免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Laminin-like immunoreactivity in the snail Helisoma: involvement of approximately 300 kD extracellular matrix protein in promoting outgrowth from identified neurons

Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis. Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, approximately 300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the approximately 300-kD snail protein. Immunofluorescence of snail ganglia with the anti- approximately 300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti- approximately 300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- approximately 300-kD antibody revealed the recognition of the same approximately 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- approximately 300-kD antibodies reveal the recognition of a soluble approximately 300-kD protein similar to the approximately 300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the approximately 300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- approximately 300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the approximately 300-kD ECM protein may be the NOPF in CM and/or functions in promoting neurite outgrowth.     

Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33.     

Laminin-like immunoreactivity in the snail Helisoma: Involvement of a ∼300 kD extracellular matrix protein in promoting outgrowth from identified neurons

Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, ～300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the ～300-kD snail protein. Immunofluorescence of snail ganglia with the anti-～300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti-～300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- ～300-kD antibody revealed the recognition of the same ～ 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- ～300-kD antibodies reveal the recognition of a soluble ～300-kD protein similar to the ～300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the ～300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- ～300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the ～300-kD ECM protein may be the NOPF in CM and /or functions in promoting neurite outgrowth.     

Antibody against extracellular vaccinia virus (EV) protects mice through complement and Fc receptors

Protein-based subunit smallpox vaccines have shown their potential as effective alternatives to live virus vaccines in animal model challenge studies. We vaccinated mice with combinations of three different vaccinia virus (VACV) proteins (A33, B5, L1) and examined how the combined antibody responses to these proteins cooperate to effectively neutralize the extracellular virus (EV) infectious form of VACV. Antibodies against these targets were generated in the presence or absence of CpG adjuvant so that Th1-biased antibody responses could be compared to Th2-biased responses to the proteins with aluminum hydroxide alone, specifically with interest in looking at the ability of anti-B5 and anti-A33 polyclonal antibodies (pAb) to utilize complement-mediated neutralization in vitro. We found that neutralization of EV by anti-A33 or anti-B5 pAb can be enhanced in the presence of complement if Th1-biased antibody (IgG2a) is generated. Mechanistic differences found for complement-mediated neutralization showed that anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice.     

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.     

Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA.

Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti-(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1-U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti-(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP-specific B" antigen (mol. wt. 28 500). Antibodies to the U2 RNP-specific A' protein (mol. wt. 31 000) were found in only one serum. The B" antigen differs slightly in mol. wt. from the U1-U6 RNA-associated B/B' antigens and can be separated from this doublet by two-dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti-B" antibody specificity also reacts with U1 RNPs which is due to cross-reactivity of the antibody with the U1 RNA-specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti-A antibodies contained in anti-(U1)RNP sera, it also shares epitopes with the U2 RNP-specific B" antigen.     

Passive enhancement of mouse skin allografts by alloantibodies is Fc dependent.

The capacity of F(ab')2 fragments of alloantibodies to enhance mouse allografts was studied in B6AF1 recipients of B10.D2 skin grafts. F(ab')2 obtained by digestion of B6AF1 anti-B10.D2 antibodies was purified by means of affinity chromatography, with anti-subclass antisera and protein A. The degree of contaminating IgG was less than 0.02%. Administration of F(ab')2 with an antigen-binding capacity similar to the IgG from which it originated, inhibited acute antibody-mediated graft rejection but was unable to induce enhancement. Even a dose that was 130 times the molar amount of the minimal enhancing dose of undigested IgG2 was ineffective. We conclude, therefore, that passive enhancement of mouse skin allografts by alloantibodies requires the Fc part.     

Direct Antiglobulin Reaction in ABO-Haemolytic Disease of the Newborn

The minimum number of IgG anti-A (or anti-B) molecules detectable on A or B red cells by the antiglobulin reaction was found to be the same—that is, about 150 molecules per red cell—with newborn as with adult cells. Furthermore, the ratio of anti-IgG bound to IgG anti-A (or anti-B) molecules was the same whether the anti-A (or anti-B) molecules were present on newborn or on adult cells and was similar to that found for anti-IgG bound to IgG anti-Rh.In 15 infants (11 group A, 4 group B) with haemolytic disease of the newborn due to ABO-incompatibility the amount of anti-A or anti-B on the red cells ranged from 0·25 to 3·5 μg antibody per ml red cells, corresponding to 90-1,320 antibody molecules per cell; only five infants had more than 0·55 μg antibody per ml of red cells. These amounts are far smaller than those found in most moderate or severe cases of Rh-haemolytic disease.It is concluded that the weak direct antiglobulin reactions observed in ABO-haemolytic disease are due simply to the fact that the number of anti-A (or anti-B) molecules on the infant''s red cells is at the lower limit of sensitivity of the test. Since ABO-haemolytic disease can be quite a severe process it seems probable that IgG anti-A and anti-B molecules are more effective than anti-Rh molecules in bringing about red cell destruction.     

The effect of alloantisera on antigen-induced T cell proliferation.

In the guinea pig, alloantisera raised by cross-immunization of strain 2 and strain 13 animals are capable of specifically inhibiting the in vitro proliferative response of (2 X 13)F1 T lymphocytes to those antigens the response to which is controlled by Ir genes linked to the genes controlling the alloantigens against which the serum is directed. However, in similar studies performed in the two parental strains, the responses to antigens not known to be under unigenic control were also markedly inhibited by the appropriate alloantisera. We have extended our studies of this "nonspecific" inhibitory effect of alloantisera on T cell proliferation and have demonstrated that the proliferative response of strain 2 and strain 13 T cells to a large number of antigens is markedly inhibited by anti-2 and anti-13 sera, respectively. Antisera to the B alloantigen, the product of a linked but distinct histocompatibility locus, which is present in both strain 2 and strain 13 animals, also produced a marked inhibition of T-lymphocyte proliferation. A number of possible explanations for the generalized inhibitory effect of alloantisera on T cell proliferation are discussed.     

Anti-immunoglobulin antibodies. II. Expression of individual and cross-reactive idiotypes on syngeneic and homologous anti-idiotype antibodies

Syngeneic anti-(anti-Id) antibodies were prepared against BALB/c anti-A48Id antibodies, BALB/c anti-460Id monoclonal antibodies, and A/J anti-J558 IdI monoclonal antibodies. With these anti-(anti-Id) antibodies we identified cross-reactive idiotypes on syngeneic and homologous anti-A48Id and anti-460Id antibodies. By contrast, tbe idiotypic determinants of A/J anti-J558 IdI monoclonal antibodies were not shared by other syngeneic, homologous, or xenogenic anti-J558 IdI or IdX antibodies. These results suggest that idiotype-antiidiotype reactions that serve as regulatory controls within the immune system are characteristic for each particular antigen system, strain, or species and that such interactions make the system self-limited with respect to the whole antild repertoire.     

Monoclonal anti-A antibody removal by synthetic A antigen immobilized on specific antibody filters

Removal of blood group anti-A and anti-B antibodies can prevent hyperacute organ rejection in ABO-incompatible transplantation. We are developing an extracorporeal-specific antibody filter (SAF) as an immunoadsorption device for direct removal of ABO blood group antibodies from whole blood, without the need for plasma separation and plasma exchange. A hollow fiber-based small scale SAF (mini-SAF) device was fabricated and synthetic A antigen, Atrisaccharide (Atri) conjugated to activated polyacrylic acid, was immobilized on the fiber lumen surface. Monoclonal antibody anti-A IgM were specifically removed up to 70% of initial antibodies using mini-SAF device. The monoclonal anti-A capture experiments on mini-SAF indicated that antibody removal relative to the initial concentration is independent of inlet concentration in the beginning; however, as the surface starts saturating with bound antibodies, removal becomes dependent on inlet concentration. No significant effect of flow rate on removal rate was observed. The radial diffusion and axial convection-based mathematical model developed for unsteady state antibody removal was in good agreement with the experimental data and showed that the antibody removal rate can be maximized by increasing the antibody-binding capacity of the SAF.     

Immunochemical studies on lactate dehydrogenase A subunit deficiencies.

In lactate dehydrogenase (LDH) A subunit deficiency, is there not only a lack of activity but also a lack of subunit production? We demonstrated three remarkable points to answer this question: There are no proteins that immunologically react with anti-A subunits. There are no heterotetramers that react with anti-B subunits. B subunits seem to be genetically produced at normal level, and all of them form only one isoenzyme, LDH-B4. From these points, we concluded that there is a complete lack of A subunit production or production of incomplete A subunits that can neither react with anti-A subunits nor form heterotetramers.     

Blood groups of apes and monkeys. VII. The human-type A-B-H factors in gelada monkeys (Therapithecus gelada)

A series of 21 gelada monkeys (Theropithecus gelada) all showed strong reactivity of their saliva for H substance, but no reactivity for either A or B. Tests on their sera in no case showed the simultaneous presence of both the agglutinins anti-A and anti-B; instead some animals had only anti-A, others had only anti-B, while the remainder had neither anti-A nor anti-B. These findings distinguish gelada monkeys from all other species of Old World monkeys tested to date. They also provide further evidence supporting the genetic independence of the H substance and the A-B-O blood groups.     

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes

Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I:A-active ganglioside II:The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.     

Immune regulation by monoclonal antibodies: failure to enhance mouse skin allografts

Monoclonal anti-H-2k alloantibodies were analyzed for their capacity to enhance the survival of B10.A skin grafted onto B10.D2 recipients. Included were five anti-class I and four anti-class II antibodies. In contrast to conventional B10.D2 anti-B10.A serum, none of the individual anti-class I or anti-class II monoclonal antibodies induced enhancement. The same negative results were obtained with various mixtures of anti-class I, anti-class II, or anti-class I + anti-class II antibodies. The failure to induce enhancement was not due to inefficient antigen binding in vivo, because monoclonal antibodies were as effective as conventional B10.D2 anti-B10.A serum in the induction of acute antibody-mediated graft rejection, and in the opsonization of 51Cr-labeled B10.A leukocytes injected into B10.D2 recipients pretreated with antibody. These results demonstrate that monoclonal antibodies cannot always substitute for conventional sera, at least not in immune regulation. They also show that although opsonization may be a prerequisite for the induction of enhancement, it does not guarantee that enhancement will invariably occur.     

The Cis AB complex of the ABO system]

Members of six unrelated families from Japan, France, Belgium and Poland were studied in parallel. Major immunological features characteristic of the phenotype produced by the Cis AB complex are the following: 1) The red cell A reactivity is close to normal, is beyond the values of agglutination scores by Helix and by anti-A from B; likewise, with percent agglutination measurements, A reactive appears hiher than that of A2B cells; one sample only is slightly detected by anti-A from Dolichos. 2) The B reactivity, on the contrary, is lower than that of normal AB cells. A single sample is detected by anti-B from A1. All samples are well detected by anti-B from AW, Aend, Ax, Am but none is detected by anti-B from ABx, Cis AB, or by an auto-anti-B. Under standard conditions, percent aggutination is around 80, very close to that of normal AB cells, thus differentiating Cis AB from AB3 (some of which only reach this figure), and from ABx which are very far from this value. 3) An abnormally high reactivity to anti-H antibody is observed, higher than that of normal A2B, similar to that of A2 red cells. 4) Among secretors, A substance is found to be normal or in excess, H substance is in excess, while B substance is only detected by Cis AB red cells inhibition. 5)An anti-B antibody was identified in the samples studied; however, we recently received from Germany a Cis AB samples, the serum of which did not contain anti-B antibody. By these main characteristics, the studied samples seem to be identical; however, agglutination kinetics and thermodynamic methods show that they differ by their reaction with a same anti-B antibody in standard conditions. The reactive structures of the various samples are indeed different from one family to another. The main point is that identical values were observed in all samples within a same family. Thus, the various Cis AB can be considered as different families mutants.     

Quantitative studies of the ABO blood group system. Quantitative changes in agglutinability of antigens A, B and H with regard to the patient's age]

The results of a comparative quantitative investigation about the agglutinability of erythrocyte antigens A, B, and H to be found in fetus (fifth to ninth lunar month), newborns, adults, and old age people are represented. In investigating with anti-H sera it could be found that A-antigens in fetus undergo the same development to be observed in newborns; it amounts to 75% of the agglutinability present in adults. After delivery the agglubinability will essentially increase up to the fifth month; it will only reach the average values of adults, however, after the seventh year of age and will remain unchanged then until the end of life. The agglutinability of B-erythrocytes with anti-B will also change in the same way. In fetus and newborns the agglutinability of A1 erythrocytes with anti-A1 sera is markedly weaker than that determined by anti-A sera. After delivery it will rapidly increase, will be stronger afterwards than the agglutinability with anti-A sera and will have the values to be found in adults after the third year of age. After the 85 year of age, however, there is a tendency of weakening the agglutinability with anti-A1 serum. With growing age the agglutinability of O-erythrocytes with anti-H serum is changed in the same way. Furthermore, it could be detected that the interaction between genes A1 and B to be found in adults will find its expression in a weakening of the agglutinability of A1B-erythrocytes with anti-A1 and anti-B sera, the agglutinability with anti-A serum remaining constant. This behaviour is valid for the whole life of man. In all stages of life an interaction between weak and strong genes can be observed in A2B-erythrocytes.     

Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.     

Cross reactions in the H-2 system of cytotoxic T-lymphocytes concentrated by the adsorption-elution method on target cell monolayers]

Cytotoxic T-lymphocytes (CTL) obtained by in vivo immunization were enriched by the absorption-elution technique, using the relevant allogeneic target cell (TC) monolayers. After the separation of C57BL anti-A (anti-KkDd) lymphocytes into anti-Kk and anti-Dd subpopulations they displayed cross killing cytotoxic effect on H-2d and H-2kTC, respectively. B10D2 anti-B10 (anti-KbDd) lymphocytes cross reacted to H-2a and H-2q TC. The results are discussed in the light of heterogeneity of CTL clones or their receptors.