Use of immunoglobulin heavy- and light-chain measurements compared with existing techniques as a means of typing monoclonal immunoglobulins

Using automated measurements of kappa and lambda light chains and IgG, IgA, and IgM, we assessed the utility of the kappa/lambda ratio and the heavy chain/light chain ratio in characterizing monoclonal immunoglobulins previously identified by sensitive electrophoresis on agarose gel and typed by immunofixation or immunoelectrophoresis. We examined 348 selected samples, of which 165 contained monoclonal components, finding that 93.4% were detected and 89% correctly typed by this approach. Eight samples were shown to contain monoclonal light chains that were not visible as bands on electrophoresis but demonstrated abnormalities of the kappa/lambda or heavy chain/light chain ratio.     

Light-chain ratios of immunoglobulins G, A, and M determined by enzyme immunoassay

We describe an enzyme-linked immunosorbent assay for determination of light-chain ratios for IgG, IgA, and IgM in serum. A commercial serum with known overall kappa and lambda concentrations was used as standard. To capture the respective immunoglobulins, we used antibodies to gamma, lambda and mu chains coated onto microtiter plates. Peroxidase-conjugated anti-kappa and anti-lambda chain antisera were reacted with light chains on the captured immunoglobulins, and the amount of enzyme bound was monitored with o-phenylenediamine and urea-hydrogen peroxide as substrates. Calculation of absorbance ratios allowed determination of kappa and lambda chain concentrations of individual immunoglobulins in the standard and samples. Within-run and between-run CVs (n = 25) ranged from 5.9% to 13.0% for "high," "normal," and "low" kappa/lambda ratios for IgG, IgA, and IgM. The thoroughness of light-chain detection, expressed as, e.g., (IgA kappa + IgA lambda)/(total IgA), for 150 sera was 91-110%. The detection limit was 1 microgram/L. Reference intervals (mean +/- SD) for kappa/lambda ratios in sera from 100 apparently healthy adults were 2.34 +/- 0.80 for IgG, 1.59 +/- 0.40 for IgA, and 1.86 +/- 0.76 for IgM.     

IgE型多发性骨髓瘤的分型检测

目的探讨用免疫固定电泳法检测IgD和IgE单克隆免疫球蛋白，在筛选罕见IgE型多发性骨髓瘤(MM)中的应用价值。方法通过检测血清kD和IgE单克隆免疫球蛋白，从148例MM病人中，筛出只有轻链而不出现IgGAM重链的25份血清样本，用抗游离轻链的单克隆抗体及抗IgD、抗IgE单克隆抗体再进行免疫固定电泳检测。结果25例MM病人中有1例为IgE-κ单克隆免疫球蛋白阳性，24例呈游离轻链阳性(κ型7例、λ型17例)；在17例λ型游离轻链病人中有3例抗λ轻链(游离和结合)病人血清样本呈双克隆带状，用抗IgD单克隆抗体检测证实，有IgD．λ单克隆免疫球蛋白并伴有游离轻链λ。结论采用免疫固定电泳对MM病人血清进行IgG、IgA、IgM和轻链的检测后，用固定免疫技术对只有轻链而不出现IgGAM重链的样本，进行抗游离轻链、抗IgD和抗IgE单克隆抗体分型检测，成功地筛出1例罕见的IgE-κ型MM，提高了对该病的检测水平。     

Light chain ratios of serum immunoglobulins in disease

We have determined the individual kappa (kappa)/lambda (lambda) ratios of serum IgG, IgA, and IgM in normal subjects and patients with rheumatoid arthritis, systemic lupus erythematosus, hepatic cirrhosis and IgA nephropathy--40 in each group. Serum samples were first screened by agarose electrophoresis to exclude paraproteinaemia. Concentrations of IgG, IgA, and IgM were determined by enzyme-linked immunosorbent assay (ELISA). The kappa and lambda chain concentrations of each immunoglobulin class were assayed by an ELISA method first developed by us for the determination of kappa/lambda ratios. Our results showed that kappa/lambda ratios of serum IgA and IgM were significantly different from that of IgG in normal subjects and the 4 groups of patients studied (p less than 0.01). The kappa/lambda ratios of individual immunoglobulins in patients with rheumatoid arthritis, systemic lupus erythematosus and liver cirrhosis were similar to those of normal subjects. However, patients with IgA nephropathy displayed a distinctly lower IgA kappa/lambda ratio, suggesting a unique antibody response in the immunopathogenesis of this disease.     

Pediatric and perinatal reference intervals for immunoglobulin light chains kappa and lambda.

In routine analysis for immunoglobulin light chains in pediatric diagnostics, the age-related reference intervals for serum kappa (kappa) and lambda (lambda) light chains were evaluated in 1543 healthy subjects (newborns to age 16 years, including 168 premature infants). Light-chain analysis was performed by rate nephelometry. IgG, IgA, and IgM were measured simultaneously, and heavy- and light-chain differences were calculated for control purposes. Results for IgG, IgA, and IgM generally agreed with reference intervals reported in the literature. kappa showed age-related changes comparable with changes in IgG concentrations, whereas lambda showed moderate fluctuations. The kappa/lambda ratio showed an almost linear increase with age, starting with 0.97 at four months and reaching the highest value of 2.21 at 15 years (mean values). Preterm infants presented with markedly low serum concentrations of IgG and corresponding light chains but with adult-type kappa/lambda ratios because of the maternal-origin IgG.     

Infectious mononucleosis: immunoglobulin synthesis by cell lines

Immunoglobulin synthesis by 16 long-term suspension cultures of mononuclear cells derived from peripheral blood of nine patients with heterophile-positive infectious mononucleosis (IM) has been demonstrated by radioimmunoelectrophoretic techniques. All cell lines synthesized molecules with IgG (gamma) heavy chain specificity. 14 cell lines produced molecules with IgM (mu) heavy chain specificity and 11 cell lines produced molecules with IgA (alpha) heavy chain specificity. No detectable synthesis of molecules with IgD (delta) heavy chain specificity was observed by these cell lines derived from peripheral blood of patients with IM. 13 cell lines produced molecules with type K (kappa) light chain specificity and 6 cell lines produced molecules with type L (lambda) light chain specificity. Of interest, 9 of 16 lines produced IgG (gamma), IgA (alpha), and IgM (mu) heavy chain molecules and 5 of these cell lines produced molecules with type K (kappa) and type L (lambda) light chain specificity as well.Further characterization by combined polyacrylamide gel filtration, immunodiffusion, and radioautography indicated the presence of newly synthesized immunoglobulin molecules with both heavy and light polypeptide chains in close association as well as free light polypeptide chain synthesis. Investigation of the localization of immunoglobulin in single cells by immunofluorescent techniques revealed that 5-22% of cells in these lines were strongly reactive with a fluorescein isothiocyanate-conjugated rabbit antisera directed against the antigenic determinants of human IgG and cross-reactive with the determinants common to IgA and IgM. No heterophile antibody, heteroagglutinin, or hemolytic antibody could be demonstrated in these cell lines derived from peripheral blood of patients with heterophile-positive infectious mononucleosis.     

Macro lactate dehydrogenase in a patient with myocarditis.

Elevated level of plasma lactate dehydrogenase activity in a patient with myocarditis was found to be due to the presence of lactate dehydrogenase-immunoglobulin complexes in circulation. The complexes were demonstrated by counter-immunoelectrophoresis. In this case, three immunoglobulins (IgG, IgA and IgM) and two types of light chains (lambda and kappa) combined with lactate dehydrogenase.     

Oligoclonal banding in serum from heart-transplant recipients

Sera from heart-transplant patients on immunosuppressive drugs frequently exhibit pronounced oligoclonal banding patterns. There is a strong association between the appearance of these immunoglobulins and infection with cytomegalovirus (CMV) in these patients. Fainter bands were seen both in patients with CMV infection and with various other infections. We saw no oligoclonal banding in electrophoretograms of sera of heart-transplant patients free of known infection.     

Double light-chain disease: a case report.

A patient with massive proteinuria was discovered to have double light-chain disease. Immunological studies demonstrated monoclonal light chains of both the lambda and kappa type in urine. The light chains were separate and distinct and were not found to be a part of any of the whole molecule immunoglobulins such as IgG, IgM, IgA, IgD, or IgE. Uniqueness of the proteins was confirmed by column chromatography. Clinical studies showed that the patient had multiple myeloma.     

Oligoclonal immunoglobulins in the sera of healthy subjects at risk for AIDS

Analysis of serum samples from 68 healthy subjects seropositive for HTLV-III antibodies was performed by high-resolution zone electrophoresis. A high incidence of oligoclonal immunoglobulin bands was detected in the sera of these subjects compared with absence of these bands in the sera of healthy seronegative controls. Identification of the immunoglobulin bands by immunofixation revealed a single heavy chain IgG, single kappa and mixed kappa and lambda light chains. The presence or absence of oligoclonal immunoglobulin bands, determined by zone electrophoresis in serum samples from healthy subjects who have been exposed to HTLV-III, may prove to be a useful biochemical marker for following the course of infection and for determining a prognosis.     

Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine

BACKGROUND: Bence Jones proteins or monoclonal immunoglobulin kappa and lambda free light chains (FLCs) are important markers for identifying and monitoring many patients with B-cell tumors. Automated immunoassays that measure FLCs in urine and serum have considerable clinical potential. METHODS: Sheep antibodies, specific for FLCs, were prepared by immunization with pure kappa and lambda molecules and then adsorbed extensively against whole immunoglobulins. The antibodies were conjugated onto latex particles and used to assay kappa and lambda FLCs on the Beckman IMMAGE protein analyzer. RESULTS: The unconjugated antibodies showed minimal cross-reactivity with intact immunoglobulins or other proteins. With latex-conjugated antibodies, kappa and lambda FLCs could be measured in normal sera and most normal urine samples. Patients with multiple myeloma had increased concentrations of the relevant serum FLC, whereas both FLCs were increased in the sera of patients with systemic lupus erythematosus. CONCLUSIONS: We developed sensitive, automated immunoassays for kappa and lambda FLC measurements in serum and urine that should facilitate the assessment of patients with light chain abnormalities.     

Peripheral T lymphocytes from rheumatoid arthritis patients recognize determinants on light chains and/or Fab fragments of immunoglobulin G.

Peripheral blood T lymphocytes from 29 of 31 patients with rheumatoid arthritis incorporated significant quantities of thymidine when cultured with pooled human immunoglobulin G (IgG). In contrast to the observation of general reactivity to pooled Igg, responses to pooled IgM were rare (3 of 26 patients). None of 11 controls responded to either IgG or IgM. Response to IgG is maximal on day 6 of culture and is dependent on concentration of IgG. The responding cells recognize determinants on monoclonal light chains and/or Fab fragments. Response to light chains follows one of three patterns: preferential response to lambda chains, preferential response to kappa chains, and essentially equal response to either kappa of lambda chains.     

The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies

Cryptococcus neoformans is a ubiquitous fungus that can cause serious infections in humans. The fungus has a polysaccharide (C. neoformans capsular polysaccharide; CNPS) capsule that contributes to its pathogenicity and can elicit an antibody response. Nevertheless, only 4 of 60 BALB/c mice chronically infected with C. neoformans had a detectable increase in serum anti-CNPS. The sera of three responder mice contained both IgM and IgG anti-CNPS antibody, and the titers of lambda and kappa anti-CNPS antibody were approximately equal. Eight IgM and one IgG3 monoclonal antibodies (mAbs) were generated from the spleen of one responder mouse, and one IgA was generated from the spleen of another mouse. Seven of the IgMs, the IgG3, and the IgA mAb had lambda light chains and were specific for serotype D CNPS. Molecular analysis confirmed that this was a highly restricted antibody response. All of the D-specific antibodies used VH441, JH3, and either V lambda 2/J lambda 2 or V lambda 1/J lambda 1, and all had the same heavy chain CDR3 amino acid sequence, even though there were differences in the nucleotide sequence of the N/D segment. One IgM mAb reacted with both serotype A and D CNPS, and this mAb used different VH and JH genetic elements and had kappa light chains. All the anti-CNPS mAbs used J proximal VH gene elements that have previously been shown to bind dextran and other polysaccharides. Sequence and Southern blot analysis indicate that the serotype-D CNPS-specific mAbs arose from only a few precursor B cells.     

Isoenzyme specificities of immunoglobulins isolated from lactate dehydrogenase-immunoglobulin complexes

Lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27, LD) specific immunoglobulins were isolated from LD-immunoglobulin complexes in sera from 11 patients and the isoenzyme specificity of the immunoglobulins was studied by sandwich electroimmunofixation (SEIF). The immunoglobulins differed in isoenzyme specificity, depending on the heavy and/or light chains. IgA kappa recombined with LD2 and LD3, and was thought to be the antibody to common structures (H2M) in LD2 (H3M) and LD3 (H2M2). IgA kappa in a IgA/G kappa lambda case recombined with LD3-5, so that the IgA kappa might be an antibody to M2 dimer in the isoenzymes. IgA lambda recombined only with LD3, and was thought to recognize the H2M2 structure of LD3. As IgG kappa and/or lambda recombined with all LD isoenzymes of LD1-5, the immunoglobulins might react with common structures in H and M subunits. IgG lambda recombined with LD2-5, particularly with LD5, so that the IgG lambda was thought to be an antibody to the M subunit in LD2-5. From the above findings and the tetrameric structure of the LD molecule, 18 types of antibodies to different antigenic determinants on LD molecules can be theoretically derived. Each of the present 11 cases of immunoglobulin corresponded to one of the 18 types, respectively. A formula which can theoretically determine the percent LD activity recombined with immunoglobulins is described.     

Strategy to diagnose monoclonal gammopathies in serum: high-resolution electrophoresis, immunofixation, and kappa/lambda quantification

Identification of monoclonal gammopathies in serum has involved electrophoresis of serum proteins, immunoelectrophoresis (IEP), and quantification of IgG, IgA, and IgM. Recent innovations in technology--including high-resolution electrophoresis (HRE), immunofixation (IFX), and quantification of kappa- and lambda-containing immunoglobulins--allow for more rapid and precise assessment of serum for monoclonal proteins. We present a series of guidelines to determine when high-resolution electrophoresis and quantification of immunoglobulins (including kappa and lambda) are sufficient and when additional IFX is required to characterize the monoclonal gammopathy. Of the samples studied, 88% were correctly diagnosed by HRE with quantification of immunoglobulins and kappa/lambda; only 12% required that IFX be performed. The guidelines allow us to detect monoclonal gammopathies quicker and more efficiently by avoiding redundant IEP or IFX testing. For the vast majority of cases, these guidelines allow for a correct diagnosis within one day. After one year of follow-up since completion of the study, no undetected cases of monoclonal gammopathy have eventuated.     

Four monoclonal immunoglobulins in a patient with chronic lymphocytic leukemia

We report here the case of a 73-year-old woman with chronic lymphocytic leukemia. Two years after the diagnosis, electrophoresis of her serum showed two monoclonal fractions, but our modified immunofixation procedure revealed four monoclonal immunoglobulins in five fractions: two IgG lambda fractions, one IgG kappa fraction, one IgA kappa fraction, and one IgG kappa fraction. This is an exceptionally high number of monoclonal immunoglobulins in a single patient. In the course of her disease the patient did not show any clinical, radiological, or laboratory signs of multiple myeloma or macroglobulinemia. The different monoclonal immunoglobulins were considered to be one expression of her B-lymphocytic malignancy.     

Reference values for immunoglobulin kappa and lambda light chains and the kappa/lambda ratio in children's serum.

We analyzed 708 serum samples from healthy children and adolescents by immunonephelometry to obtain reference values for the immunoglobulin kappa (kappa) and lambda (lambda) light chains and for their ratio at a time of life when immunoglobulin synthesis is maturing and continually being stimulated. The lambda chain concentration that is to be maintained throughout the child's life is reached very early, just after 1 year, whereas the concentration of the kappa chains, which increases gradually, reflects the concentration of the immunoglobulins as a whole. These reference values may be useful for studying kappa and lambda chains in illnesses involving the immune system in children.     

免疫固定电泳检查在多发性骨髓瘤中的应用

目的探讨免疫固定电泳检查在多发性骨髓瘤患者血清单克隆免疫球蛋白（M蛋白）中的应用,并对M蛋白的特性进行分型鉴定。方法对39例血清蛋白电泳异常的患者血清进行免疫固定电泳、血清蛋白和免疫球蛋白分析。结果39例多发性骨髓瘤患者中,IgG型20例（51.3%）,其中κ轻链型9例,λ轻链型11例;IgA型11例（28.2%）,其中κ轻链型5例,λ轻链型6例;IgM型2例（5.1%）,均为λ轻链型,单纯轻链型6例（15.4%）,λ轻链型3例,κ轻链型3例。血清总蛋白升高者20例,球蛋白升高者24例。免疫球蛋白和轻链检测表明同型免疫球蛋白和轻链其含量多明显升高并常伴有其他组份的异常。结论应用免疫固定电泳对多发性骨髓瘤患者的M蛋白进行分型鉴定特异性好、灵敏度高,对多发性骨髓瘤的诊断分型和临床分期及预后具有重要意义。     

Selective deposition of immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus.

To further characterize the IgA deposits found in glomeruli of patients with IgA nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus, renal biopsies from patients with these disorders were stained by immunofluorescence with monoclonal anti-IgA subclass reagents, anti-light chain reagents and anti-J chain. The mesangium and peripheral capillary were brightly stained for IgA1 and were negative for IgA2. IgA1 and, to a lesser extent, IgA2 were contained in tubular casts. Both kappa and lambda light chains were found in all deposits. The intensity of J chain staining correlated with the intensity of IgM and not IgA staining. Biopsies brightly stained for IgA but negative for IgM were negative for J chain. These results indicate that glomerular IgA deposits in these disorders consist predominantly of monomers of IgA1.     

The predictive power of serum kappa/lambda ratios for discrimination between monoclonal gammopathy of undetermined significance and multiple myeloma.

The predictive power of serum kappa/lambda ratios on initial presentation of immunoglobulin G (IgG) or IgA monoclonal component was studied to differentiate between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. The retrospective study involved 145 patients clinically diagnosed with monoclonal gammopathy of undetermined significance or multiple myeloma, who had serum M-protein IgG <35 g/L or IgA <20 g/L at M-protein detection. Serum light chains kappa and lambda were measured by fixed-time nephelometry. Test performance indices, predictive values and likelihood ratios were calculated according to the Weissler recommendation. MM patients were considered as diseased and MGUS patients as non-diseased in order to estimate the performance characteristics of serum kappa/lambda ratios. There was a statistically significant difference in kappa/lambda ratios distribution between both groups of patients, in both M-protein kappa-type (Mann-Whitney U=168, p<0.001) and in M-protein lambda-type (Mann-Whitney U=143, p<0.001). Negative likelihood ratios at threshold levels of 0.6 and 4.2 were 2.17- and 3.32-fold greater, respectively, than positive likelihood ratios, so that the predictive power of a serum kappa/lambda ratio within these limits is better in ruling out (negative predictive power) than ruling in disease (positive predictive power). The post-test characteristics of a serum kappa/lambda ratio interval between 0.6 and 4.2 in discriminating MGUS from MM in our geographic population were: sensitivity 0.96 (0.93-0.99 95% CI); specificity 0.70 (0.63-0.77); positive predictive value 0.68 (0.64-0.73); negative predictive value 0.96 (0.94-0.99); likelihood ratios (+)LR 3.23 (2.68-4.04); and (-)LR 17.16 (11.00-63.00). Thus, serum M-protein with a kappa/lambda ratio between 0.6 and 4.2 increases the posterior probability of MGUS from 0.60 to 0.96 in asymptomatic patients, for whom only monitoring may be suggested when the serum kappa/lambda ratio is within these limits.