A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Duck immune responses to Riemerella anatipestifer vaccines

Riemerella anatipestifer (Ra) infection is probably the most economically important infectious disease of farm ducks worldwide but the immune responses to natural infection and vaccines are poorly understood. We have used the lymphocyte transformation test (LTT) to study the expression of cell-mediated immunity (CMI), and the enzyme-linked immunosorbent assay to monitor antibody (Ab) production following administration of formalin-inactivated and live attenuated serotype 2 (= G) Ra vaccines. Lymphocytes (8x10(5) in 200 microl of RPMI + 10% duck serum, in 96 well trays) were stimulated with Ra antigen, prepared by freeze-thaw and sonication; optimum responses were obtained with antigen at 6.25 microg/ml. Cells were cultured for 3 days at 41.6 degrees C/5% CO(2), prior to assessing 3H-thymidine uptake. Ra bacterin, incorporating aluminium hydroxide as adjuvant, stimulated strong but transient (about 4 weeks) LTT response; there was some cross-reaction of the LTT to proteins derived from other serotypes of Ra. Revaccination stimulated slightly stronger responses with the same time course. The Ab response to each vaccination was longer-lived than the LTT response. Vaccination with a live, attenuated strain of Ra stimulated weaker but longer lasting LTT responses, but similar Ab responses compared to the bacterin. It is apparent, therefore, that the transient protection reported using Ra bacterins is due to the fact that the CMI response to these vaccines is transient; and that it is possible for ducks to have detectable levels of serum Ab at times when CMI is not detectable by LTT. These observations are important in terms of our understanding of immunopathogenesis, immunoprophylaxis, and immunodiagnosis in Ra.     

Bacteria-Immune System Interactions. VIII. Modulation of Human Cytotoxic Reactions by Crude Lipoteichoic Acid Extracts of Bacillus Globigii

Crude lipoteichoic acid (LTA)¹ extracts were prepared from Bacillus globigii (Bg-LTA). The addition of Bg-LTA to mixed lymphocyte reactions (MLR) produced a dose dependent inhibition of specific cytotoxic reactions and of tritiated thymidine (³H-TdR) incorporation. Much less Bg-LTA was needed to inhibit the initiation (addition at day 0) than the effector phase (addition at day 6) of the cytotoxic reactions. None of the LTA quantities used had a toxic effect on the lymphocytes. LTA pretreated stimulator cells also had a modulatory effect on the cytotoxic reactions. Stimulators pretreated with very low or very high LTA concentrations had no effect; however, pretreatment with intermediate concentrations induced a drastic inhibition of cytotoxicity. LTA pretreated stimulator cells did not significantly affect ³H-TdR Incorporation. Suppression of the cytotoxic reaction could also be obtained by LTA-pretreatment of the effector cells.     

Antibody and Cell-Mediated Target Cell Lysis of Technetium-99m Labeled Erythrocytes

An assay for cell-mediated lysis of sheep erythrocytes has been developed using technetium-99m (^99mTc) as a radioisotopic label. Spleen cells obtained from sensitized BALB/c mice in combination with either non-immune or immune mouse anti-sheep RBC serum produced maximum release (100%) of ^99mTc. Non-immune spleen cells in combination with immune serum produced significantly greater release of ^99mTc compared to that observed with non-immune cells in the presence of non-immune serum. The kinetics of antibody-mediated complement dependent release of ^99mTc as a function of time and temperature paralleled the release of ⁵¹Cr and hemoglobin as an indicator of erythrocyte membrane injury. Because of its high specific activity, low spontaneous release, and ready availability in those institutions where diagnostic scanning is performed, ^99mTc appears to be particularly well suited for the assessment of both antibody and cell-mediated lysis of erythrocytes.     

In vitro detection and characterization of drug hypersensitivity using flow cytometry

Background:  The lymphocyte transformation test (LTT) is the only in vitro test for detecting drug sensitization at the cellular level irrespective of the reaction's phenotype. However, the LTT includes working with radioactive substances and is considered impracticable for routine laboratory investigation.
Objective:  The aim of this study was to assess drug-specific cytokine production by means of flow cytometry as an alternative nonradioactive approach which may be more appropriate for routine testing and may provide in addition more information about the pathophysiology of the reaction than proliferation-based assays, like the LTT.
Method:  Peripheral blood mononuclear cells of 19 patients were incubated with culprit drugs ( n  = 28) or irrelevant antigens ( n  = 10). Ten healthy persons served as controls for all different drugs ( n  = 15). Intracellular interleukin (IL)-5, interferon (IFN)-γ and IL-10 production was investigated using flow cytometry. Accuracy of the flow cytometry test system was confirmed using different statistical tests, i.e. receiver operating characteristic curve and Mann–Whitney rank test. In addition, drug-specific secretion of IL-5, IL-2 and IFN-γ were analysed using enzyme-linked immunosorbent assay (ELISA).
Results:  Drug-specific cytokine production could be demonstrated in 75% of the patients using flow cytometry and in 79% using ELISA respectively. Combining ELISA and flow cytometry increased the sensitivity to 100%. Analysis of involved T-cell subsets [e.g. CD4⁺ or CD8⁺; T helper (TH) 1 or TH 2] allowed characterization of the in vitro lymphocyte reactivity pattern.
Conclusions:  Analysis of drug-specific cytokine production by means of flow cytometry proved a useful and reliable approach for the in vitro detection and characterization of drug hypersensitivities.     

Immunostimulation-Inhibition of Tumor Cell Growth in Vitro Utilizing Tumor Target Cells Labeled with 125I-Iodo-Deoxyuridine

Immune stimulation-inhibition of normal and sensitized syngeneic, allogeneic and xenogeneic lymphocytes with the B16 melanoma was tested by an in vitro assay. Various numbers of lymphocytes were mixed with non-labeled or with ¹²⁵IUDR-labeled B₁₆ cells incubated for 2 hours on a rotating platform and plated into culture dishes. One, 2 or 3 days later the dishes were fixed and viable tumor cells counted either by microscopy or by radioactive monitoring. Sensitized but not normal lymphocytes at ratios up to 1:1000 repeatedly and significantly enhanced the plating efficiency and growth of the target cells. At higher lymphocyte doses, colony inhibition was evident. Numbers of viable target cells calculated from radioactive monitoring agreed closely with visual counts of the cultures. It appears that the technique utilizing ¹²⁵IUDR-labeled cells affords a relatively easy, fast and accurate assay for in vitro studies of immune stimulation-inhibition of target growth.     

ELISpot: a new tool for the detection of nickel sensitization

BACKGROUND: The standard assay for the clinical detection of contact allergy against nickel is the patch test. For research purposes, the in vitro lymphocyte transformation test (LTT) is mostly applied. However, the in vivo application of allergens includes the potential of sensitization, whereas the LTT demands 6 days of cell culture and radioactive labelling procedures. OBJECTIVE: The purpose of this study was to validate an enzyme-linked immunospot (ELISpot) assay to specifically detect cellular response against nickel sulphate in correlation with patch test, anamnestic data, and LTT results. METHODS: In probands with positive (n=60) and negative patch test results (n=19), cytokine ELISpot and LTT were performed in parallel using various concentrations of nickel sulphate, various numbers of responding peripheral blood mononuclear cells (PBMC), and various durations of cell cultures. RESULTS: Concentrations of 50 and 75 microM nickel sulphate were found to be optimal to stimulate in the ELISpot 4x10(5) and in the LTT 2x10(5) PBMC, respectively. An increase in ELISpot sensitivity was reached by pre-incubation with nickel sulphate for 24 h prior to transfer to ELISpot plates. In nickel-sensitive probands, an average precursor cell frequency of 19x10(5), 1.7x10(5), and 0.7x10(5) could be defined for IFN-gamma, IL-2, and IL-4 producing PBMC, respectively. In non-sensitive probands IFN-gamma producing cells were detectable, but with significantly lower frequency (2x10(5); P=0.004). The prior performance of patch tests had no significant effect on cytokine production or lymphocyte proliferation. Overall, the parameters patch test, anamnestic nickel allergy, ELISpot, and LTT results were positively correlated (P<0.05). CONCLUSIONS: Based on these experiments, the quantitative in vitro detection of cell-mediated reactions towards nickel as well as other heavy metal ions should be possible utilizing the above-described ELISpot assay.     

d-Penicillamine in Vivo Enhances Lymphocyte DN A Synthesis: Role of Macrophages

Administration of d -penicillamine (50 mg/kg/day orally) for 4 days increased the uptake of ³H-thymidine (³H-TdR) in unstimulated and concanavalin -A-stimulated unseparated lymph node and spleen cells from Lewis rats. Increased ³H-TdR incorporation was also found in cultures depleted of adherent cells. d -Penicillamine treatment did not increase the incorporation of ³H-TdR in lymph node and spleen cells from rats concomitantly treated with the selective macro-phage toxin silica. in contrast, treatment with d -penicillamine during the last 4 days of silica treatment sometimes resulted in a marked decrease in ³H-TdR incorporation. It is suggested that d -penicillamine treatment in vivo is able to enhance the responsiveness of the lymphocytes, dependent on the presence of functionally intact macrophages. The increased response vanished after 2-3 weeks, even with continuous administration of d -penicillamine.     

冷冻预处理温度对胎兔许旺细胞活性的影响

背景：许旺细胞作为周围神经损伤修复的重要桥接材料,其活性对临床修复周围神经损伤成功与否具有重要影响。 目的：观察不同维持温度冷冻预处理超深低温保存对胎兔许旺细胞活性的影响。 方法：设计胎兔许旺细胞冷冻维持温度为-30~-70 ℃,每间隔-5 ℃为一组,以10%二甲基亚砜为低温保护剂,对胎兔许旺细胞进行上述温度冷冻预处理,液氮中保存48 h,快速复温后继续培养48 h,透射电镜观察细胞超微结构改变,MTT法和³H-TdR掺入法检测细胞活性。 结果与结论：透射电镜下见-45 ℃处理的细胞超微结构良好,其余各温度组细胞均出现细胞器肿胀、坏死等轻重不同的冷冻损伤表现。MTT法和³H-TdR检测结果均显示-45 ℃冷冻预处理对许旺细胞的生长抑制率最低,分别为(3.83±0.56)%、(4.41±0.71)%,和其余各温度组之间相比,差异有显著性意义(P < 0.05)。提示-45 ℃是胎兔许旺细胞冷冻预处理的最佳维持温度值,对细胞活性影响最小,可较好保持其生物学活性。     

Comparison of protein and DNA synthesis assays of guinea pig spleen lymphocytes after stimulation with influenza virus antigen and phytohemagglutinin

Two in vitro-methods for demonstration of cell-mediated immune response are compared: Protein and DNA synthesis for detection of in vitro influenza virus antigen- and mitogen-induced lymphocyte stimulation. Guinea pig spleen lymphocytes sensitized with influenza virus antigen were tested in a microadaptation of the lymphocyte transformation test using 14C- or 3H-leucine and 3H-thymidine. As a positive control for T-cell stimulation PHA-induced lymphocyte stimulation was measured. The following results were obtained: 1. Kinetics of the incorporation of 14C-leucine and 3H-thymidine in lymphocytes incubated with optimal and suboptimal PHA-doses respectively are quantitatively similar but different in time. 2. The results of the protein- and DNA-synthesis stimulation assays were correlated against influenza virus antigens, this could not be described by the comparison of cellular and single humoral parameters. 3. The administration of influenza virus antigens in CFA induced a more intensive cell-mediated reaction than injections of antigens in aqueous suspensions, but the results of both methods of CMI were correlated. 4. The optimal CMI under the experimental conditions described is induced by an administration of 30 to 50 microgram virus protein per animal and by a combined intramuscular--intraperitoneal immunization procedure. 5. The measurement of the early stimulation of protein synthesis in the PSS-test is substantially more rapid than for the classical LTT.     

The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo

Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.     

Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.     

Elastase-induced hydrolysis of synthetic solid substrates: poly(ester-urea-urethane) and poly(ether-urea-urethane)

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) were incubated with two radiolabelled model poly(urethane)s, a poly(ester-urea-urethane) containing [¹⁴C]toluene diisocyanate ([14C]TDI), poly(caprolactone) (PCL) and ethylenediamine (ED), and a poly(ether-urea-urethane) containing [¹⁴C]TDI, poly(tetramethylene oxide) (PTMO) and ED. Ten-fold more radioactive carbon was released when PPE was incubated with [¹⁴C]TDI/PCL/ED than when HNE was used. The PPE-induced radioactive carbon release was significantly reduced by a specific elastase inhibitor. Ten-fold less radioactive carbon was released when [¹⁴ was incubated with PPE as compared to [¹⁴C]TDI/PCL/ED. Since neutrophils, which contain elastolytic activity, are present during the inflammatory response, the stability of biomaterials used in implanted devices may be affected.     

Dye Dilution Proliferation Assay: Application of the DDPA to Identify Tumor-Specific T Cell Precursor Frequencies in Clinical Trials

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8⁺ cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8⁺ lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8⁺ as well as CD4⁺ lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8⁺ and CD4⁺ precursor frequency (PF) data in patients being treated on novel immunotherapy trials.     

供体凋亡淋巴细胞预输注对猪肝移植受体CD4+T、CD8+T、CD4+CD25+调节性T细胞的影响

背景：凋亡细胞能够主动调节机体的免疫功能,并能通过调节机体细胞免疫和体液免疫的途径诱导免疫耐受,但这些结果只在大鼠肝脏移植模型中证实。 目的：探讨通过⁶⁰Co γ射线体外处理后的供体淋巴细胞预输注诱导猪肝移植特异性免疫耐受的作用中,对淋巴细胞亚群的影响。 方法：建立非转流小型猪原位肝移植模型。将受体猪随机摸球法均分为2组：空白对照组,受体猪无特殊处理,行肝移植;淋巴细胞组：受体猪在肝移植前7 d经耳静脉注射⁶⁰Co γ射线处理过的5×10⁸个供体淋巴细胞。观察两组受体猪移植后的存活时间,移植后T淋巴细胞亚型CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr变化及病理。 结果与结论：移植后3 d,两组病理活检均呈急性中、重度排斥反应;移植后6 d,两组均呈急性重度排斥反应。移植后1,3,6 d CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr升降趋势,两组间差异无显著意义(P > 0.05)。提示,⁶⁰Co γ射线体外处理过的淋巴细胞预输注未能够诱导猪同种异体肝移植特异性免疫耐受,未能引起T淋巴细胞亚群变化有关。     

Varicella-zoster virus-specific cell-mediated immunity in subjects with herpes zoster

Though cell-mediated immunity (CMI) against varicella-zoster virus (VZV) is critical for prevention of the onset of herpes zoster (HZ), clinicians currently lack a simplified procedure to monitor CMI. We have recently developed an assay, called the IFN-γ release assay, and showed that it is a simple and reliable method to determine VZV-specific CMI. In the present study, we applied an IR assay to measure the VZV-specific CMI of patients with HZ. VZV-specific CMI levels were significantly high at the onset of the disease, but were decreased several weeks later. In contrast, CMI VZV-specific antibody titers increased in convalescent phase compared to those in acute phase. Thus, this technology is likely to be very useful in monitoring ongoing VZV-specific immune status.     

An enzyme-linked double antibody immunoassay to measure murine immunoglobulins--its application to determine the specific activity of radiolabeled monoclonal antibodies

A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with radioactive isotopes. A combination of radioactive counting and measurement of the absorbance of a peroxidase catalyzed reaction permits accurate determination of the specific radioactivity of labeled monoclonal antibodies.     

A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [H]thymidine incorporation assay

A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [³H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.A1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [³H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [³H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [³H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [³H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.     

Cell mediated lympholysis; a modified technique using indium-oxine-labelled targets

The isotope ⁵¹Cr generally used in the cell mediated lympholysis (CML) assay suffers from the disadvantage of low specific activity, poor incorporation and high spontaneous release, limiting the CML assay to 4–6 h. We have labelled PHA derived human lymphoblasts with the isotope ¹¹¹indium (using ¹¹¹indium-oxine) and evaluated these cells as targets in CML. The level of ¹¹¹In-oxine incorporation decreased rapidly in the presence of serum; in the absence of serum approximately 85% of the available isotope in the supernatant was incorporated into the blasts. Under the labelling conditions used, spontaneous release was 1.6–2%/h on average allowing an effector phase of 18 h. About 5–8% of the released isotope was reutilized by the effector cells during an 18 h incubation period. Extending the CML assay from 6 to 18 h greatly increased the cytotoxicity. At an effector to target ratio of 25:1, the average per cent specific release increased from 15 to 50%. The use of ¹¹¹In-oxine labelled targets in the CML therefore increases the sensitivity of the test and allows fewer effector and target cells to be used as compared with ⁵¹Cr techniques.     

The Rna Binding Subset of Adenosine Antibodies

Hapten specific antibodies were elicited against adenosine-BSA conjugate (Ado*-BSA) in rats. By radiohapten assay, two major populations of hapten specific antibodies were identified. They were directed against either adenosine trialcohol(A^ox-red) or morpholino-adenosine(Morph-Ade). The association constant (Ka) for the ³H-A^ox-red binding population was determined to be 1.02 × 10⁷ M^-1. Majority of the A^ox-red specific antibodies were crossreactive with Morph-Ade, but they did not cross-react at all with adenosine(A) or deoxyadenosine(dA). ³H-A binding subsets were actually Morph-Ade specific and constitute less than 10% of the hapten specific antibodies. The Ka value for the ³H-A binding was determined to be 4.5 × 10⁶ M^-1. A very minor and highly cross-reactive subset of Morph-Ade specific antibodies participate in RNA binding. Though a larger proportion of Morph-Ade specific antibodies interact with ³H-A, only a fraction of them bind to RNA. The extreme crossreactivity and hence, the highly adaptive nature of the binding sites of the antibodies interacting with RNA, might be a stringent requirement for recognising adenine residues of nucleic acids in solution.