Human Antiganglioside Autoantibodies

Validation of ELISA
MEPUR H. RAVINDRANATH, SAKUNTHALA MUTHUGOUNDER, THIRUVERKADU S. SARAVANAN, NAFTALI PRESSER, AND DONALD L. MORTON Laboratory of Glycoimmunotherapy, John Wayne Cancer Institute, Santa Monica, California, USA

ABSTRACT: Gangliosides have a hydrophilic sugar chain that contains antigenic determinants and a hydrophobic ceramide. In humans, gangliosides elicit a Tcell independent IgM response; antiganglioside IgM autoantibodies may be pentameric or polymeric. A correlation between specific neuropathies and antiganglioside autoantibodies has been confirmed. Although many neurologists attempt to lower titers of antiganglioside autoantibodies, oncologists are developing strategies to augment production of IgM antibodies that will remove immunosuppressive gangliosides from the circulation of patients and target gangliosides and kill tumor cells. Antiganglioside IgM antibodies can cause leakage of the blood–nerve barrier in a concentration-dependent and complementindependent manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker of axonal damage in neuropathies such as multiple sclerosis. They are also a promising biomarker of early prostate cancer. There is a need to validate the protocol for enzyme-linked immunosorbent assay (ELISA) of antiganglioside IgM autoantibodies. This validation must consider the purity of gangliosides from different commercial sources, the coating of gangliosides onto a solid matrix in a manner that maximizes exposure of oligosaccharide epitopes to IgM paratopes, techniques to minimize background noise and eliminate nonspecific antibody binding, and carefully defined positive and negative controls. The validated protocol also must include a simple formula to estimate titers for several replicas. Finally, antibody titers must be converted to natural logs for statistical appraisal. KEYWORDS: antiganglioside; autoantibodies; ELISA; gangliosides; human; titer; validate; tumor; antigen

Gangliosides are amphophilic molecules with atomic mass units (AMU) ranging from 1300 to 3000. They have a hydrophilic sugar chain with one or more sialic acids and a hydrophobic ceramide with sphingosine and a long-chain fatty acid.1,2 Diversity in the gangliosides is created by the number and nature of their sugars, the number and length of glycosidic linkages of sialic acids, the number of double bonds, and the extent of fatty-acid hydroxylation. Gangliosides are expressed on the outer layer
Address for correspondence: Mepur H. Ravindranath, Laboratory of Glycoimmunotherapy, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404–2302. Voice: 310-449–5263. ravindranathm@jwci.org Ann. N.Y. Acad. Sci. 1050: 229–242 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1313.024 229

4Glcβ1Cer 2144 of the bilayered lipid membrane of every human cell.8NeuAcα2.3GalNAcβ1. In our ELISA system. which prevents direct and specific recognition by conventional specific T cells.4Glcβ1Cer NeuAcα2.8 Oligosaccharide residues of gangliosides are unable to bind into the groove of MHC molecules.4Galβ1. ethanol suspension is employed to coat polystyrene plates in an enzyme-linked immunosorbent assay (ELISA) to measure antiganglioside antibodies. although antiganglioside IgG (commonly IgG2a or IgG3) antibodies are found in mice and rabbits.4Glcβ1Cer Galβ1.3NeuAc)Galβ1.3Galβ1. and they do not require T-cell help. extraneural. there is no evidence for isotype switching of antiganglioside IgM or any atypical memory responses mediated by T . Neural tissues and malignant cells overexpress gangliosides.8NeuAcα2.4 Antigen–antibody interactions also may involve salt linkages.3Galβ1. the molecules aggregate and form irregular micelles. CMC increases.4Galβ1.5–7 Because ganglioside antigens fail to induce a memory response in humans. micelle formation requires a higher critical micellar concentration (CMC).4 nm of paratope. In ethanol.3 During drying in vacuo. Gangliosides elicit consistent and specific humoral responses without T-cell help.3)Galβ1.3)Galβ1. that is.4Glcβ1Cer GalNAcβ1. it is 6 sugar residues for antiganglioside antibodies.4(NeuAcα2.4(NeuAcα2.3NeuAc)Galβ1.3GalNAcβ1. we rarely encounter IgG antibodies to gangliosides in the sera of patients who have been immunized with ganglioside-based vaccines.8NeuAcα2.3GalNAcβ1. The upper limit of epitope size is determined by the variable region of the CCS.4Glcβ1Cer GalNAcβ1. gangliosides are not presented in the context of MHC molecules. The epitope is the antigenic determinant that binds to the paratope. Therefore.4Glcβ1Cer NeuAcαGalβ1.3GalNAcβ1.5 nm of sugar residues and 30.230 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 1.4(α2.3Galβ1.3GalNAcβ1. and malignant human cells.4Glcβ1Cer NeuAcαGalβ1. Structure of gangliosides found in normal and malignant human tissues Glycolipid GM3 GM2 GD3 GD2 GM1a GM1b GD1a GD1b GD1c GT1b NeuAcα2. Fifteen amino acids of the antibody establish 90 van der Waals forces and 9 hydrogen bonds. TABLE 1 illustrates some of the most common antigenic determinants of gangliosides in neural.5–8 They do not elicit specific or consistent cellular immune responses.3)Galβ1.4Glcβ1Cer NeuAcα2. causing attachment of the tail group to the polystyrene plates. To our knowledge.8NeuAcα2.8NeuAcα2. The contact areas involve 25.3)Galβ1.4(NeuAcα2. which transport antigen fragments to the cell surface and present them to T cells as well as to B cells to generate primary and secondary antibody responses.4(α2. In aqueous media. The antigenic determinants of common gangliosides are the sugar chains on the lactosylceramide backbone.3Galβ1.4Glcβ1Cer Galβ1.4(NeuAcα2. Although protein or peptide antigens are processed by intracellular enzymes and major histocompatibility complex (MHC) molecules. the complementary combining site (CCS) of an antibody.3GalNAcβ1.4Glcβ1Cer Structure AMU 1236 1385 1545 1694 1547 1547 1838 1838 1838 NeuAcα2. antiganglioside antibodies are invariably IgM.

14. it is far from clear whether the antibodies cause axonal damage or are a result of axonal damage. or other polymers.11 Although earlier studies suggested that exogenous adjuvants were necessary to induce an antibody response to ganglioside antigens. provoking a specific antiganglioside antibody response.9 The polymeric IgM is potentially useful in cancer patients who develop high titers of antiganglioside antibodies. These findings suggest that glyco-immunomic studies may lead to the development of specific antiganglioside IgM as early biomarkers of human cancer. antiganglioside IgM biomarkers may not be . necrotic cells released tumor gangliosides into circulation. Many laboratories carry out antiganglioside IgM assays without analyzing the rationale and suitability of each step of the assay system. suggesting that gangliosides released during necrosis induced antiganglioside IgM without any exogenous adjuvants. It is possible that necrosis would have acted as a natural adjuvant or would release endogenous adjuvants such as heat shock proteins to stimulate the ganglioside-specific antibody response.21 However. including multiple sclerosis. There is a need to validate the assay protocol for antiganglioside IgM antibodies.RAVINDRANATH et al. Some antiganglioside IgM antibodies can increase the permeability of the blood–nerve barrier in a concentration-dependent and complement-independent manner. The increase in antibody levels was followed by a decrease in serum gangliosides. Researchers also must monitor changes in the profile of antiganglioside IgM antibodies during different stages of cancer.15 Although many neurologists attempt to lower the titer of the antiganglioside antibodies in neuropathies. the possible pathogenesis induced by some of the antiganglioside IgM antibodies should caution oncologists against indiscriminate boosting of the antiganglioside IgM response. polymeric IgM may not have a J chain.10 Accruing evidence suggests that antiganglioside IgM may be secreted by a separate class of B cells that express the CD5 T-cell marker. heptamers. A disease-specific correlation between specific neuropathies and antiganglioside antibodies has been confirmed.16 and some may bind to neuronal gangliosides to create a neuromuscular block.22 and our unpublished observations revealed antiganglioside IgM antibodies in serum and ascites of patients with epithelial ovarian cancer.13 In this study of patients with advanced colon cancer. we found that cryoablation of metastatic lesions caused tumor necrosis. This is important to understand and to identify the homeostatic mechanism by which the host eliminates gangliosides that are recognized by the immune system as danger signals. Antiganglioside IgM antibodies may be pentamers.: HUMAN ANTIGANGLIOSIDE AUTOANTIBODIES 231 cells. Without proper validation of the assay to monitor the antiganglioside IgM. Unlike conventional pentameric IgM. Any IgG antiganglioside antibodies reported in humans could be anti-antiidiotypic antibodies or antibodies directed against peptides that mimic ganglioside epitopes. The development of clinically effective ganglioside vaccines against specific cancers requires biochemical and immunochemical definition of gangliosides associated with specific tumor types. hexamers. We recently identified a significant antiganglioside antibody response to earlystage (organ-confined) prostate cancer.12 our recent study of antiganglioside responses to cryosurgical ablation of liver metastases indicates otherwise: exogenous adjuvants are not required to elicit an antiganglioside IgM response. oncologists are developing strategies to boost antiganglioside IgM responses to tumor-associated gangliosides. because antibodies without a J-chain fix complement 20-fold more efficiently than do conventional pentamers.17–20 While antiganglioside IgM can serve as a marker of axonal damage in neuropathies.

it may detect autoimmune neuropathies associated with peripheral neuropathies. cytomegalovirus. (2) Coating of gangliosides onto microtiter plates. we have developed a standard operating procedure and validated the ELISA for antiganglioside IgM antibodies. Helicobacter pylori. (9) Converting antibody titers to natural logs. In cancer patients. and check the level of tumor necrosis in situ. abortion. In infectious disease. Chaga’s disease. (3) Blocking to eliminate background noise. HIV. celiac disease. epilepsy. (6) Taking precautions at the final steps. This ELISA for antiganglioside IgM antibodies has a wide range of clinical and investigative applications in neoplastic and nonneoplastic disease. and neuroborreliosis. or multiple sclerosis. and stroke. (8) Applying a formula to estimate titers for several replicas.3 Since then. evaluate danger signals corresponding to very early stages of disease and ascertain their correlation with prognosis. Our first publication in this direction appeared a decade ago. VALIDATION OF PROTOCOL FOR ANTIGANGLIOSIDE ANTIBODY ELISA Since our previous report. it can be a tool for monitoring rheumatoid arthritis. Finally. vaccinia. No biomarker assay can be proposed for clinical use unless it is properly and rigorously validated. rotavirus. it can be used to monitor endogenous immune responses to early or localized disease. Our standard operating procedure entails the following: (1) Characterizing the purity of ganglioside antigens in different batches and from different commercial sources. We screened . the ELISA can be used to determine the specificity of antiganglioside monoclonal antibodies and to develop microchip arrays of antiganglioside IgM for human diseases. MillerFischer/Bickerstaff’s encephalitis. (4) Maximizing epitope–paratope interaction. assess spontaneous and therapeutically induced regression. In neuropathic disease.232 ANNALS NEW YORK ACADEMY OF SCIENCES identified or introduced into clinical laboratories.3 we repeatedly validated our standard operating procedures to obtain high-resolution titers of 1 or more antiganglioside IgM antibodies in sera from patients with cancer or other diseases and in sera from healthy controls. Guillain-Barré syndrome. diabetes mellitus I and II. amyotrophic lateral sclerosis. (7) Establishing positive and negative controls. (5) Eliminating nonspecific binding of antibodies. the ELISA can be used to check sequelae associated with Campylobacter jejuni. HPTLC is critical because there is no standard definition of purity for gangliosides from different commercial sources. Characterizing the Purity of Ganglioside Antigens in Different Batches and from Different Commercial Sources We routinely use high-performance thin-layer chromatography (HPTLC) to examine the purity of gangliosides stained with resorcinol or with ganglioside-specific murine monoclonal antibodies. Mycoplasma pneumoniae. atherosclerosis. Further. amoebiasis.

and not detectable for GD1a from Sigma. (B) Staining of GD1a (2 nmol) from 4 different commercial sources (Calbiochem. Contaminating glycolipids are indicated by the thin lines on the right. Advanced Immunochemical. Resorcinol staining identified a GM1-like contaminant that was distinct for GD1a from Accurate. Alexis. Interestingly.G2a). Alexis. Alexis. Ganglioside purity was assessed by high-performance thin-layer chromatography (HPTLC). and Sigma) by resorcinol and antiganglioside monoclonal antibodies against GD1a (GMR17) and GD2 (14. The GD1b from Alexis was free of contamination and hence used for ELISA. (A) Staining of human and bovine brain GD1b (3 nmol) from 4 different commercial sources (Sigma.G2a identified a contaminant in GD1a from Calbiochem. less apparent for GD1a from Calbiochem and Alexis.RAVINDRANATH et al. Chromatograms were stained by resorcinol-HCl and immunostained with ganglioside-specific murine monoclonal antibodies (MAbs). . Accurate. and Calbiochem). the anti-GD2 monoclonal antibody 14.: HUMAN ANTIGANGLIOSIDE AUTOANTIBODIES 233 FIGURE 1.

because this contaminant level was very low in Sigma GD1a and because this GD1a did not contain GM1-like contaminant. Base treatment for 2 h or 4 h removed the top fraction. but the O-acetyl group of the internal sialic acid was deacetylated only by treatment with 7 N ammonium hydroxide overnight. However. Accurate Chemical and Scientific (Westbury. CA). (E) Two different lots of GD2 from Advanced Immunochemical. positions of the contaminating glycolipids are indicated by the thin lines on the right. If it is O-acetylated GD2. and Sigma. Alexis USA (San Diego. Alexis.22 FIGURE 1A shows our experience with GD1b purified from human and bovine brain by Sigma (St. including O-AcGD2. and we selected a few of these monoclonal antibodies to test the purity of gangliosides on HPTLC. NY). the monoclonal antibody for GD2. and Sigma was free of contaminants stainable by either resorcinol or anti-GD3 monoclonal antibodies. Because the contaminant level was very low in Sigma GD1a and because this GD1a did not contain the GM1-like contaminant. Our second choice was Calbiochem. Staining with MAb 14. Calbiochem.234 ANNALS NEW YORK ACADEMY OF SCIENCES murine monoclonal antibodies for their monospecificity using our ELISA protocol. 1 or both sialic acids in the GD2 can be O-acetylated. (D) Two-dimensional chromatogram of GD2 from Advanced Immunochemical shows 2 forms of O-acetyl GD2 before and after treatment with ammonium hydroxide at different time intervals. In the resorcinolstained chromatograms. Additional base treatment with 7 N ammonium hydroxide for and Sigma. the new lot is totally devoid of the contaminants after strong base treatment. GD2 from Advanced Immunochemical (which is reasonably priced) contains alkali-susceptible GD2. Sigma no longer produces GD3. and Calbiochem (San Diego. We selected GD3 from Calbiochem for ELISA. . which could be GD2-lactone or O-acetylated GD2. and Sigma was free of contaminants by staining with resorcinol and monoclonal antibodies. no commercially obtained GD2 is free of contamination of other ganglioside fractions. FIGURE 1B shows contamination in purified GD1a obtained from Calbiochem. Treatment of GD2 with 2. We selected GD3 from Calbiochem because Sigma stopped its supply. which by its position could be GD2 with an O-acetyl group in the inner sialic acid (FIG. Calbiochem. we selected GD1a from Sigma for ELISA. CA). identified a contaminant at the position of GD2 in the preparations from Calbiochem. Chromatograms were stained by resorcinol and by monoclonal antibodies for all gangliosides including GD1a (GMR17). which would correspond to lactones or O-acetyl groups in the terminal sialic acid of GD2. MO). (C) GD3 from Alexis. Base treatment with ammonia (3N) may remove both lactone and O-acetylated derivatives. base treatment also introduced a new fraction midway between GD2 and the top O-acetyl GD2 band.5 N ammonium hydroxide de-O-acetylated the terminal sialic. The old lot shows base-labile contaminants. However. 1D). we chose GD1a from Sigma. Unfortunately. FIGURE 1C shows that GD3 from Alexis. Our preferred source for GD1b is Alexis because its GD1b shows no evidence of contamination. FIGURE 1D shows the result of exposing GD2 to ammonium hydroxide. Louis. and Sigma. Alexis. Resorcinol staining showed a distinct GM1-like contaminant in the preparation from Accurate. CA). This contaminant was less apparent in GD1a from Calbiochem and Alexis and was not detectable in GD1a from Sigma. The scale in the figure shows the position of the O-acetylated GD2 in the untreated preparation. Advanced Immunochemical (Long Beach.G2a.

310 0.315 0.5 N ammonium hydroxide for 4 h (FIG. Anti-GD2 IgM values (expressed in absorbency of sera diluted 1/500) in 40 patients with regional (stage III) metastatic melanoma Treatment None (old lot) Background correction Minimum Maximum Median Mean No Yes NH4OH (2. Gamma irradiation at 40 kRad (Mark 1-30 irradiator.642 1. a major fraction of GD2 had both sialic acids O-acetylated.038 0. Base-treated gangliosides show significantly (P < . Absorbency higher than 0.294 0.316 0. TABLE 2 shows the reactivity of sera from 40 patients with stage III melanoma against (1) GD2 (Advanced Immunochemical) treated in the laboratory with 2. suggesting that the sera may contain IgM antibodies to O-acetyl GD2.031 0. In the first (early) lot. which compares 2 lots of GD2 from Advanced Immunochemical.RAVINDRANATH et al.045 0.016 . Base treatment abolished the upper band but not the lower band.466 STD 0.030 0. 1E.0019 .595 1.330 2.154 0.100 at a serum dilution of 1/200 is not suitable.002) higher values because 100% of 3 nmol of new lot is GD2. 1D) and (2) new lot of GD2 (FIG. In summary. Coating of Gangliosides onto Microtiter Plates The way in which the solid matrix of a microtiter plate is coated with ganglioside will determine how efficiently the ganglioside’s sugar domains (epitopes) are exposed for immune recognition. The new lot showed significantly (P < .168 0.609 1. 2 h failed to remove the fraction. which may contain O-acetyl group in the inner sialic acid.021 1.261 0. The second (new) lot is free of the contamination and is used for routine analyses of sera for anti-GD2 IgM.081 (NS) .228 0. Optimal plates were those that had a low reactivity to antibody without gangliosides in the presence of excipient used for gangliosides. CA).489 0. it is critical to obtain pure ganglioside and to use a precise concentration (3 nmol) of ganglioside per well before measuring the titers of antiganglioside IgM antibodies in ELISA.278 0.195 0.5 N for 4 h) New lot No Yes No Yes 0. JL Shepherd and Associates) can lower background reactivity. The new lot provided by Advanced Immunochemical represents the final product (FIG. Although microtiter plates are treated with gamma . 1E).: HUMAN ANTIGANGLIOSIDE AUTOANTIBODIES 235 TABLE 2.484 . In a previous report. but the fraction is eliminated entirely after overnight treatment in 7 N ammonium hydroxide.318 0.02) lower values than untreated.322 0.3 we coated ganglioside antigens onto different polystyrene microtiter plates. The importance of base treatment before assessing anti-GD2 titers is shown in FIGURE 1E.0011 P valuea NOTE: All three batches of GD2 were from Advanced Immunochemical (Long Beach.035 0.285 0.487 0. new lot).632 2. The lower values obtained with the base-treated old lot are due to the presence of 80% or less of GD2/well.341 0. aPaired-sample test between untreated and other treatment group or new lot.310 0.

We strongly discourage using gelatin. the background noise should be <<< 0. 1C). This HSA is superior in performance and electrophoretic albumin/nonalbumin ratio to other preparations. Clinical-grade HSA is available from Baxter Healthcare Corporation (Glendale. milk protein. the surface of the wells should be checked under a dissection microscope for any evidence of peeling. possibly because of differential irradiation or lack of quality control by the manufacturers. We selected Falcon 3915 as the best plate for antiganglioside IgM studies. investigators should irradiate the plates in a gamma irradiator and ascertain that the absorbency of the background noise (at dual wavelength. The oligosaccharide residues should cluster on the plate in a fashion that simulates their appearance on the cell surface and facilitates maximal attachment of IgM. as cited in the literature.4) for 90 min will block nonspecific binding of the serum proteins. In countries where these plates are not available. After several such investigations. or body fluids obtained during clinical investigations are usually stored in liquid nitrogen or in subzero (−35°C) freezers. in part. Coating the plates with 4% HSA in phosphate-buffered saline (pH 7. and the degree of hydroxylation. Desiccation can be extended up to 1 month if undisturbed.236 ANNALS NEW YORK ACADEMY OF SCIENCES irradiation during manufacture.S. if the titer is determined to be 6400. Because lot numbers for Falcon 3915 constantly change. vide infra) is less than 0.599. The contaminants in some preparations can significantly lower purity. because they introduce various anomalies.100 at 1/3200. we attach the fatty acid residues of the ganglioside to the plate by 24-h vacuum desiccation. and other antibodies. Maximizing Epitope–Paratope Interaction Serum. 20030049692). rather than methanol or chloroform or buffer.3 In contrast to other prevailing methods (U. Blocking with HSA yields a baseline or background value (primary negative control) that should not exceed an absorbency of 0. These characteristics depend. Known concentrations of this HSA should be subjected to polyacrylamide gel electrophoresis under reducing or native conditions to determine the nature and number of contaminating proteins. in recent months. we determined that 3 nmol of ganglioside is the optimal concentration per well and upon coating in vacuo. Spain). The major problems encountered are ganglioside differences in the length of fatty acids. after 1 week. We selected 3 nmol as the optimal concentration per well.1. the dose and duration of treatment are not available. patent 6. For example. we found that some of the lots of Falcon 3915 have differed markedly in their background values. 1/1600. plasma. on whether the cell is normal or neoplastic. they also depend on the commercial source (FIG. We use 200-proof ethanol as an excipient. CA) and Instituto Grifols (Barcelona. there is a need to test background noise for each lot before analysis of antiganglioside IgM antibodies. the number of double bonds. Blocking to Eliminate Background Noise Intermolecular spaces and ganglioside-free zones of the polystyrene plate are blocked with a buffer that contains 4% human serum albumin (HSA). Empirically. . and 1/400. IgM. bovine serum albumin. or other xenogenic proteins for blocking or washing.756. Because immunoglobulins. 1/800. however.100 at titer dilution or 4 dilutions below titer dilution. we selected 20% HSA manufactured by Instituto Grifols (stored at room temperature).

Depending on the nature of the disease. up to 12 dilutions). Although we do not use lyophilized serum. others have used it to obtain accurate measurements of antiganglioside titers (personal communication. Taking Precautions at the Final Steps Precautions at the final steps involve conditions to optimize the oxidation of substrates on the solid matrix and the use of a dual wavelength to correct for noise from the solid matrix.1% HSA (Grifols) in PBS (pH 7. If Tween-20 is stored for a long time. we determined that this step is critical to recover IgM precipitated in frozen sera. Diluted sera (100 µL) are overlaid onto antigen-coated (or noncoated) microtiter plates either vertically (from rows A to H. i. 1/1600.e. Sera can be aliquoted for analyses soon after vortexing. GD3.4) with 0.3 We studied varying concentrations of Tween-20 to optimize the concentration required to minimize background noise (optical density below 0.4).1% of Tween-20 (not .800 using detergent-free blocking buffer with 4% HSA in PBS. Wells overlaid with serum antibody are washed with a freshly prepared buffer comprising 0. The incubation time for maximal resolution is 1 h. Yuki). CA).4] at 37°C for 30 min. it becomes ineffective for lowering background noise. either manually or by an automated washer (BioRad Model 1575 Immunowasher.RAVINDRANATH et al. The lyophilized sera are reconstituted using blocking buffer that contains 4% HSA (Grifols). the serum can be diluted up to 1/204. Cryopreserved sera should be extensively vortexed immediately upon thawing to resuspend IgM cryoglobulins. 1/400. before further dilution and addition to microtiter wells. N. The volume of blocking buffer will be the same as the volume of the original serum used for lyophilizing. Most IgM antibodies against major gangliosides (GM2. 1/800. the plates are incubated at 37°C for 2 h. up to 8 dilutions) or horizontally (from rows 1 to 12. Empirically.. GD1a and GD1b) are not affected by 5 freeze-thaw cycles (unpublished data). The secondary antibody that is conjugated to serum IgM is a peroxidase-conjugated rabbit antihuman Fc5µ IgM suspended in 4% HSA (Grifols) in PBS (pH 7.100). i. Wells overlaid with serum antibody were washed with a buffer containing 0.: HUMAN ANTIGANGLIOSIDE AUTOANTIBODIES 237 particularly IgM cryoglobulins. We do not store Tween-20 longer than 1 month after opening. Serum is further diluted to 1/200. 1/6400. For maximal resolution.4).800.1% of Tween-20. and 1/12. Eliminating Nonspecific Binding of Antibodies We previously published a table to show the nature and concentration of detergents that have been used to eliminate nonspecific binding of serum antibodies. measurements based on fluid from the top of the tube will underestimate true antibody titers. 1/3200.1% HSA (Grifols) in PBS (pH 7. Wells are washed manually with a multichannel pipette that is also used to remove the sera. precipitate during thawing of frozen fluid specimens. BioRad. This antibody is diluted 1/5000 in 4% HSA (Grifols) in PBS (pH 7. The most critical step required after reconstitution is incubation of aliquoted sera diluted 1/100 [with 4% HSA (Grifols) in phosphate-buffered saline (PBS) pH 7. The aliquoted sera can be refrozen or lyophilized using an accelerated freeze-drying procedure. Hercules. The washing step is repeated five times..e.4) containing 0.

ELISA failed. ELISA is repeated on different days with the same positive and negative control sera. When control values peaked. It is not correct to measure absorbency at A490 without adding 6 N H2SO4 or at A400 after adding 6 N H2SO4. After exactly 45 min of incubation in the dark. .00) with 21 µL of 30% hydrogen peroxide (Sigma. Sigma) in citrate-phosphate buffer (pH 5. it is A405. used within 1 month after opening the bottle)] is added to wells. and wash buffer is manually added to wells (using 150 µL/well). During validation studies. cryopreserved specimens are thawed. age is important because the titers of most antiganglioside antibodies are low in younger patients. For a valid assay. The absorption maximum after adding 6 N H2SO4 is A492. FIGURE 2. Negative controls are obtained by pooling sera from adult males older than 50 years. Serum is removed from wells with a multichannel pipette. Establishing Positive and Negative Controls Positive or reference controls for antiganglioside IgM vary with the type of disease or cancer. Absorbency is measured after 10 min in an ELISA microtiter plate reader at dual wavelength [A490 − A650] to correct for anomalies in the background and titer plates. The figure illustrates anti-GM2 IgM values in positive control sera pooled from different specimens of a patient with AJCC stage III melanoma (top) and negative control sera pooled from healthy volunteers older than 50 years (bottom). Consistency of antiganglioside ELISA. enzymatic oxidation of substrate is arrested by 120 µL of 6 N H2SO4. the coefficient of variation for positive and negative controls must be less than 15%.14 Again. and vortexed to obtain 10 to 15 mL of sera. Then. 100 µL of substrate [50 mL of ortho-phenylenediamine dihydrochloride (2 × 25 mg tablets. which is further split into 10-µL aliquots in 500-µL Eppendorf (color) vials. Serum aliquots are obtained on different days from 1 or more patients who have high antibody titers. about 10 to 15 mL of pooled sera is divided into 10-µL aliquots in 500-µL Eppendorf (colorless) vials. without 6 N H2SO4. The BioRad washer removes the buffer and automatically adds and removes the wash buffer five times.238 ANNALS NEW YORK ACADEMY OF SCIENCES stored for more than 1 month). pooled.

Type “) +” and then highlight A2 (highest dilution factor). type “/” and highlight B3 (slope).1 is calculated by the following formula: (0.RAVINDRANATH et al. highlight cells A1 and A2 (X values). and close parenthesis. FIGURE 2 shows the values obtained by a single investigator (SM) who repeated the ELISA daily for 50 days. paste the data (Ctrl + V).082. To find the titer at 0. Highlight the B4 cell and right click. in the chart below. and there should always be a comma between the X and Y values: Cell column 1 2 3 4 X 1600 3200 Slope Titer Y 0.082 −0. insert a comma. Ravindranath. “= (A1 − A2)”].1710 0. H.1 − lowest absorbency value)/slope) + highest dilution factor (which corresponds to the lowest absorbency value). the absorbency values of the microtiter plates can be calculated by the following procedure (developed by D.000055625 • The titer at 0. The slope should always be negative.000055625 2876.1 absorbency (A490nm − A650nm).082 −0. cell column 1 has a dilution (X ) of 1600 and absorbency (Y ) of 0. Round up the titer value by changing the number of decimals to 0.1 − B2)/B3) + A2.171. choose “format cells” and click on “number”. unpublished): • After reading a plate. then change the decimal value to 0: Cell column 1 2 3 4 A 1600 3200 Slope Titer B 0. highlight B2 (lowest absorbency value) and close parenthesis. After opening “Excel” program. type “= ((0. cell column 2 has a dilution of 3200 and an absorbency of 0.1 − “. Click on B4 cell. After clicking B3. only 2 readings are assessed. This procedure is repeated for every plate. type in the cell “= slope (“.g. For example.404494 .. Cell column B and row 3 in Excel (slope value) is used (clicked) to determine slope (change in Y. In the menu. Applying a Formula to Estimate Titers for Several Replicas With Microsoft Excel software.171 0. and highlight cells B1 and B2 (Y values). • Correct experimental values against background noise by using the subtraction command [e. use the “copy window (Ctrl + C)” command in Softmax to copy the data in “Window”. Soh with M.: HUMAN ANTIGANGLIOSIDE AUTOANTIBODIES 239 One vial each of positive and negative control is analyzed simultaneously with each study specimen. change in X ). likewise in the chart: = ((0.

61 4.07 ± 0.18 ± 1.88 ± 0.78 ± 0.022 <.61 4.19 4.181 .18 5.16 ± 0.020 . TABLES 3A and 3B summarize the log titers of different gangliosides obtained from healthy volunteers and patients with benign prostate hyperplasia.65 T3/4 CaP (n = 7) ANOVA Mean ± P SD Median value 5.86 5. and anti-GD1a IgM antibodies in sera from healthy controls and patients with benign prostatic hyperplasia (BPH) or prostate cancer (CaP) Healthy (n = 11) IgM Mean ± SD Median target GM1 GM2 GM3 GD3 GD2 GD1a 4.74 ± 1.61 4.61 6.63 4.99 5.90 ± 0.58 5.91 ± 1. and unconfined prostate cancer (stage T3/T4).638 .00 5.24 5.61 4.28 ± 0.70 4.99 4.74 ± 1.68 ± 0.97 4.21 4.45 ± 0.61 ± 0.16 ± 0.61 4.61 5.61 T1/2 CaP (n = 20) Mean ± SD Median 5.29 4.91 4.09 4.61 5.19 ± 1.61 4.09 GT1b 5. we convert values to natural log titers.48 ± 1. anti-GD2.25 4.23 TABLE 3A.20 ± 1.007 .76 ± 0.27 5.75 ± 1.15 ± 1.61 4.19 5.55 5.30 6.1.240 ANNALS NEW YORK ACADEMY OF SCIENCES • If the copy-and-paste function is used to find slope and titer values.181 GD1b 5.61 4.04 ± 1.61 5.01 4.77 4. the 2 dilution values must be above and below 0.073 .22 5.60 6. but the titer equation must be changed to 0. Further elaboration of the same data with an expanded sample size is shown elsewhere. The same procedure also can be used to find titers at a different absorbency such as 0.70 5.61 5.74 ± 1.99 4.61 ± 0.83 4.00 5. To overcome this problem. organ-confined prostate cancer (stage T1/T2).61 4.46 ± 1.07 5.26 5.19 ± 1.19 4.98 5. Converting Antibody Titers to Natural Logs Comparison of the antiganglioside IgM antibody titers for different gangliosides and in relation to treatment or stages of disease cannot use mean or median values because of the large standard deviation.61 5.55 .43 ± 0.22 4.51 ± 0.17 6.06 ± 0.27 BPH (n = 10) Mean ± SD Median 4.61 4.2.90 ± 1.93 ± 0.58 5.2 and all other dilution and absorbency values must be above and below 0.37 4.04 .70 5. ANOVA assessment of P values showing significant differences in log titers of anti-GD3.61 4.61 4.62 ± 1.65 4.07 ± 1.61 5.2.04 6.

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