To Develop A New Method To Measure Immunoglobulin Free Light Chain κ And λ In The Serum And Urine

Objective:To develop a new detection method to measurement of immunoglobulin free light chain κ and X in the samples of serum and urine.Methods:The anti-human immunoglobulin free light chain κ and λ monoclonal antibody was cultivated in vitro and purified. Then monoclonal antibody was covalently attached to the beads and its connectivity was determined simultaneously. The biotin was connected to immunoglobulin free light chain κ and X and the binding effect of biotin on the light chain was determined. The micro beads containing anti-free light chain κ and anti-free light chain λ were mixed and then the free light chain κ and X with biotin were added along with the samples under test. The principle of competition was used in the test. After a certain period of reaction, the readings of the content of free light chain κ and X in the samples were obtained in the Luminex200instrument. The specificity, sensitivity, reproducibility and stability of the method of measuring immunoglobulin free light chain were assessed. The levels of the free light chain κ and X in200normal human serum and600myeloma patients’serum were measured and compared with turbidimetry.Results:When the monoclonal antibody was connected to the beads, the anti-mouse IgG from goat coupled with the R-phycoerythrin (R-PE) was able to be combined with antibody specificity on the beads. When it was detected on the Luminex200, the mean fluorescence intensity on the beads connected with the anti-free light chain κ and λ antibody was significantly higher than those not connected to the antibody. We conjugated the biotin to the immunoglobulin free light chain κ and λ, took the beads connected with antibody of anti-free light chain κ and λ, and let it to react with biotin free light chain-specificity. Then it would react with Streptavidin R-avidin phycoerythrin conjugate (SAPE). When it was detected in the Luminex200, the free light chain κ and λ conjugated with biotin have higher mean fluorescence intensity.The anti-free light chain κ antibody was attached to a coded micro bead and the anti-free light chain λ antibody was connected to another coded bead. The two beads connected to antibody were mixed and a small amount of test sample was added. The sample can be used to measure the levels of immunoglobulin free light chain κ and λ. Micro beads containing antibodies react with free light chain κ and λ and with the bound light chain κ and λ. The result is the micro beads having anti-free light chain k antibody reacted with biotin free light chain κ specificity. But the biotin free light chains X and biotin bound light chain κ and λ will have no cross-reactivity. The micro beads with anti-free light chain λ antibody can only specifically react with biotin free light chains λ but not cross-react with biotin free light chain κ and biotin bound light chain k and X.When the concentrations of free light chain k are3.75,32.24and104mg/L, the coefficients of variation under determination CV are2.2%,3.4%,4.2%, respectively and the inter-assay coefficients of variation CV are9.7%,10.4%,12.4%, respectively. When concentration of free light chain X are2.70,34.51and107.79mg/L, the coefficients of variation under determination CV are3.2%,4.4%,5.2%, respectively and the inter-assay coefficients of variation CV are9.8%,12.7%,14%respectively. The minimum detectable concentration of the free light chain κ and λ was0.4mg/L. The micro beads connected with antibody were stored in the refrigerator at4℃and its valid period is six months. If we put the monoclonal antibody stored in the refrigerator at-20℃, when we connected the antibody to the corresponding micro beads again12months later, the antibody is still functioning. Mix the micro beads containing the anti-free light chain k and λ antibodies and let them react with the samples to simultaneously test the concentrations of free light chain k and X in the samples. The tests between these two kinds of beads do not interfere with each other.We tested the free light chain κ and λ in the serum of200normal people and600myeloma patients with the mixed beads method and tested the same samples with the turbidimetric method. Comparing these two methods we can get the free light chain κ and λ values. We found that there is a blank range in the low zone using the turbidimetric method because this method requires dilution of samples resulting an area of values that cannot be measured but there is no such a problem using the mixed-beads method. The turbidimetric method cannot determine the free light chain κ and λ values simultaneously, while the mixed two beads method can do. Therefore, it can reduce the sample testing, improve the detection efficiency, and reduce testing costs. One of the most important features in the mixed beads method is that, according to our needs, we can add more indicators to be measured into the beads, such as albumin, globulin determination etc., and use a small amount of sample to obtain all results of all available indicators to be determined in a single test.Conclusion:The mixed beads method to determine the levels of immunoglobulin free light chain κ and λ is a simple, quick, and very sensitive method with high specificity and reproducibility and accuracy. This method will have a very good development prospects to be applied to clinical diagnosis of disease.