Human Tamm-Horsfall Protein Research Articles

Measures of high-density lipoprotein function in men and women with severe aortic stenosis

BackgroundCalcification of the aortic valve is a common heart valve disorder, in some cases leading to clinically impactful severe aortic stenosis (AS). Sex-specific differences in aortic valve calcification (ACV) exist, with women having a lower burden of calcification than men as measured by computed tomography; however, the pathophysiological mechanism that leads to these differences remains unclear.MethodsUsing cultured human Tamm-Horsfall protein 1 (THP-1) macrophages and human aortic valve interstitial cells, the effects of high-density lipoprotein (HDL) particles isolated from the plasma of men and women with severe AS were studied for cholesterol efflux capacity (CEC).ResultsHDL-CEC was assessed in 46 patients with severe AS, n = 30 men, n = 16 women. ATP-Binding Cassette A1 (ABCA1)-mediated HDL-CEC was measured from human cultured THP-1 macrophages to plasma HDL samples. Women with severe AS had more ABCA1-mediated HDL-CEC, as compared to men (8.50 ± 3.90% cpm vs. 6.80 ± 1.50% cpm, P = 0.04). HDL pre-β1 and α-particles were higher in woman than in men by spectral density, (pre-β1 HDL, 20298.29 ± 1076.15 vs. 15,661.74 ± 789.00, P = 0.002, and α-HDL, 63006.35 ± 756.81 vs. 50,447.00 ± 546.52, P = 0.03). Lecithin-cholesterol acyltransferase conversion of free cholesterol into cholesteryl esters was higher in women than men (16.44 ± 9.11%/h vs. 12.00 ± 8.07%/h, P = 0.03).ConclusionsSex-specific changes in various parameters of HDL-CEC were found in patients with severe AS. Sex-based modifications in HDL functionality by HDL-CEC might account for the reduced burden of calcification in women vs. men with severe AS. Therefore, future studies should target sex-related pathways in AS to help to improve understanding and treatment of AS.Graphical abstractSex specifc differences in AVC and differences associated with HDL function in men and women with severe AS. When compared to men, women had higher preβ-HDL and α-HDL migrating particles, higher cholesterol efflux to HDL, and higher lecithin cholesterol acyl transferase (LCAT) activity, possibly indicating that improved reverse cholesterol transport may be protective against worsened calcification.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4–40 μg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 μg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65–107 and 108–149) and three small oxalate-binding domains (residues 199–207, 361–368 and 601–609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation.

Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement.

Tamm‐Horsfall Protein Binds Properdin

The purpose of this study was to determine if Tamm‐Horsfall protein (THP), an abundant urinary protein that interacts with various molecules of the immune system, binds to properdin (Factor P), a positive regulator of complement activation which stabilizes the alternative pathway C3 convertase. Enzyme‐linked immunosorbent assays (ELISA) were used to evaluate the binding of soluble human properdin to immobilized human THP. In low ionic strength buffers containing magnesium and calcium, THP bound properdin strongly (KD ~ 10‐8 M). Removing divalent cations from the buffer markedly reduced the ability of THP to bind properdin. Ionic strength also was important in this interaction. Properdin binding to THP was stronger in low ionic strength (20mM NaCl) and intermediate ionic strength buffers (90mM NaCl), than in physiologic strength (154mM NaCl) buffers. By ELISA, soluble THP effectively competed with immobilized THP for binding to properdin, with 0.1µM soluble THP inhibiting 50% of 0.05µM properdin's binding to immobilized THP. Western blot analysis demonstrated that soluble THP could bind to properdin after the properdin had been separated by SDS‐PAGE and transferred to nitrocellulose paper. In conclusion, this study shows that THP binds to properdin in the presence of divalent cations. This interaction is influenced by NaCl concentration, suggesting that electrostatic interactions between the two molecules are important. Properdin appears to recognize THP in solution, as well as immobilized THP, and THP binds to soluble properdin, as well as properdin that has been partially denatured by SDS during electrophoresis and Western blotting. The functional significance of this interaction remains to be evaluated. Funded by the PNWU Research Seed Program.

Effect of Mouse and Human Tamm-Horsfall Protein on Adhesion of Uropathogens to Human Uroepithelial Cells

Tamm-Horsfall Protein (THP) is the normal, conserved protein excreted in mammalian urine. For experimental studies, mice are preferred animals. The present study was undertaken to compare effect of coating of human THP (hTHP) and mouse THP (mTHP) on uropathogens and uroepithelial cells (UECs). Three common species of bacteria associated with UTIs were employed in planktonic and biofilm cell forms. On the whole, effect of hTHP and mTHP was comparable. Biofilm cells of all the three strains adhered to UECs in higher numbers in comparison to their planktonic counterparts in all four set of experiments. Findings provide the basis for future in vivo studies to be carried-out in mice where relevant conditions matching with humans can be created.

Tamm‐Horsfall protein binds to factor H of the alternate complement pathway

The function of Tamm-Horsfall protein (THP)(uromodulin), an abundant urinary protein of renal origin, remains in question. We previously have shown that THP interacts with the classical complement pathway by binding to C1q. The purpose of the present study was to determine whether THP also interacts with the alternate complement pathway, specifically with factor H (FH). Binding of THP to native and reduced human FH was assessed by transferring SDS-PAGE-separated FH to nitrocellulose and incubating the immobilized FH with human THP. Bound THP was detected with sheep anti-human THP and HRP-coupled donkey anti-sheep IgG antibodies. THP bound strongly to native FH, but did not bind to reduced FH. The interaction between THP and FH was explored further using an ELISA, where THP-coated wells were incubated with FH (1.0 to 250 μg/ml). Bound FH was detected with goat anti-human FH and Protein A/G-alkaline phosphatase. FH bound to THP with a KD of 9.6 × 10−7 M. An inhibition ELISA was performed. Soluble THP inhibited FH binding to immobilized THP in a dose-dependent manner. These results indicate that THP specifically binds to native FH, and thus suggests that THP may interact with the alternate complement pathway. Supported by the Warner/Fermaturo and ATSU Board of Trustees Research Funds.

Vitamin A excreted in the urine of canines is associated with a Tamm-Horsfall like protein.

Under physiological conditions canines transport vitamin A in blood plasma primarily as retinyl esters bound to lipoproteins and excrete substantial amounts of vitamin A as retinol and retinyl esters with urine. In the aqueous environment of urine, the hydrophobic vitamin A has to be associated with a protein. This vitamin A-protein complex was purified to homogeneity, prepared by preparative ultracentrifugation (density 1.21 g/mL), native polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography. The vitamin A-protein complex has a high molecular mass of > 5,000 kDa under native conditions. SDS PAGE under reduced conditions revealed a single band with a molecular mass of about 100 kDa for the protein moiety. Peptides obtained after limited proteolysis with trypsin from the 100 kDa protein were characterised by MALDI-TOF mass spectrometry and showed amino acid sequence homology to the human Tamm-Horsfall Protein (THP). This was further confirmed by a positive immunoreaction of the isolated protein with crossreacting human THP antibodies. The localisation of THP in dog kidneys was determined by using immunohistology. The reaction was strong along the entire thick ascending limb ofthe Henle loop and distal convoluted tubule. Our data point to the possibility that THP functions as a novel carrier for vitamin A in the urine of canines.

Role of polymeric Tamm-Horsfall protein in cast formation: Oligosaccharide and tubular fluid ions

In acute tubular necrosis (ATN), distal tubules are obstructed by casts formed by tubular debris, cells, and Tamm-Horsfall protein (THP). Since there are Arginine-Glycine-Aspartate (RGD) and Leucine-Aspartate-Valine (LDV) adhesive sequences in human THP, there may be direct integrin-mediated binding of tubular cells to THP. Alternatively, polymerization of THP may result in entrapment of the cells in its gel. Adhesion of LLC-PK(1) cells to THP-coated wells was directly measured. THP concentrate was dissolved in solutions which mimic urine from ATN (ATN-S), distal convoluted tubule (DCT-S), collecting duct (CD-S), and monomeric buffer (M buffer). THP was also denatured by either boiling or N-glycanase digestion. Gel formation of THP was then measured. Inhibition of LLC-PK(1) cell adhesion to collagen type I was measured with each solution, as well as after the collagen was pretreated with either monomeric (mTHP) or polymeric (pTHP) THP. The effect of pTHP on the settling rate of LLC-PK(1) cells in suspension was also measured. LLC-PK(1) cells did not directly adhere to THP, a finding against integrin-mediated binding as a mechanism for in vivo tubular cell/THP cast formation. The high electrolyte concentration of ATN-S and CD-S, however, was associated with pTHP gel formation. Moreover, cells suspended in pTHP remained in suspension. In cell adhesion studies, mTHP attenuated cell adhesion by binding to the matrix, whereas pTHP attenuated cell adhesion by trapping cells in its gel. An active process was involved since both pTHP gel formation and attenuation of cell adhesion were abolished by boiling or oligosaccharide removal with N-glycanase digestion. With renal ischemia and proximal tubule cell shedding, ATN and collecting duct fluid composition enhance THP gel formation and thus favor tubular cast formation and obstruction. The present in vitro results indicate the importance of oligosaccharide residues in mediating the effect of the THP on gel formation and potential cast formation in ATN.

Tamm-Horsfall Protein Binds to Type 1 Fimbriated Escherichia coli and Prevents E. coli from Binding to Uroplakin Ia and Ib Receptors

The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Biochemical and quantitative analysis of Tamm Horsfall protein in rats

The involvement of Tamm Horsfall protein (THP) in nephrolithiasis is currently under investigation in several laboratories. Although rat is a commonly used species as an in vivo model for such studies, there is only limited information available about the biochemical properties and excretion profile of THP in normal rats. In order to characterize rat THP, we purified and analyzed normal male rat THP, and compared it with normal human male urinary THP by gel electrophoresis. Both THPs migrated at approximately 90 KDa, and stained similarly for protein (Coomassie blue) as well as carbohydrates (periodic acid Schiff reagent). Compositional analysis revealed that rat THP was largely similar to human THP in amino acid and carbohydrate contents but showed differences in the individual sugar components from other mammals. There was considerable variation in the day-to-day urinary excretion of THP in normal rats, with values ranging from 552.96 micrograms to 2865.60 micrograms and a mean value of 1679.54 micrograms per 24 h. It was concluded from this study that rat THP did not contain any unusual biochemical components and was primarily similar to human THP in composition and mean urinary concentration.

Determining the Origin of Hematuria by Immunocytochemical Staining of Erythrocytes in Urine for Tamm-Horsfall Protein

Purpose We analyzed the feasibility of immunocytochemical staining of urinary erythrocytes for Tamm-Horsfall protein to differentiate renal from nonrenal hematuria. Materials and Methods Urine samples collected from 74 patients with hematuria were evaluated. Erythrocytes in urine were inspected morphologically and the same samples were immunocytochemically stained with antibody against human Tamm-Horsfall protein. Results Erythrocytes in urine were clearly stained for Tamm-Horsfall protein in renal hematuria. Tamm-Horsfall protein immunocytochemistry was more consistent with clinical diagnosis than morphology. Conclusions Immunocytochemical staining of urine erythrocytes for Tamm-Horsfall protein appears to be a reliable tool to differentiate renal from nonrenal hematuria.

Tamm-Horsfall glycoprotein binds IgG with high affinity

Tamm-Horsfall protein (THP), a monomeric glycoprotein (M(r) 80 to 100 kDa), is produced by the mammalian kidney's thick ascending limb of Henle cells and excreted into the urine. The function of THP is uncertain. Here we report that a high molecular weight contaminant in sheep THP (sTHP) preparations was identified as sheep IgG by its positive reaction with donkey anti-sheep IgG antibody and with protein G. To answer the question of whether sTHP and sheep IgG co-purified because of a physical interaction between the two proteins, an enzyme-linked immunosorbent assay (ELISA) using immobilized sTHP and soluble sheep IgG was performed. Analysis of the ELISA data identified the presence of two sets of binding sites: a high affinity site (Kd 10(-8) to 10(-9) M) and a lower affinity site (Kd 10(-6) to 10(-7) M) [corrected]. The ELISA detected a similar high affinity interaction between human THP (hTHP) and human IgG. The binding of sheep IgG to immobilized sTHP was inhibited by soluble sTHP. These observations suggest an additional factor to be considered in studies addressing THP's potential immunoregulatory function.

Distribution of immunoreactive Tamm-Horsfall protein in various species in the vertebrate classes

A sheep antibody to human Tamm-Horsfall protein, the major protein in normal urine, was used in an immunohistological study of organs of 48 species of vertebrate animals, representing the classes Mammalia, Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes. Immunoreactivity was shown in the thick limb of the loop of Henle in the kidney of mammals, but there was no reactivity with tissues of birds or reptiles. Superficial layers of the skin of several amphibians and fish, superficial layers of the oral mucosa and gills of fish, and the distal tubules of the kidney of some amphibians, reacted with the antibody. Immunoreactivity with mammalian kidney was removed by passage of the antibody down an immunoadsorption column coated with human Tamm-Horsfall protein, and amphibian immunoreactivity was removed by incubation of the antibody with material prepared from frogs in the same way as Tamm-Horsfall protein. These findings suggest that immunoreactive Tamm-Horsfall protein appeared early in vertebrate phylogeny, initially in skin and gills and later in kidney, and that although conserved in evolution, it shows antigenic differences between amphibians and mammals. Its distribution is consistent with the hypothesis that is acts as a waterproofing agent.

Localizing the Site of Hematuria by Immunocytochemical Staining of Erythrocytes in Urine

We have further explored the immunocytochemical staining method to discriminate renal and nonrenal hematuria, reported by Abrass and Laird (Am J Kidney Dis 1987;9: 44-50). After fixation on slides with acetone, erythrocytes in urine were stained with antiserum against human Tamm-Horsfall protein. Reactions were made visible by using either a fluorescent second antibody or a biotinylated second antibody, avidin, and biotinylated horseradish peroxidase, producing an insoluble reaction product. The staining methods were validated with material from clinically diagnosed cases of hematuria of renal or nonrenal origin. In material from kidney transplantation patients, in samples from the catheter that were presumed to contain renal erythrocytes, 84.7% and 80.1% of the erythrocytes stained by the immunofluorescence and immunoperoxidase methods, respectively, whereas in samples from the catheter that were supposed to contain nonrenal erythrocytes, 9.3% and 13.1% of the cells stained. In a group of nontransplantation patients with various causes of renal hematuria, 87.3% and 89.8% of the erythrocytes in urine stained with the immunofluorescence and immunoperoxidase methods, respectively, whereas in samples from patients with hematuria of known nonrenal origin, 12.9% and 12.4% of the cells stained. Staining of erythrocytes in renal and nonrenal hematuria was significantly different (P less than 0.001) and better discriminated between renal and nonrenal hematuria than did inspection of the morphology of erythrocytes in urine.

Immunoreactive Tamm-Horsfall protein in the kidney and skin of the frog Rana temporaria

Tamm-Horsfall protein (THP) is the main protein in normal human urine, and is found in the thick limb of the Loop of Henle in human kidney, and in other mammalian species. The skin of the frog. Rana temporaria, has similar physiological properties to this mammalian kidney tissue. In the present study, an immunohistological method involving an antibody to human THP was used to investigate the distribution of this distinctive protein in frog kidney and skin, and to compare its distribution with that found in the kidney tubules of rat and rabbit. THP-positive material was detected in the distal renal tubules and nephric duct of frogs, and was also located in the superficial epidermis of skin. It is suggested that its presence in amphibian skin is consistent with the hypothesis that THP is an important component of tissues that absorb sodium and chloride ions, but remain impermeable to water.

Antisera to a Rabbit Urinary Tract Antigen also React with Human Bladder and Kidney Tissue

The mucin layer covering the bladder transitional cell mucosa appears to function as a primary defense mechanism against bacterial infection. We have previously prepared a glycoprotein fraction (GP1) from the urinary bladder mucosa of NZW rabbits and raised murine antisera against it. These antisera react with bladder, ureter and kidney tissue from rabbits, rats, guinea pigs, and hamsters. We now show that a similar substance occurs in human kidneys and bladder. In order to remove antibodies reactive with the Tamm-Horsfall protein (THP), the antisera were initially absorbed with an immunoadsorbent composed of purified human THP covalently bound to Sepharose CL-4B gel. Using an enzyme linked immunosorbent assay (ELISA) it could be shown that the absorbed antisera did not react with THP but retained a high titer in binding to GP1. Immunohistochemical procedures involving avidin-biotin-immunoperoxidase staining demonstrated that the absorbed anti-GP1 reacted well with six human urinary bladder biopsy specimens and two kidney autopsy specimens while normal murine sera showed little or no binding. Although this reactivity was not as strong as that found with homologous tissue (rabbit) these studies suggest that GP1, an antigen common to several animal species, is also related to a human urinary tract component. (J. Urol., 143: 414–417, 1990)

Ultrastructural localization of Tamm-Horsfall protein in human kidney using immunogold electron microscopy.

The ultrastructural localization of Tamm-Horsfall protein (THP) was studied in paraformaldehyde-fixed human renal biopsies. Pre-embedding and post-embedding immunogold labelling techniques were developed utilizing a monoclonal antibody specific for human urinary THP. With the pre-embedding technique, membrane contrast was enhanced by osmification thus allowing precise localization of gold particles. Reasonable tissue penetration of antibodies was achieved without compromising ultrastructural detail. The hydrophilic resin LR White was used for post-embedding labelling to ensure maximum penetration of antibodies. However, sections had only mild osmification and consequently localization of label was less certain. Both labelling techniques gave similar results. THP was found to be associated with two renal cell types. Epithelial cells lining the thick ascending limb of Henle's loop had gold label closely associated with the whole cell plasmalemma, with some of these cells having an apparently random distribution of label throughout the cytoplasm. Only the luminal plasmalemma of epithelial cells lining distal convoluted tubules were found to be labelled. Basolateral membranes and the cytoplasm of these cells were negative. The use of a monoclonal antibody of defined specificity combined with the two immunolabelling procedures represents a precise reliable method for studying ultrastructural localization of THP in the human kidney.

Tamm-Horsfall protein coating of free cells in urine.

The origin of free cells in urine is difficult to determine in the absence of cellular casts. Using fluorescein-conjugated antibody to human Tamm-Horsfall protein (THP), it was demonstrated that some free cells in urine are coated with THP. To evaluate the usefulness of this observation in the differentiation of upper from lower urinary tract disease, the presence of THP coating of cells was correlated with the clinical diagnosis in 141 subjects. The percentage of THP-coated cells for each group (mean +/- SD) was Healthy volunteers (n = 10), 4% +/- 2%; hospitalized controls (n = 20), 3% +/- 3%; glomerulonephritis (n = 21), 61% +/- 6%; chronic interstitial nephritis (n = 26), 56% +/- 5%; other renal parenchymal diseases (n = 14), 50% +/- 8%; bladder disease (n = 14), 8% +/- 2%; and hypertension (n = 36), 24% +/- 33%. Based on the results from the bladder disease group, 12% coating was set as the 95% confidence limit for lower urinary tract disease. The results in this group were not different from control subjects. By analysis of variance and chi 2 analysis, subjects with renal parenchymal disease could be distinguished from those with hypertension and bladder disease (P less than .001 and P less than .0001, respectively). The presence of cellular coating by THP in renal parenchymal disease and its absence in bladder disease suggests that this simple test may be of use in determining the origin of free cells observed in the routine microscopic urinalysis.

Interaction of Tamm–Horsfall protein with bacterial extracts

Crude extracts of uropathic Escherichia coli have been reported to inhibit the binding of human Tamm-Horsfall protein (THP) to homologous and heterologous anti-THP antibody in immunoassays. This phenomenon was believed to be due to immunologic cross reactivity between THP and the bacterial antigens for the same antibody. Our attempts to further purify and characterize these "cross reactive" antigens with ion exchange and molecular sieve chromatography were unsuccessful. When purified anti-THP antibody was conjugated to sepharose beads forming an immunoadsorption column capable of isolating THP and cross reactive antigens from solution, the bacterial extracts did not react with the affinity column. However, binding between THP and the bacterial extracts and between THP and whole bacteria were demonstrated. These findings suggest that the cross reactivity seen in the immunoassays is caused by the interaction between the bacterial extracts and THP, and is not representative of true immunologic cross reaction for a common antibody.