocols (1, 2), Marques et al. (9) measured total IgM and IgG in IC (and sera) by first concentrating IC at 2:1 after the polyethylene glycol (PEG) precipitation step and then making dilutions (IC, 1:10; sera, 1:100) and measuring them by nephelometry. After correcting for concentration and dilution, they arrived at an IC having 3.2-fold more IgM (and 50% less IgG) than unprocessed sera. In the Discussion, they attribute the larger amount of immunoglobulin in IC to extra immunoglobulin contributed by antibody present in unprocessed sera adding to that contributed by IC, to justify their experimental finding (9). More likely, “overmanipulation” of the IC sample to be measured (first concentrating—for no obvious reason—then diluting and measuring the final dilution by nephelometry) is the probable cause for the inconsistent measurement. They confused my protocol (which used a 2:1 concentration) with the one I used for antigen detection (1, 2), where enrichment of IC (by volume reduction before adsorption with beads) may be important for antigen detection.  Recognizing the importance of total IgM in IC and whole sera (2) in early infections, we developed a quantitative IgM assay. We coated plates with an excess (10 μg/ml) of anti-IgM that reacted with dilutions of an IgM standard (whole-molecule human IgM myeloma; Jackson ImmunoResearch), compared to dilutions of unknowns (IC, not previously concentrated, and unprocessed sera), and calculated the values of unknowns that fell in the linear portion (50 to 500 ng/ml) of the standard curve by linear regression (using anti-human IgM-horseradish peroxidase second antibody reporter), as in typical protein assays. There was a range of IgM concentrations three- to sevenfold greater in sera than IC for all LD and non-LD sample pairs tested. This was confirmed independently by nephelometry (SmithKline Beecham). These values were used to normalize total IgM in sera and IC and demonstrated superior sensitivity of IC for detecting LD patient antibodies in immunoblots (see Fig. 2c in reference 2). This was a more rigorous method for measurement. Even if some free antibody was trapped in their PEG precipitate (as they claim in their Discussion to account for their result of higher immunoglobulin content in IC), the validity of their measurement seems unlikely. Unless PEG precipitation somehow adds IgM, their result violates conservation of mass. Digeon et al. have already shown only neglible amounts of free antibody are precipitated with 3.5% PEG (5).  An even greater objection is to their attempts to detect antigen in IC (9). They used a 1:10 dilution of monoclonal antibody H5332 supernatant obtained from Alan Barbour. They first tried a semidry transfer with a sensitive chemiluminescent reagent and got multiple bands, including the 31-kDa band, for both patients and controls. They state that using several types (e.g., GammaBind-G-Sepharose, mannan-binding protein, protein-l-agarose) of antibody binding beads “failed to remove the nonspecific binding.” That is not the intended purpose of using these beads, which simply react with the Fc portion of antibodies of their respective isotype. They lowered the H5332 concentration to 1:100, which did not remove nonspecificity. They dissociated the IC with acid of pH 3.5 (possibly detrimental to the complex, which is why we used a high pH, 10.2). They tried a less sensitive chemiluminescent reagent with no success. Finally they used the more sensitive wet transfer with the less sensitive colorimetric detection, whereupon they obtained no bands. The main problem is their weak H5332 supernatant, which was either freeze-thawed, poorly stored, overpassaged, or otherwise improperly handled, and/or possibly incorrect dilution of their secondary antibody. They changed multiple variables (reagents and methods) simultaneously. They neglected to troubleshoot nonspecific bands with an unrelated MAb. They showed no immunoblots.  We purchased the original H5332 hybridoma cells from Alan Barbour in the late 1980s. We propagated these ourselves, using low-passage fresh supernatant (never frozen) at a 1:1,000 dilution (compared to their 1:10 dilution). The secondary antibody, horseradish peroxidase-goat anti-mouse IgG Fab specific (A-2304; Sigma), was used at a 1:150,000 dilution, along with Renaissance-plus (NEN) chemiluminescent reagent (no longer available). This gave one specific band at 31 kDa in Lyme disease patients and no bands in controls. The technique was used on 24 samples (patients of various LD manifestations and some controls) in one report (2), and one seronegative patient in another (1), and we showed immunoblots.  The techniques and materials used by Marques et al. are clearly so different from those I developed that there can be no direct comparison between their results and ours. Further, the authors may have been unaware of several studies where pathogen-specific antibody has been found in IC in infectious diseases prior to LD (5-8, 11).  Attempts to disprove the concept of IC by merely claiming to repeat a procedure with poor materials, techniques, and multiple simultaneous methodological changes should be discounted. Their conclusions are invalid, counterproductive, and a disservice to patients with or suspected of LD.

Report Refuting Value of Immune Complexes To Diagnose Lyme Disease Is Invalid